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it is the leading cause of bacterial sexually transmitted disease in both developed and developing countries with more than 90 million new cases of genital c. trachomatis infections occurring each year . in the past years , an increase in the number of stis and in particular of c. trachomatis infections has been observed in many , if not all , european countries . this increase might be attributed to changes in attitudes , increased awareness of healthcare workers , and improved diagnostics . in the genital tract , infection with c. trachomatis is propagated within the single cell columnar layer of the epithelium in the urethra of men and the endocervix of women . within the epithelial cells , c. trachomatis undergoes a unique biphasic developmental cycle consisting of an infectious , but metabolically inert , elementary body ( eb ) and a noninfectious , but metabolically active , reticulate body ( rb ) . after completion of the developmental cycle , the ebs are released and infect neighboring epithelial cells , thereby spreading the infection . infection can result in acute inflammation characterized by redness , edema , and mucosal discharge and is diagnosed clinically as mucopurulent cervicitis in women and non - gonococcal urethritis in men [ 3 , 4 ] . in women , infection can manifest as abnormal vaginal discharge and/or postcoital bleeding , while the infection is limited to the lower genital tract and irregular uterine bleeding and/or pelvic discomfort once the infection ascends to the upper genital tract . symptoms in males are generally limited to dysuria and moderate clear to whitish discharge . while these symptoms signify an infection , the absence of such symptoms does not necessarily indicate the absence of infection . up to 75% of women and 50% of men infected with c. trachomatis are asymptomatic [ 5 , 6 ] , and these infected people do not seek medical attention . if the infection remains untreated , it often results in pelvic inflammatory disease ( pid ) , tubal scarring , ectopic pregnancy , and chronic pelvic pain in women which could lead to infertility , epididymitis in men , and infant pneumonia in children [ 710 ] . although very effective antimicrobial therapy is available , a vaccination program is considered to be the best approach to reduce the prevalence of c. trachomatis infections . it would be much cheaper and have a greater impact on controlling c. trachomatis infections worldwide than a screening program or treating infections with antibiotics . long - term induced immunity against stis such as c. trachomatis would be preferable . however , since stis have the highest incidence at the reproductive age , even short- to medium - term immunity would be of great benefit . therefore , a c. trachomatis vaccine protecting at least women in their fertile period against complications would be a valuable tool in achieving a higher level of public health . currently , there are no vaccines available against a c. trachomatis genital infection despite the many efforts that have been made throughout the years to develop a protective c. trachomatis vaccine . in this paper the many attempts to develop a protective vaccine against a genital c. trachomatis infection will be reviewed . the most used animal model to study c. trachomatis female tract infections is the mouse model . the mouse is susceptible to c. muridarum mouse pneumonitis ( mopn ) , formerly known as the mouse biovar of c. trachomatis , and to human genital tract isolates of c. trachomatis . the genomes of these species share remarkable similarities in the content and order of genes and in the presence of putative virulence factors . an important difference between the species is the absence and presence of a tryptophan operon in the genome of c. trachomatis and c. muridarum , respectively [ 12 , 13 ] . consequently , these biovars have differential sensitivity to ifn- , a cytokine which plays an important role in the early clearance of chlamydia from the genital tract . most likely the c. muridarum and the c. trachomatis strain will also differ in response to other cytokines . in contrast to human isolates , c. muridarum is able to cause severe upper genital tract pathology and a high incidence of infertility after a single infection in mice . in addition , the developmental cycle of c. muridarum is more rapid , its duration being approximately half that of human strains , and the strain is more prolific . chlamydia muridarum can infect mice of various strains nearly equally , while infection of mice with c. trachomatis is highly dependable on the mouse strain . overall , lower shedding and minimal to moderate inflammation can be noticed in mice infected with c. trachomatis . this is in accordance to the fact that upper genital tract progression followed by pathology , usually resulting from multiple infections , is only seen in a small percentage of women . in contrast to c. muridarum infection , c. trachomatis infection was unaltered in the absence of cd4 t cells . mice infected with c. trachomatis developed protective immunity to rechallenge , but unlike c. muridarum infection , optimum resistance required multiple infectious challenges despite the generation of adaptive serum and local chlamydial specific immune responses . thus , understanding the chlamydial pathogenic and host immunologic factors that result in a diminished protective role for cd4 t cells in c. trachomatis murine infection might lead to new insights important to human immunity and vaccine development . it has been demonstrated that strong adaptive immune responses are generated when mice are infected with c. trachomatis serovars [ 3 , 18 , 19 ] , but it has also been shown that these infections in mice can resolve in the absence of adaptive immunity , suggesting that innate immune responses alone can resolve infection . this is not necessarily a reason to invalidate the use of c. trachomatis serovars in murine studies of genital tract infection or in vaccine development . it could be that the rather mild infection seen in murine studies utilizing human c. trachomatis biovars may replicate some aspects of human infection . in order to resolve the murine c. muridarum genital infection and to protect against reinfection , adaptive immune responses are absolutely indispensable . as there are considerable differences between the c. muridarum and the c. trachomatis murine model , it is difficult to make direct comparisons . in order to understand the pathogenesis of human chlamydial infections completely , it is absolutely necessary to thoroughly investigate chlamydial infection in its natural human host . information on the immune mechanisms of clearance of infection and resistance to reinfection has been provided in particular by mouse models of genital infection . t cells , especially major histocompatibility complex ( mhc ) class ii - restricted cd4 t cells , are required for protective immunity [ 2124 ] . mhc class i - restricted cd8 t cells , on the other hand , are not necessary for infection resolution or immunity to reinfection [ 2124 ] . the protective role of antibody is less easily discernible than that of the cellular response , but important to vaccine development , it is as protective as cd4 t cells in immunity to reinfection [ 22 , 25 ] . furthermore , th1 cytokines , specifically ifn- and interleukin-12 ( il-12 ) , are essential to induce a protective response [ 13 , 26 , 27 ] . in women , cd4 t cells are indeed recruited to the cervix during active infection ; however , cd8 and dentritic cells are also recruited , and the relative proportions of these cells may be situational . different studies involving women have confirmed that local th1 cytokines , mainly ifn- , are associated with c. trachomatis infection ( reviewed by ) although these studies have not been able to determine which specific responses lead to infection resolution versus persistence [ 2931 ] . serum and genital mucosal igg and iga antibodies to specific c. trachomatis proteins and to chlamydial ebs are usually detected during active infection in women [ 3234 ] . these antibody responses in humans infected with c. trachomatis , including those measured in endocervical secretions , have not been found to correlate with protective immunity but appear to be markers of prior infection . when developing a vaccine against genital c. trachomatis infections , it is important to take into account the unique properties of the genital tract . this mucosal site is unique among mucosal effector tissues , as it lacks organized lymphatics which can result in a delayed systemic response relative to other sites . furthermore , the female genital tract is also subjected to hormonal regulation , and the effectiveness of intravaginal vaccination has been shown to be influenced by the phase of the menstrual cycle [ 3537 ] . the immunological characteristics of the genital tract and the tropism of chlamydia for mucosal epithelial cells show that a c. trachomatis vaccine has to induce both mucosal and systemic protective responses . initial attempts to develop an effective vaccine for controlling both animal and human chlamydial infections began with the use of inactivated or live , attenuated whole organism preparations in the 1950s . common problems are the cost and the complexity of production , the requirement for cold storage , the presence of antigens which can induce autoimmunity or immunopathology , and the limited efficacy in neonates with high levels of maternal antibodies . the first vaccines that were used against chlamydiaceae were live vaccines . with this method of immunization , attenuated or the development of attenuated strains usually happens by a number of passages of the wild - type strain in different types of cell cultures or by chemical mutagenesis . due to the passages , one or more mutations could arise , resulting in a nonvirulent attenuated strain . live attenuated vaccines can elicit humoral and cellular immunity , because they replicate in a manner analogous to the target pathogen , promoting the processing and presentation of antigens in a way that is most similar to the natural infection . on the other hand , they can also revert to the virulent wild - type strain resulting in disease or persistent infection . whole - organism vaccination is unlikely to be attempted in the near future , because there is a risk of immunopathology , the large - scale production of pure chlamydiae is extremely difficult and because of the possible spread of live chlamydiaceae in the environment . in the 1960s , unsuccessful vaccination trials with live attenuated vaccines against trachoma were performed in humans and primates . four decades later , several authors have explored the possibility to vaccinate with live attenuated bacteria against genital c. trachomatis infection . immunized mice intranasally or intraperitoneally with viable c. trachomatis , serovar e. mice immunized intranasally with live c. trachomatis exhibited significant protection upon a vaginal infection , while intraperitoneally immunized mice did not . however , the protection was not complete . performed an experiment in mice to investigate the ability of a live attenuated c. trachomatis vaccine to prevent genital infection . results showed that a self - limiting subclinical infection of the murine genital tract with c. trachomatis is as efficient as a clinically apparent acute infection in generating a protective anti - chlamydial immune response . based on these results , the authors concluded that a live attenuated vaccine would be useful for the prevention of chlamydial stis . evaluated the protective immunity of the attenuated c. trachomatis l2 ( 25667r ) strain in a murine model . they concluded that intravaginal vaccination with the live - attenuated strain l2 is safe , induces a systemic antibody and a cd4 th1-based immune response , but its protective efficacy is limited to reducing chlamydial burden at early time periods after - infection . vaccinated mice intranasally with live c. muridarum with or without cpg - containing oligodeoxynucleotide 1862 . protection was correlated with the frequency of multifunctional t cells coexpressing ifn- and tnf- with or without il-2 . these results suggest that ifn- producing cd4 t cells that highly coexpress tnf- may be the optimal effector cells for protective immunity . in view of the safety aspects ( possible return to the virulent wild type strain ) and the risk for immunopathological damage , it seems unlikely that a live attenuated c. trachomatis vaccine will be allowed in humans . because live vaccines are not always safe or available , research switched to the use of killed or inactivated organisms . they may contain undesirable components like bacterial endotoxins , that can cause detrimental side effects , or nonprotective components that may reduce the degree of protection that is required . their major disadvantage is that they are not able to replicate anymore , which stresses the need to revaccinate and to use adjuvants . another consequence of their inability to replicate is that they are poor inducers of cell - mediated immunity although they can induce and adequate level of humoral immunity . because a strong cell - mediated immunity is needed for clearance of chlamydial infections , inactivated or killed organisms seem to be less suitable for vaccine development against chlamydiaceae . studies on inactivated or killed organism vaccines against genital c. trachomatis infection are rare . in this study , peterson et al . failed to elicit a protective response to a vaginal c. trachomatis infection in mice immunized intranasally and intraperitoneally with 1 10 uv inactivated inclusion forming units of c. trachomatis serovar e. in order to avoid harmful effect of the preparations containing the whole organism , it was proposed that a subunit vaccine was needed . subunit vaccines are safer , they can not revert to a virulent form , and undesirable antigens , which can induce immunopathology or inflammatory damage , can be avoided . vaccine candidate antigens , or parts of antigens , may be represented as purified proteins , recombinant proteins or as synthetic proteins . but subunit vaccines have also some disadvantages . like inactivated vaccines , they are poor inducers of cell - mediated immunity , which is very important in the defense against chlamydial infections . furthermore , the use of adjuvants is being recommended . following the identification of the major outer membrane protein ( momp ) as the structurally and immunologically dominant protein in the chlamydial outer membrane , vaccine research mainly focused on this protein . pal et al . found that a chlamydial outer membrane complex ( comc ) preparation of c. muridarum could induce significantly protective immunity in mice against a genital challenge , while purified momp preparations could not . some years later , the same research group immunized mice with a purified and refolded preparation of the c. muridarum momp in combination with freund 's adjuvants . a significant level of protection was conferred in the vaccinated mice against a genital challenge . demonstrated the protective potential of native momp of a c. muridarum serovar in combination with novel adjuvants , the nontoxic subunit b of cholera toxin ( ctb - cpg ) . immunization elicited a significant antigen - specific antibody and cell - mediated immune response as well as protection against a pulmonary challenge with c. muridarum . could demonstrate that immunization of mice with purified c. muridarum momp could induce neutralizing antibodies which leaded to reduced numbers of infected mice . therefore , it is important to keep in mind that immunity can potentially induce pathology and this should be considered when designing vaccines . igietseme and murdin prepared a momp - iscom vaccine based on momp extracted from c. trachomatis serovar d. this vaccine was able to produce a th1 antigen - specific immune response , and immunized mice cleared a vaginal infection within one week . from these studies , it is clear that some preparations can induce more protection than others . this is probably due to the difference in extraction method which can influence the preservation of conformational momp epitopes , necessary for protection . although vaccination with refolded , purified momp preparations have been reasonable successful , the major drawbacks of these vaccines are that they are very expensive and there are problems to grow chlamydia in bulk , which renders these kinds of vaccines commercially non - viable . nowadays , it is possible to produce high amounts of bacterial proteins by recombinant dna technology which is cheaper and more cost effective . the genes , coding for protective antigens , will be expressed in prokaryotic or eukaryotic cells that will produce the desired recombinant protein . for chlamydial vaccines , recombinant momp ( rmomp ) however , the expression of full - length rmomp in prokaryotic expression systems is generally toxic , and it is also difficult to produce rmomp in a native form with intact , conformationally relevant epitopes . different attempts were made to elicit protection against a c. trachomatis infection by rmomp vaccination . transcutaneous immunization with momp in combination with the cholera toxin and cpg oligodeoxynucleotides elicits igg and iga antibody response in the vaginal and cervical lavage fluid and an igg antibody response in the serum . the immunization protocol resulted in enhanced clearance of c. muridarum following intravaginal challenge of mice . pal et al . demonstrated that immunisation with purified c. muridarum momp , co - administered with borrelia burgdorferi outer surface protein ( osp ) a as adjuvant , can induce significant protection in mice against a c. muridarum genital infection . sun et al . compared vaccines based on recombinant ( rmomp ) and native momp ( nmomp ) . the recombinant preparation based on c. muridarum momp can elicit a protective immune response in mice against an intranasal challenge . however , the degree of protection obtained with the rmomp was not as robust as that achieved with an nmomp preparation indicating that the structural conformation of the momp is important for inducing protection . hickey et al . showed that transcutaneous immunization of mice with rmomp incorporated in lipid c , induces partial protection of both the respiratory and genital mucosae against challenge with c. muridarum . the efficacy of a recombinant vaccine is not only defined by the protein that is used but also by the administration routes . it has been proven that a combined systemic and mucosal vaccination with rmomp provides better protection against a challenge with c. muridarum than either systemic or mucosal immunization alone . systemic immunization of mice with rmomp from c. trachomatis could reduce the number of animals developing severe salpingitis but failed to reduce chlamydial colonization of the lower genital tract . mice , immunized with rmomp directly into the peyer 's patches ( to stimulate mucosal immunity ) , shed fewer chlamydiae from the vagina , but showed little reduction in oviduct damage . although in both cases specific igg and iga antibody responses could be observed , they could not completely protect the mice . although most recombinant vaccines are based on momp , other proteins can also be viable vaccine candidates . in 2007 , a novel vaccination strategy using a secreted protein , chlamydial protease - like activity factor ( cpaf ) was developed by murthy et al . . intranasal immunization using recombinant cpaf ( rcpaf ) accompanied by interleukin-12 ( il-12 ) was used to assess the protective immunity against genital c. muridarum infection in balb / c mice . rcpaf + il-12-vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and accelerated resolution of infection compared to mock - immunized animals . moreover , rcpaf + il-12-immunized animals exhibited protection against pathological consequences of chlamydial infection . these results demonstrate for the first time that a secreted chlamydial protein , cpaf , is a viable vaccine candidate that should be considered for induction of efficacious , antichlamydial immunity . the chlamydial proteins omcb and rl16 have been identified as human b and t cell targets during chlamydial infections in humans [ 65 , 66 ] . vaccination of mice with a fusion protein ( cth1 ) composed of those two antigens promoted a cd4 t - cell dependent protective response but lacks a cd4 independent protective mechanism for complete protection . today , computer - based methods to predict antigenic domains or epitopes are available . synthetic production of these epitopes makes it possible to produce synthetic peptides which correspond with the important immunogenic domains on the antigens . on the other hand , we have to take into account that a lot of antigenic determinants need conformational or three - dimensional structures , like in the complete protein , to elicit an immune response . preliminary studies in mice indicated that intradermal injection of a peptide from a conserved region of the momp of c. trachomatis , conferred some protection against the development of salpingitis . in contrast to these findings , su et al . found that parenteral immunization of mice with an alum - adsorbed synthetic oligopeptide of the c. trachomatis momp , was ineffective in preventing chlamydial genital tract infection although mice produced high levels of antichlamydial serum igg neutralizing antibodies . therefore , dna vaccination which induces both humoral and cellular immune responses can be an alternative method to protect animals from chlamydial infections . injection of plasmid dna encoding a foreign gene of interest can result in the subsequent expression of the foreign gene product and the induction of an immune response within the host . they encode multiple immunogenic epitopes and evoke both humoral and cell - mediated immune responses . therefore , dna vaccines exhibit the advantages of attenuated vaccines without the safety problems associated with the in vivo replication and possible reversion to a virulent form . due to the endogenous production of the antigen large - scale manufacturing procedures are available and the dna can be easily and inexpensively purified to homogeneity , resulting in lower costs to develop and manufacture this type of vaccine [ 42 , 72 ] . this makes this strategy applicable as a human vaccine approach in underdeveloped countries and as a veterinary vaccine strategy , where the cost per dose is of major economic concern . in addition , dna is more thermostable than vaccine strategies which require a cold chain for storage , and it should exhibit a longer shelf - life because of the improved stability . dna also allows a more simplified and effective quality control process that provides additional cost benefits . this has not been proven yet , and it is thought that the chance that this will happen is lower than the spontaneous mutation frequency . immune responses to dna occur in autoimmune diseases , and the possibility exists that bacterial dna injection could induce an immune response that might cross - react with host dna . thirdly , long - term expression of injected dna into muscle cells may have an effect on immune responses to subsequent vaccination with different dna , and the immune responses to protective epitopes associated with this second immunization can be compromised . the fourth disadvantage is that dna vaccination strategies are unsuccessful when evaluating non - protein - based antigens , such as bacterial polysaccharides and lipids . other possible disadvantages are the low transfection and expression efficiency of dna vaccines , certainly in large animals and humans . however , by using various combinations of delivery systems and different adjuvants , the immune response can be enhanced . in the past , different studies have evaluated the protective potential of dna vaccines against chlamydial infections . the first attempt to generate an momp - based dna vaccine against a genital chlamydial challenge was disappointing . only modest immune response was elicited , but no protection could be established against infection or disease . because dna immunization alone did not generate immune responses or protection to the same extent as those induced by using live organisms , combinational vaccines were evaluated . dna priming followed by boosting with immune - stimulating complexes ( iscom ) of momp protein ( momp iscom ) in mice resulted in higher protection when compared to mice given momp iscom immunization alone . in 2010 and 2011 , schautteet et al . [ 78 , 79 ] studied the ability of a dna vaccine based on c. trachomatis momp to protect against genital c. trachomatis infection in a recently developed pig model . when administrating the vaccine to the vaginal mucosa , a cellular immune response was induced which elicited significant protection in pigs . the infection could not be cleared completely . when the dna vaccine was administered combined to the nasal and vaginal mucosa of the pig , both cellular and humoral immune responses were induced which contributed to the significant protection of pigs against a genital c. trachomatis infection . since a couple of years , other genes than ompa were evaluated for their potential as vaccine candidates . dna immunization with the pgp3 gene of c. trachomatis could inhibit the spread of the infection from the lower to the upper genital tract . the pgp3 gene encodes a 28 kda polypeptide found on the pct plasmid of c. trachomatis which may provide a function related to chlamydial cell physiology . ifere et al . developed a dna vaccine composed of momp and the porin b protein ( porb ) of c. trachomatis . a recombinant vibrio cholerae ghost ( rvcg ) was used as carrier and delivery system . significant higher levels of th1 response and secretory iga and igg2a were induced by immunization . furthermore , all animals which were immunized with the multisubunit vaccine completely resolved the infection two weeks after challenge . in 2008 , a porf5 dna vaccine was evaluated for its protective immunity in a mouse model of genital chlamydial infection . the vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and an accelerated resolution of the infection . these results demonstrate the potential of the porf5 dna vaccine to elicit protective immunity against a genital chlamydial challenge . recently , a mathematical model has been developed that simulates transmission in a heterosexual population by linking the within - host biology of susceptibility and the chlamydia - infected individuals to their sexual behavior and partnership dynamics . the model tracks the infection time course , disease progression , and dynamic infectiousness of infected individuals and the transmission to others . the authors have demonstrated that if a fully protective vaccine is available , and this will be administered to adolescents before their sexual debut , epidemics of chlamydia infection could be eradicated within 20 years . furthermore , it is likely that targeting 100% of one sex ( females ) will have a greater epidemiological impact than administering vaccines to 50% of both sexes . if lifelong sterilizing immunity can not be achieved , a chlamydia vaccine should be effective for at least 10 years in order to lead to population - level eradication . based on the information generated by this mathematical model , the candidate vaccines should protect individuals by raising the infectiousness threshold and secondary reduce the peak load and the duration of the infection in vaccinated individuals who become infected . vaccination could be substantially more effective than other biomedical interventions in controlling epidemics of chlamydia infection . currently , the best public health intervention available is increasing the rate of screening and treating infected individuals . administrating a protective vaccine to adolescents before their first sexual experience could induce a significant reduction in prevalence which could not be obtained by screening teenagers , even with a coverage of 100% . unfortunately , no protective vaccines , either fully or partially , are available although there have been many attempts to develop one . the reasons for the variability in success are still unclear but are probably a consequence of different immunization protocols and a reflection of the different protective mechanisms required for the different infections .

chlamydia trachomatis is a gram - negative obligate intracellular bacterium . it is the leading cause of bacterial sexual transmitted infections ( stis ) . world health organization figures estimated that over 90 million new cases of genital c. trachomatis infections occur worldwide each year . a vaccination program is considered to be the best approach to reduce the prevalence of c. trachomatis infections , as it would be much cheaper and have a greater impact on controlling c. trachomatis infections worldwide rather than a screening program or treating infections with antibiotics . currently , there are no vaccines available which effectively protect against a c. trachomatis genital infection despite the many efforts that have been made throughout the years . in this paper , the many attempts to develop a protective vaccine against a genital c. trachomatis infection will be reviewed .

1. Introduction 2. 3. Protective Immune Responses to 4. Whole Organism VaccinesFirst-Generation 5. Subunit VaccinesSecond-Generation 6. DNA VaccinesThird-Generation 7. Impact of a 8. Conclusions

it is the leading cause of bacterial sexually transmitted disease in both developed and developing countries with more than 90 million new cases of genital c. trachomatis infections occurring each year . in the past years , an increase in the number of stis and in particular of c. trachomatis infections has been observed in many , if not all , european countries . this increase might be attributed to changes in attitudes , increased awareness of healthcare workers , and improved diagnostics . in the genital tract , infection with c. trachomatis is propagated within the single cell columnar layer of the epithelium in the urethra of men and the endocervix of women . within the epithelial cells , c. trachomatis undergoes a unique biphasic developmental cycle consisting of an infectious , but metabolically inert , elementary body ( eb ) and a noninfectious , but metabolically active , reticulate body ( rb ) . after completion of the developmental cycle , the ebs are released and infect neighboring epithelial cells , thereby spreading the infection . while these symptoms signify an infection , the absence of such symptoms does not necessarily indicate the absence of infection . up to 75% of women and 50% of men infected with c. trachomatis are asymptomatic [ 5 , 6 ] , and these infected people do not seek medical attention . although very effective antimicrobial therapy is available , a vaccination program is considered to be the best approach to reduce the prevalence of c. trachomatis infections . it would be much cheaper and have a greater impact on controlling c. trachomatis infections worldwide than a screening program or treating infections with antibiotics . long - term induced immunity against stis such as c. trachomatis would be preferable . however , since stis have the highest incidence at the reproductive age , even short- to medium - term immunity would be of great benefit . therefore , a c. trachomatis vaccine protecting at least women in their fertile period against complications would be a valuable tool in achieving a higher level of public health . currently , there are no vaccines available against a c. trachomatis genital infection despite the many efforts that have been made throughout the years to develop a protective c. trachomatis vaccine . in this paper the many attempts to develop a protective vaccine against a genital c. trachomatis infection will be reviewed . the most used animal model to study c. trachomatis female tract infections is the mouse model . the mouse is susceptible to c. muridarum mouse pneumonitis ( mopn ) , formerly known as the mouse biovar of c. trachomatis , and to human genital tract isolates of c. trachomatis . the genomes of these species share remarkable similarities in the content and order of genes and in the presence of putative virulence factors . an important difference between the species is the absence and presence of a tryptophan operon in the genome of c. trachomatis and c. muridarum , respectively [ 12 , 13 ] . most likely the c. muridarum and the c. trachomatis strain will also differ in response to other cytokines . in addition , the developmental cycle of c. muridarum is more rapid , its duration being approximately half that of human strains , and the strain is more prolific . chlamydia muridarum can infect mice of various strains nearly equally , while infection of mice with c. trachomatis is highly dependable on the mouse strain . overall , lower shedding and minimal to moderate inflammation can be noticed in mice infected with c. trachomatis . this is in accordance to the fact that upper genital tract progression followed by pathology , usually resulting from multiple infections , is only seen in a small percentage of women . in contrast to c. muridarum infection , c. trachomatis infection was unaltered in the absence of cd4 t cells . mice infected with c. trachomatis developed protective immunity to rechallenge , but unlike c. muridarum infection , optimum resistance required multiple infectious challenges despite the generation of adaptive serum and local chlamydial specific immune responses . thus , understanding the chlamydial pathogenic and host immunologic factors that result in a diminished protective role for cd4 t cells in c. trachomatis murine infection might lead to new insights important to human immunity and vaccine development . it has been demonstrated that strong adaptive immune responses are generated when mice are infected with c. trachomatis serovars [ 3 , 18 , 19 ] , but it has also been shown that these infections in mice can resolve in the absence of adaptive immunity , suggesting that innate immune responses alone can resolve infection . this is not necessarily a reason to invalidate the use of c. trachomatis serovars in murine studies of genital tract infection or in vaccine development . it could be that the rather mild infection seen in murine studies utilizing human c. trachomatis biovars may replicate some aspects of human infection . in order to resolve the murine c. muridarum genital infection and to protect against reinfection , adaptive immune responses are absolutely indispensable . as there are considerable differences between the c. muridarum and the c. trachomatis murine model , it is difficult to make direct comparisons . in order to understand the pathogenesis of human chlamydial infections completely , it is absolutely necessary to thoroughly investigate chlamydial infection in its natural human host . information on the immune mechanisms of clearance of infection and resistance to reinfection has been provided in particular by mouse models of genital infection . t cells , especially major histocompatibility complex ( mhc ) class ii - restricted cd4 t cells , are required for protective immunity [ 2124 ] . the protective role of antibody is less easily discernible than that of the cellular response , but important to vaccine development , it is as protective as cd4 t cells in immunity to reinfection [ 22 , 25 ] . furthermore , th1 cytokines , specifically ifn- and interleukin-12 ( il-12 ) , are essential to induce a protective response [ 13 , 26 , 27 ] . different studies involving women have confirmed that local th1 cytokines , mainly ifn- , are associated with c. trachomatis infection ( reviewed by ) although these studies have not been able to determine which specific responses lead to infection resolution versus persistence [ 2931 ] . serum and genital mucosal igg and iga antibodies to specific c. trachomatis proteins and to chlamydial ebs are usually detected during active infection in women [ 3234 ] . these antibody responses in humans infected with c. trachomatis , including those measured in endocervical secretions , have not been found to correlate with protective immunity but appear to be markers of prior infection . when developing a vaccine against genital c. trachomatis infections , it is important to take into account the unique properties of the genital tract . this mucosal site is unique among mucosal effector tissues , as it lacks organized lymphatics which can result in a delayed systemic response relative to other sites . furthermore , the female genital tract is also subjected to hormonal regulation , and the effectiveness of intravaginal vaccination has been shown to be influenced by the phase of the menstrual cycle [ 3537 ] . the immunological characteristics of the genital tract and the tropism of chlamydia for mucosal epithelial cells show that a c. trachomatis vaccine has to induce both mucosal and systemic protective responses . initial attempts to develop an effective vaccine for controlling both animal and human chlamydial infections began with the use of inactivated or live , attenuated whole organism preparations in the 1950s . common problems are the cost and the complexity of production , the requirement for cold storage , the presence of antigens which can induce autoimmunity or immunopathology , and the limited efficacy in neonates with high levels of maternal antibodies . whole - organism vaccination is unlikely to be attempted in the near future , because there is a risk of immunopathology , the large - scale production of pure chlamydiae is extremely difficult and because of the possible spread of live chlamydiaceae in the environment . four decades later , several authors have explored the possibility to vaccinate with live attenuated bacteria against genital c. trachomatis infection . immunized mice intranasally or intraperitoneally with viable c. trachomatis , serovar e. mice immunized intranasally with live c. trachomatis exhibited significant protection upon a vaginal infection , while intraperitoneally immunized mice did not . however , the protection was not complete . performed an experiment in mice to investigate the ability of a live attenuated c. trachomatis vaccine to prevent genital infection . results showed that a self - limiting subclinical infection of the murine genital tract with c. trachomatis is as efficient as a clinically apparent acute infection in generating a protective anti - chlamydial immune response . based on these results , the authors concluded that a live attenuated vaccine would be useful for the prevention of chlamydial stis . evaluated the protective immunity of the attenuated c. trachomatis l2 ( 25667r ) strain in a murine model . these results suggest that ifn- producing cd4 t cells that highly coexpress tnf- may be the optimal effector cells for protective immunity . in view of the safety aspects ( possible return to the virulent wild type strain ) and the risk for immunopathological damage , it seems unlikely that a live attenuated c. trachomatis vaccine will be allowed in humans . they may contain undesirable components like bacterial endotoxins , that can cause detrimental side effects , or nonprotective components that may reduce the degree of protection that is required . because a strong cell - mediated immunity is needed for clearance of chlamydial infections , inactivated or killed organisms seem to be less suitable for vaccine development against chlamydiaceae . studies on inactivated or killed organism vaccines against genital c. trachomatis infection are rare . in this study , peterson et al . failed to elicit a protective response to a vaginal c. trachomatis infection in mice immunized intranasally and intraperitoneally with 1 10 uv inactivated inclusion forming units of c. trachomatis serovar e. in order to avoid harmful effect of the preparations containing the whole organism , it was proposed that a subunit vaccine was needed . furthermore , the use of adjuvants is being recommended . found that a chlamydial outer membrane complex ( comc ) preparation of c. muridarum could induce significantly protective immunity in mice against a genital challenge , while purified momp preparations could not . some years later , the same research group immunized mice with a purified and refolded preparation of the c. muridarum momp in combination with freund 's adjuvants . a significant level of protection was conferred in the vaccinated mice against a genital challenge . demonstrated the protective potential of native momp of a c. muridarum serovar in combination with novel adjuvants , the nontoxic subunit b of cholera toxin ( ctb - cpg ) . immunization elicited a significant antigen - specific antibody and cell - mediated immune response as well as protection against a pulmonary challenge with c. muridarum . therefore , it is important to keep in mind that immunity can potentially induce pathology and this should be considered when designing vaccines . igietseme and murdin prepared a momp - iscom vaccine based on momp extracted from c. trachomatis serovar d. this vaccine was able to produce a th1 antigen - specific immune response , and immunized mice cleared a vaginal infection within one week . from these studies , it is clear that some preparations can induce more protection than others . although vaccination with refolded , purified momp preparations have been reasonable successful , the major drawbacks of these vaccines are that they are very expensive and there are problems to grow chlamydia in bulk , which renders these kinds of vaccines commercially non - viable . nowadays , it is possible to produce high amounts of bacterial proteins by recombinant dna technology which is cheaper and more cost effective . the genes , coding for protective antigens , will be expressed in prokaryotic or eukaryotic cells that will produce the desired recombinant protein . for chlamydial vaccines , recombinant momp ( rmomp ) however , the expression of full - length rmomp in prokaryotic expression systems is generally toxic , and it is also difficult to produce rmomp in a native form with intact , conformationally relevant epitopes . different attempts were made to elicit protection against a c. trachomatis infection by rmomp vaccination . the immunization protocol resulted in enhanced clearance of c. muridarum following intravaginal challenge of mice . demonstrated that immunisation with purified c. muridarum momp , co - administered with borrelia burgdorferi outer surface protein ( osp ) a as adjuvant , can induce significant protection in mice against a c. muridarum genital infection . sun et al . the recombinant preparation based on c. muridarum momp can elicit a protective immune response in mice against an intranasal challenge . however , the degree of protection obtained with the rmomp was not as robust as that achieved with an nmomp preparation indicating that the structural conformation of the momp is important for inducing protection . hickey et al . showed that transcutaneous immunization of mice with rmomp incorporated in lipid c , induces partial protection of both the respiratory and genital mucosae against challenge with c. muridarum . it has been proven that a combined systemic and mucosal vaccination with rmomp provides better protection against a challenge with c. muridarum than either systemic or mucosal immunization alone . systemic immunization of mice with rmomp from c. trachomatis could reduce the number of animals developing severe salpingitis but failed to reduce chlamydial colonization of the lower genital tract . intranasal immunization using recombinant cpaf ( rcpaf ) accompanied by interleukin-12 ( il-12 ) was used to assess the protective immunity against genital c. muridarum infection in balb / c mice . these results demonstrate for the first time that a secreted chlamydial protein , cpaf , is a viable vaccine candidate that should be considered for induction of efficacious , antichlamydial immunity . the chlamydial proteins omcb and rl16 have been identified as human b and t cell targets during chlamydial infections in humans [ 65 , 66 ] . synthetic production of these epitopes makes it possible to produce synthetic peptides which correspond with the important immunogenic domains on the antigens . on the other hand , we have to take into account that a lot of antigenic determinants need conformational or three - dimensional structures , like in the complete protein , to elicit an immune response . preliminary studies in mice indicated that intradermal injection of a peptide from a conserved region of the momp of c. trachomatis , conferred some protection against the development of salpingitis . found that parenteral immunization of mice with an alum - adsorbed synthetic oligopeptide of the c. trachomatis momp , was ineffective in preventing chlamydial genital tract infection although mice produced high levels of antichlamydial serum igg neutralizing antibodies . injection of plasmid dna encoding a foreign gene of interest can result in the subsequent expression of the foreign gene product and the induction of an immune response within the host . therefore , dna vaccines exhibit the advantages of attenuated vaccines without the safety problems associated with the in vivo replication and possible reversion to a virulent form . due to the endogenous production of the antigen large - scale manufacturing procedures are available and the dna can be easily and inexpensively purified to homogeneity , resulting in lower costs to develop and manufacture this type of vaccine [ 42 , 72 ] . this has not been proven yet , and it is thought that the chance that this will happen is lower than the spontaneous mutation frequency . thirdly , long - term expression of injected dna into muscle cells may have an effect on immune responses to subsequent vaccination with different dna , and the immune responses to protective epitopes associated with this second immunization can be compromised . however , by using various combinations of delivery systems and different adjuvants , the immune response can be enhanced . the first attempt to generate an momp - based dna vaccine against a genital chlamydial challenge was disappointing . dna priming followed by boosting with immune - stimulating complexes ( iscom ) of momp protein ( momp iscom ) in mice resulted in higher protection when compared to mice given momp iscom immunization alone . [ 78 , 79 ] studied the ability of a dna vaccine based on c. trachomatis momp to protect against genital c. trachomatis infection in a recently developed pig model . when the dna vaccine was administered combined to the nasal and vaginal mucosa of the pig , both cellular and humoral immune responses were induced which contributed to the significant protection of pigs against a genital c. trachomatis infection . dna immunization with the pgp3 gene of c. trachomatis could inhibit the spread of the infection from the lower to the upper genital tract . the pgp3 gene encodes a 28 kda polypeptide found on the pct plasmid of c. trachomatis which may provide a function related to chlamydial cell physiology . developed a dna vaccine composed of momp and the porin b protein ( porb ) of c. trachomatis . furthermore , all animals which were immunized with the multisubunit vaccine completely resolved the infection two weeks after challenge . in 2008 , a porf5 dna vaccine was evaluated for its protective immunity in a mouse model of genital chlamydial infection . the vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and an accelerated resolution of the infection . these results demonstrate the potential of the porf5 dna vaccine to elicit protective immunity against a genital chlamydial challenge . the authors have demonstrated that if a fully protective vaccine is available , and this will be administered to adolescents before their sexual debut , epidemics of chlamydia infection could be eradicated within 20 years . furthermore , it is likely that targeting 100% of one sex ( females ) will have a greater epidemiological impact than administering vaccines to 50% of both sexes . if lifelong sterilizing immunity can not be achieved , a chlamydia vaccine should be effective for at least 10 years in order to lead to population - level eradication . based on the information generated by this mathematical model , the candidate vaccines should protect individuals by raising the infectiousness threshold and secondary reduce the peak load and the duration of the infection in vaccinated individuals who become infected . vaccination could be substantially more effective than other biomedical interventions in controlling epidemics of chlamydia infection . currently , the best public health intervention available is increasing the rate of screening and treating infected individuals . administrating a protective vaccine to adolescents before their first sexual experience could induce a significant reduction in prevalence which could not be obtained by screening teenagers , even with a coverage of 100% . unfortunately , no protective vaccines , either fully or partially , are available although there have been many attempts to develop one .

acrylamide ( acr ) is monomeric precursor for many industrially important polymeric products ; however , the incidental formation of acr during the cooking process of many common starchy foods is the primary source of human exposure in the range of 1 g / kg / day ( 1 ) . the likely major pathway in the formation of acrylamide in food cooked at high temperature involves the maillard browning reaction between reducing sugars and asparagine , an amino acid present in potatoes and cereals ( 2 ) . in fact , the maillard reaction is considered desirable in the production of starchy food because it imparts a palatable taste , even though it reduces the bioavailability of some amino acids , including taurine and lysine and increase acr formation ( 3 ) . acrylamide is metabolized by hepatic cyp 2e1 produces an epoxide , glycidamide ( ga ) , which is reactive toward protein and nucleic acid nucleophiles ( 4 ) . ga had been reported to be 100 - 1000 times more reactive with dna than acrylamide , and considered as genotoxic in a variety of test systems ( 5 ) . many studies were done to test the effects of oral acrylamide administration on the testicular functions and the effects include : severe testicular damage , significant reduction in mating , fertility , and pregnancy by formation of abnormal sperm and a decrease in sperm count which could be a result of alkylation of sh groups in the sperm nucleus and tail , depletion of gsh , or dna damage in the testis ( 6 ) . testicular toxicity had been reported in rodents exposed to acr , changes in gonadal and pituitary hormones had been associated with histopathological and gene expression changes ( 7 ) . one effective approach against environmental agents - induced toxicity is prevention , so identification and development of effective chemopreventive agents that block activation or enhance detoxification of environmental agents is an important aspect . alpha - lipoic acid ( la ) is a naturally occurring nutraceutical , whose therapeutic effect has been related to its antioxidant activity and its ability to repair oxidative damage ( 8) . it is readily distributed and accumulates in several tissues where it is rapidly converted to its more potent antioxidant form dihydrolipoic acid ( 9 ) . because of its small size and high lipophilicity , it crosses biological membranes easily and quenches free radicals in both lipid and aqueous environments ( 10 ) . pretreatment of spermatic cord torsion with la significantly protects against ischemia / reperfusion injury by decreasing oxidative stress ( 11 ) . la is used as a protector against lipopolysaccharide - induced oxidative stress in adult rat sertoli cells in vitro ( 12 ) . the idea that free radical scavengers are protective against pollutant induced toxicity ( 13 ) has provoked the present study to investigate the potential protective effect of la against acrylamide - induced oxidative toxicity in the reproductive system as represented by changes in sex hormone levels , and oxidative stress and antioxidant status in male rats . to the best of the author s knowledge , the effect of la on acrylamide treatment with respect to reproductive markers has not yet been established . acrylamide ( cas number 79 - 06 - 1 ) and alpha - lipoic acid were obtained from sigma chemicals co. ( st . quentin - fallavier , france ) , 5,5- dithiobis-2-nitrobenzoic acid , and 1- chloro-2,4-dinitrobenzene , reduced glutathione , thiobarbituric acid , and butylated hydroxytoluene were purchased from fluka ( buchs , switzerland ) , and testosterone , progesterone and estradiol hormones elisa kit from cayman chemical company . all the other reagents were of analytical , high - performance liquid chromatography , or the best available pharmaceutical grade . forty male rats weighing 210 7 g were housed 10 per cage with wood shavings as bedding , and were maintained under a controlled environment at 25 2c , 55 5% relative humidity , and a 12 h/12 h light / dark cycle throughout the experimental period . the animal experiments were carried out in accordance with the national institutes of health guidelines for the care and use of laboratory animals , and the study protocol was approved by the local authorities . after 2 weeks of acclimatization , the rats were randomly divided into four groups of 10 rats each . group i was the control group received normal basal diet , group ii and group iv were administered acrylamide 0.05% ( w / v ) in drinking water for 21 days , group iv received beside acrylamide alpha - lipoic acid 1% mixed with diet 7 day before and along with acrylamide treatment , and group iii were received alpha - lipoic acid 1% mixed with diet for 28 days . twenty - four hours after the last administration of alpha - lipoic acid or acr , the rats were anesthetized with diethyl ether . then , blood samples were collected from the inner canthus of the eye by heparinized capillary tube into clean test tubes . following standing in room temperature for at least 30 min , the blood was centrifuged at 3400 xg for 10 min and the serum was separated , transferred to eppendorf tubes , and stored at -20c prior till biochemical analysis . immediately after the collection of blood samples , the animals were euthanized , and their testis and epididymis were quickly excised , rinsed in ice - cold saline and used immediately or stored frozen at -80c until analysis . elisa procedure was used for quantitative determination of serum total testosterone , progesterone and estradiol levels ( dima gesellschaft fur diagnostika [ gmbh ] , germany ) according to manufacturer s instructions . tissue homogenates were separately prepared from frozen testis and epididymis samples in 10 volumes of 0.1 m tris edta buffer ( ph 7.4 ) and centrifuged at 8000 xg for 30 min at 4c . aliquots of the supernatant were utilized for the spectrophotometrical assessment of the levels of the following : lipid peroxidation ( lpo ) , assessed as the production of the thiobarbituric acid reactive substances ( tbars ) in the presence of bht ( 14 ) ; reduced glutathione , by using ellman s reagent ( 15 ) ; glutathione s - transferase ( gst , ec 2.5.1.18 ) activity , as the rate of gsh conjugation of cdnb ( 16 ) ; glutathione peroxidase ( gpx , ec 1.11.1.9 ) activity was determined using reduced glutathione and cummene hydroperxide as substrate by the modified method of ( 17 ) and glutathione reductase ( gr ) activity was measured according to the method of ( 18 ) . the excised testes and epididymis were fixed in bouin s solution , for 24 h and processed using standard laboratory procedures for histology . the tissue was embedded in paraffin blocks , sectioned perpendicular to the longest axis of the testis with 5-cm thickness , and stained routinely with hematoxylin and eosin . stained sections were examined using light microscopy at power 200 and 400 for detection of histopathological changes ( 19 ) . results are expressed as mean sem with the experiment repeated at least three times . statistical evaluations were done using the analysis of variance ( anova ) . a p value of < 0.05 was considered significant . acrylamide ( cas number 79 - 06 - 1 ) and alpha - lipoic acid were obtained from sigma chemicals co. ( st . quentin - fallavier , france ) , 5,5- dithiobis-2-nitrobenzoic acid , and 1- chloro-2,4-dinitrobenzene , reduced glutathione , thiobarbituric acid , and butylated hydroxytoluene were purchased from fluka ( buchs , switzerland ) , and testosterone , progesterone and estradiol hormones elisa kit from cayman chemical company . all the other reagents were of analytical , high - performance liquid chromatography , or the best available pharmaceutical grade . forty male rats weighing 210 7 g were housed 10 per cage with wood shavings as bedding , and were maintained under a controlled environment at 25 2c , 55 5% relative humidity , and a 12 h/12 h light / dark cycle throughout the experimental period . the animal experiments were carried out in accordance with the national institutes of health guidelines for the care and use of laboratory animals , and the study protocol was approved by the local authorities . after 2 weeks of acclimatization , the rats were randomly divided into four groups of 10 rats each . group i was the control group received normal basal diet , group ii and group iv were administered acrylamide 0.05% ( w / v ) in drinking water for 21 days , group iv received beside acrylamide alpha - lipoic acid 1% mixed with diet 7 day before and along with acrylamide treatment , and group iii were received alpha - lipoic acid 1% mixed with diet for 28 days . twenty - four hours after the last administration of alpha - lipoic acid or acr , the rats were anesthetized with diethyl ether . then , blood samples were collected from the inner canthus of the eye by heparinized capillary tube into clean test tubes . following standing in room temperature for at least 30 min , the blood was centrifuged at 3400 xg for 10 min and the serum was separated , transferred to eppendorf tubes , and stored at -20c prior till biochemical analysis . immediately after the collection of blood samples , the animals were euthanized , and their testis and epididymis were quickly excised , rinsed in ice - cold saline and used immediately or stored frozen at -80c until analysis . elisa procedure was used for quantitative determination of serum total testosterone , progesterone and estradiol levels ( dima gesellschaft fur diagnostika [ gmbh ] , germany ) according to manufacturer s instructions . tissue homogenates were separately prepared from frozen testis and epididymis samples in 10 volumes of 0.1 m tris edta buffer ( ph 7.4 ) and centrifuged at 8000 xg for 30 min at 4c . aliquots of the supernatant were utilized for the spectrophotometrical assessment of the levels of the following : lipid peroxidation ( lpo ) , assessed as the production of the thiobarbituric acid reactive substances ( tbars ) in the presence of bht ( 14 ) ; reduced glutathione , by using ellman s reagent ( 15 ) ; glutathione s - transferase ( gst , ec 2.5.1.18 ) activity , as the rate of gsh conjugation of cdnb ( 16 ) ; glutathione peroxidase ( gpx , ec 1.11.1.9 ) activity was determined using reduced glutathione and cummene hydroperxide as substrate by the modified method of ( 17 ) and glutathione reductase ( gr ) activity was measured according to the method of ( 18 ) . the excised testes and epididymis were fixed in bouin s solution , for 24 h and processed using standard laboratory procedures for histology . the tissue was embedded in paraffin blocks , sectioned perpendicular to the longest axis of the testis with 5-cm thickness , and stained routinely with hematoxylin and eosin . stained sections were examined using light microscopy at power 200 and 400 for detection of histopathological changes ( 19 ) . results are expressed as mean sem with the experiment repeated at least three times . statistical evaluations were done using the analysis of variance ( anova ) . a p value of < 0.05 was considered significant . compared to control rats , acrylamide treated rats exhibited a significant decrease in serum total testosterone and progesterone levels while serum estradiol was significantly increased ( p<0.05 ; table 1 ) . alpha - lipoic acid non - significantly increased serum total testosterone , progesterone and estradiol levels . administration of alpha - lipoic acid together with acrylamide significantly increased serum total testosterone level , non - significantly increased serum progesterone level and significantly increased serum estradiol level as compared to acrylamide treated rats . values are expressed as mean se the values with different letter within the same raw significantly differ at p < 0.05 in relation to control rats , acrylamide treated rats had greater level of mda in both testis and epididymis ( p<0.05 ) . acrylamide caused a reduction in enzymatic activities of gst , gpx and gr and level of gsh in both organs ( table 2 , 3 ) . alpha - lipoic acid treatment resulted in significant reduction in mda level with induction in enzymatic and non enzymatic antioxidant status in examined tissues ( p<0.05 ) . the rats treated with lipoic acid and acrylamide had a significant reduction in mda level and increased in gst , gpx and gr enzymatic activities and gsh content in testis and epididymis as compared to acrylamide treated rats ( table 2 , 3 ) . effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s testis . the values with different letter within the same raw significantly differ at p < 0.05 effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s epididymis . the values with different letter within the same raw significantly differ at p < 0.05 testicular sections of rats received lipoic acid had normal seminiferous tubules with complete spermatogenesis and normal interstitial connective tissue ( fig . however , testicular tissue of rats received acrylamide showed degenerative changes of majority of seminiferous tubules as incomplete spermatogenesis where seminiferous tubules were almost devoid of spermatids and spermatozoa and vacuolar degeneration of spermatogonia and sertoli cells ( fig . . degenerated germinal epithelial cells were sloughed in the lumina of most seminiferous tubules and multiple giant cell formations ( fig . other tubules showed coagulative necrosis and depletion of germinal epithelium with hyalinization of luminal contents ( fig . the majority of seminiferous tubules lost all germinal epithelium cells which evoked on cessation of spermatogenesis ( fig . conversely , rats received acrylamide together with lipoic acid showed slight vacuolization of germinal epithelial cells . a few numbers of seminiferous tubules had sloughed necrotic germinal epithelium in their lumina in addition to marked improvement of spermatogenesis , evidenced by presence of elongated spermatids and spermatozoa in majority of seminiferous tubules ( fig . the caput and cauda epididymis of rats received lipoic acid showed normal histological structure with normal sperm density ( fig . acrylamide treated rats exhibited histopathological alterations in the cauda epididymis as edema and moderate numbers of mononuclear cell infiltration of interstitial connective tissue beside the majority of epididymal ducts had no or low numbers of spermatozoa in their lumina ( fig . sloughing of lining epithelial cells and giant cells was evident in some ducts of caput epididymis ( fig . rats received acrylamide plus lipoic acid showed moderate enhancement in sperm density and epididymal structural integrity in caput ( e ) and cauda ( f ) epididymis . photomicrograph of rat testes stained with he : rats received lipoic acid ( 1% ) showing normal histoarchitecture of testes ( a x.160 ) . rats received acrylamide ( 0.05% ) exhibited vacuolar degeneration of the germinal epithelium and sertoli cells ( arrows ) ( b x.250 ) , sloughing of the germinal epithelium ( arrows ) with giant cell formations ( arrowheads ) in the lumen of seminiferous tubules ( c x.160 ) , depletion of germinal cells and hyalinization of the luminal contents ( arrows ) ( d x.160 ) and some seminiferous tubules loss of all germinal epithelium cells ( stars ) ( e x.160 ) . improvement of spermatogenesis in testes of rats treated with acrylamide + lipoic acid ( f x.160 ) . photomicrograph of rat epididymis stained with he ( x. 160 ) : caput and cauda epididymis of rats received lipoic acid showing normal histological structure with normal sperm density ( a & b ) . rats received acrylamide , the caput epididymis ( c ) showing the most of epididymal ductules were free from spermatozoa beside sloughed germ cells ( arrows ) while cauda epididymis ( d ) exhibited giant cells ( arrowheads ) and sloughed germ cells in their lumina . rats treated with acrylamide plus lipoic acid nearly normal structure and sperm density ( e & f ) . compared to control rats , acrylamide treated rats exhibited a significant decrease in serum total testosterone and progesterone levels while serum estradiol was significantly increased ( p<0.05 ; table 1 ) . alpha - lipoic acid non - significantly increased serum total testosterone , progesterone and estradiol levels . administration of alpha - lipoic acid together with acrylamide significantly increased serum total testosterone level , non - significantly increased serum progesterone level and significantly increased serum estradiol level as compared to acrylamide treated rats . values are expressed as mean se the values with different letter within the same raw significantly differ at p < 0.05 in relation to control rats , acrylamide treated rats had greater level of mda in both testis and epididymis ( p<0.05 ) . acrylamide caused a reduction in enzymatic activities of gst , gpx and gr and level of gsh in both organs ( table 2 , 3 ) . alpha - lipoic acid treatment resulted in significant reduction in mda level with induction in enzymatic and non enzymatic antioxidant status in examined tissues ( p<0.05 ) . the rats treated with lipoic acid and acrylamide had a significant reduction in mda level and increased in gst , gpx and gr enzymatic activities and gsh content in testis and epididymis as compared to acrylamide treated rats ( table 2 , 3 ) . effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s testis . the values with different letter within the same raw significantly differ at p < 0.05 effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s epididymis . testicular sections of rats received lipoic acid had normal seminiferous tubules with complete spermatogenesis and normal interstitial connective tissue ( fig . however , testicular tissue of rats received acrylamide showed degenerative changes of majority of seminiferous tubules as incomplete spermatogenesis where seminiferous tubules were almost devoid of spermatids and spermatozoa and vacuolar degeneration of spermatogonia and sertoli cells ( fig . . degenerated germinal epithelial cells were sloughed in the lumina of most seminiferous tubules and multiple giant cell formations ( fig . other tubules showed coagulative necrosis and depletion of germinal epithelium with hyalinization of luminal contents ( fig . the majority of seminiferous tubules lost all germinal epithelium cells which evoked on cessation of spermatogenesis ( fig . conversely , rats received acrylamide together with lipoic acid showed slight vacuolization of germinal epithelial cells . a few numbers of seminiferous tubules had sloughed necrotic germinal epithelium in their lumina in addition to marked improvement of spermatogenesis , evidenced by presence of elongated spermatids and spermatozoa in majority of seminiferous tubules ( fig . the caput and cauda epididymis of rats received lipoic acid showed normal histological structure with normal sperm density ( fig . acrylamide treated rats exhibited histopathological alterations in the cauda epididymis as edema and moderate numbers of mononuclear cell infiltration of interstitial connective tissue beside the majority of epididymal ducts had no or low numbers of spermatozoa in their lumina ( fig . sloughing of lining epithelial cells and giant cells was evident in some ducts of caput epididymis ( fig . rats received acrylamide plus lipoic acid showed moderate enhancement in sperm density and epididymal structural integrity in caput ( e ) and cauda ( f ) epididymis . photomicrograph of rat testes stained with he : rats received lipoic acid ( 1% ) showing normal histoarchitecture of testes ( a x.160 ) . rats received acrylamide ( 0.05% ) exhibited vacuolar degeneration of the germinal epithelium and sertoli cells ( arrows ) ( b x.250 ) , sloughing of the germinal epithelium ( arrows ) with giant cell formations ( arrowheads ) in the lumen of seminiferous tubules ( c x.160 ) , depletion of germinal cells and hyalinization of the luminal contents ( arrows ) ( d x.160 ) and some seminiferous tubules loss of all germinal epithelium cells ( stars ) ( e x.160 ) . improvement of spermatogenesis in testes of rats treated with acrylamide + lipoic acid ( f x.160 ) . photomicrograph of rat epididymis stained with he ( x. 160 ) : caput and cauda epididymis of rats received lipoic acid showing normal histological structure with normal sperm density ( a & b ) . rats received acrylamide , the caput epididymis ( c ) showing the most of epididymal ductules were free from spermatozoa beside sloughed germ cells ( arrows ) while cauda epididymis ( d ) exhibited giant cells ( arrowheads ) and sloughed germ cells in their lumina . rats treated with acrylamide plus lipoic acid nearly normal structure and sperm density ( e & f ) . the present study clearly demonstrates that alpha - lipoic acid has marked protective effect against the development of acrylamide induced testicular oxidative stress through its antioxidant properties . furthermore , yang et al . ( 6 ) proved that level of testosterone hormone in rats was severely affected in different doses of acrylamide and this led to the spermatogenic effects and decreased the leydig cell viability . also , hamdy et al . ( 7 ) indicated that testis is a target organ of acrylamide action as it caused severe damage in seminiferous tubules and caused decrease in plasma free and total testosterone in a dose dependent manner . ( 22 ) reported that the subchronic exposure of acrylamide could affect the normal development of sperm , cause changes of the activity of some enzymes in the testis and directly damage leydig cells and affects the endocrine function of the testis , as confirmed by histopathological changes . the inhibition of some members of the kinesin and dynein cytoskeletal motor protein families may be the common site of action of acr and ga , which could explain both neurotoxicity and male reproductive toxicity observed in animals ( 23 ) . rat s leydig cells utilize both serum uptake and denovo synthesis of cholesterol from acetate to produce testosterone ( 24 ) . it is possible that cytoskeletal inhibition by acrylamide could result in diminished uptake of cholesterol and consequent reduced testosterone levels . in addition , putative acrylamide - induced cytoskeletal dysfunction could inhibit the synthesis and/or transport of lh receptors to the cell membrane of leydig cells , thereby indirectly decreasing testosterone biosynthesis ( 25 ) . estradiol is secreted by the ovarian follicles although there is evidence that the adrenal gland is believed to secrete minute quantities of estrogen ( 26 ) . the decreased levels of estradiol and progesterone reported in the acr treated group may indicate that acr affects the ovarian follicles directly and/or indirectly through the effects on the pituitary gland and decrease its secretion of fsh which stimulates follicle growth and regulates the conversion of androstendione to estradiol ( 27 ) as confirmed by mannaa et al . ( 28 ) who reported that acrylamide induced significant decrease in the levels of estrogen and progesterone in rats . treatment with lipoic acid improved the changes in serum male sex hormones induced by acrylamide . ( 29 ) who demonstrated that lipoic acid sustained the levels of cholesterol and sex hormone binding globulin ( shbg ) to those seen in the control rats . this finding might be attributed to antioxidant milieu of la which maintained a fine tuning of signal transduction mechanisms necessary for normal function of hypothalamus - testicular axis leading to normal secretion of testosterone and sperm production . our results revealed regarding oxidative stress and antioxidant status were in agreement with alturfan et al . ( 30 ) who mentioned that rats treated with acrylamide 40 mg / kg b.wt/day for 10 days exhibited an increased tissue mda level with decreased level of gsh . enhancement of lipid peroxidation is a consequence of depletion of gsh to certain critical levels . there was a significant decrease in thiol groups in lungs , kidney , brain , testis and liver homogenates in male rats treated with acrylamide in a dose dependent manner ( 31 ) . acr is capable of interacting with vital cellular nucleophiles possessing -sh , -nh2 or oh group . therefore , it reacts with gsh in a similar manner and forms glutathione s - conjugates , which is the initial step in the biotransformation of electrophiles into mercapturic acids ( 32 ) . in the present study , decreased gsh content and increased lipid peroxidation in tissues can be explained by the reaction of acr with gsh , which in turn causes the depletion of gsh and the enhancement of lipid peroxidation . ( 28 ) showed that acrylamide caused a significant decrease in total antioxidant and significant increase in lipid peroxidation in brain homogenate in female rats . the levels of oxidative stress indices and lipid peroxidation ( mda ) in the plasma of rats treated with acrylamide were significantly increased while total antioxidant activity was significantly decreased compared with their levels in the controls ( 5 ) . treatment of rats with la showed an increase in the activities of antioxidant enzymes and reduced glutathione level with a decrease in lipid peroxidation which might be attributed to the antioxidant and oxidative damage repairing ability of la ( 8) . also , othman et al . ( 29 ) reported a decrease in the levels of tbars and protein carbonyl with normalized levels of endogenous antioxidants and protection of reproductive markers in the lipoic acid octylphenol treated rats compared to the control rats . ( 33 ) reported that co - administration of lipoic acid 20 mg / kg orally for 14 days together with bisphenol resulted in decreasing lipid peroxidation and increasing enzymatic and non - enzymatic antioxidants in rats . the current study confirms the results of other studies ( 34 ) who demonstrated that la regulated the gst activity in the hypothalamus and epididymal sperm and corrected their deficient thiol status by increasing the level of gsh . because la is a dithiol it can scavenge several ros and also bind ferrous ions to inhibit the generation of hydroxyl radicals ( 9 ) . in addition , la can stimulate the recycling of tocopherol and ascorbic acid ( 35 ) and in combination with its scavenging ability , maintain levels of protein thiol and therefore modulated endogenous antioxidants in the hypothalamus and epididymal sperm . exposure to environmental toxicants can lead to impairment of this barrier and , in turn , can induce the generation of ros leading to testicular oxidative dna damage and oxidative infertility . since la is water and fat soluble antioxidant , therefore , it is distributed in both the cellular membranes and the cytosol . our study is compatible with the earlier findings by others who reported that la has protective effect against adriamycin and lipopolysaccharide - induced oxidative disruption of blood testis barrier and testicular histological changes ( 8) . in conclusion , our results demonstrate for the first time that in acr induced oxidative stress , alpha - lipoic acid by inhibiting the changes in male sex hormones and balancing oxidant - antioxidant status protected the tissues and thus , supplementation of alpha - lipoic acid may be useful in individuals who are at risk to acr toxicity .

introduction : acrylamide is very toxic to various organs and associated with significant increase of oxidative stress and depletion of antioxidants . alpha - lipoic acid enhances cellular antioxidant defense capacity , thereby protecting cells from oxidative stress.aim of the study : this study aimed to evaluate the protective role of alpha - lipoic acid on the oxidative damage induced by acrylamide in testicular and epididymal tissues.material and methods : forty adult male rats were divided into four groups ( 10 rats each ) . control group ; acrylamide treated group administered acrylamide 0.05% ( w / v ) in drinking water for 21 days ; alpha - lipoic acid group received basal diet supplemented with 1% alpha - lipoic acid and forth group was exposed to acrylamide and treated with alpha - lipoic acid at the same doses and treatment regimen mentioned before.results:the administration of acrylamide resulted in significant elevation in testicular and epididymal malondialdehyde level ( mda ) and significant reduction in the level of reduced glutathione ( gsh ) and the activities of glutathione - s - transferase ( gst ) , glutathione peroxidase ( gpx ) and glutathione reductase ( gr ) . also , acrylamide significantly reduced serum total testosterone and progesterone but increased estradiol ( e2 ) levels . treatment with alpha - lipoic acid prior to acrylamide induced protective effects and attenuated these biochemical changes.conclusion:alpha-lipoic acid has been shown to possess antioxidant properties offering promising efficacy against oxidative stress induced by acrylamide administration .

1. INTRODUCTION 2. MATERIAL AND METHODS 2.1. Chemicals and Reagents 2.2. Animals and experimental design 2.3. Hormonal assays 2.4. Indices of antioxidant and oxidative stress 2.5. Histopathology 2.6. Statistical analysis 3. RESULTS 3.1. Serum sex hormone levels 3.2. Antioxidant and oxidative stress indices 3.3. Histopathological findings 4. DISCUSSION 5. CONCLUSION

the likely major pathway in the formation of acrylamide in food cooked at high temperature involves the maillard browning reaction between reducing sugars and asparagine , an amino acid present in potatoes and cereals ( 2 ) . acrylamide is metabolized by hepatic cyp 2e1 produces an epoxide , glycidamide ( ga ) , which is reactive toward protein and nucleic acid nucleophiles ( 4 ) . many studies were done to test the effects of oral acrylamide administration on the testicular functions and the effects include : severe testicular damage , significant reduction in mating , fertility , and pregnancy by formation of abnormal sperm and a decrease in sperm count which could be a result of alkylation of sh groups in the sperm nucleus and tail , depletion of gsh , or dna damage in the testis ( 6 ) . testicular toxicity had been reported in rodents exposed to acr , changes in gonadal and pituitary hormones had been associated with histopathological and gene expression changes ( 7 ) . alpha - lipoic acid ( la ) is a naturally occurring nutraceutical , whose therapeutic effect has been related to its antioxidant activity and its ability to repair oxidative damage ( 8) . the idea that free radical scavengers are protective against pollutant induced toxicity ( 13 ) has provoked the present study to investigate the potential protective effect of la against acrylamide - induced oxidative toxicity in the reproductive system as represented by changes in sex hormone levels , and oxidative stress and antioxidant status in male rats . to the best of the author s knowledge , the effect of la on acrylamide treatment with respect to reproductive markers has not yet been established . acrylamide ( cas number 79 - 06 - 1 ) and alpha - lipoic acid were obtained from sigma chemicals co. ( st . quentin - fallavier , france ) , 5,5- dithiobis-2-nitrobenzoic acid , and 1- chloro-2,4-dinitrobenzene , reduced glutathione , thiobarbituric acid , and butylated hydroxytoluene were purchased from fluka ( buchs , switzerland ) , and testosterone , progesterone and estradiol hormones elisa kit from cayman chemical company . the animal experiments were carried out in accordance with the national institutes of health guidelines for the care and use of laboratory animals , and the study protocol was approved by the local authorities . after 2 weeks of acclimatization , the rats were randomly divided into four groups of 10 rats each . group i was the control group received normal basal diet , group ii and group iv were administered acrylamide 0.05% ( w / v ) in drinking water for 21 days , group iv received beside acrylamide alpha - lipoic acid 1% mixed with diet 7 day before and along with acrylamide treatment , and group iii were received alpha - lipoic acid 1% mixed with diet for 28 days . twenty - four hours after the last administration of alpha - lipoic acid or acr , the rats were anesthetized with diethyl ether . then , blood samples were collected from the inner canthus of the eye by heparinized capillary tube into clean test tubes . following standing in room temperature for at least 30 min , the blood was centrifuged at 3400 xg for 10 min and the serum was separated , transferred to eppendorf tubes , and stored at -20c prior till biochemical analysis . elisa procedure was used for quantitative determination of serum total testosterone , progesterone and estradiol levels ( dima gesellschaft fur diagnostika [ gmbh ] , germany ) according to manufacturer s instructions . aliquots of the supernatant were utilized for the spectrophotometrical assessment of the levels of the following : lipid peroxidation ( lpo ) , assessed as the production of the thiobarbituric acid reactive substances ( tbars ) in the presence of bht ( 14 ) ; reduced glutathione , by using ellman s reagent ( 15 ) ; glutathione s - transferase ( gst , ec 2.5.1.18 ) activity , as the rate of gsh conjugation of cdnb ( 16 ) ; glutathione peroxidase ( gpx , ec 1.11.1.9 ) activity was determined using reduced glutathione and cummene hydroperxide as substrate by the modified method of ( 17 ) and glutathione reductase ( gr ) activity was measured according to the method of ( 18 ) . acrylamide ( cas number 79 - 06 - 1 ) and alpha - lipoic acid were obtained from sigma chemicals co. ( st . quentin - fallavier , france ) , 5,5- dithiobis-2-nitrobenzoic acid , and 1- chloro-2,4-dinitrobenzene , reduced glutathione , thiobarbituric acid , and butylated hydroxytoluene were purchased from fluka ( buchs , switzerland ) , and testosterone , progesterone and estradiol hormones elisa kit from cayman chemical company . the animal experiments were carried out in accordance with the national institutes of health guidelines for the care and use of laboratory animals , and the study protocol was approved by the local authorities . after 2 weeks of acclimatization , the rats were randomly divided into four groups of 10 rats each . group i was the control group received normal basal diet , group ii and group iv were administered acrylamide 0.05% ( w / v ) in drinking water for 21 days , group iv received beside acrylamide alpha - lipoic acid 1% mixed with diet 7 day before and along with acrylamide treatment , and group iii were received alpha - lipoic acid 1% mixed with diet for 28 days . twenty - four hours after the last administration of alpha - lipoic acid or acr , the rats were anesthetized with diethyl ether . then , blood samples were collected from the inner canthus of the eye by heparinized capillary tube into clean test tubes . elisa procedure was used for quantitative determination of serum total testosterone , progesterone and estradiol levels ( dima gesellschaft fur diagnostika [ gmbh ] , germany ) according to manufacturer s instructions . tissue homogenates were separately prepared from frozen testis and epididymis samples in 10 volumes of 0.1 m tris edta buffer ( ph 7.4 ) and centrifuged at 8000 xg for 30 min at 4c . aliquots of the supernatant were utilized for the spectrophotometrical assessment of the levels of the following : lipid peroxidation ( lpo ) , assessed as the production of the thiobarbituric acid reactive substances ( tbars ) in the presence of bht ( 14 ) ; reduced glutathione , by using ellman s reagent ( 15 ) ; glutathione s - transferase ( gst , ec 2.5.1.18 ) activity , as the rate of gsh conjugation of cdnb ( 16 ) ; glutathione peroxidase ( gpx , ec 1.11.1.9 ) activity was determined using reduced glutathione and cummene hydroperxide as substrate by the modified method of ( 17 ) and glutathione reductase ( gr ) activity was measured according to the method of ( 18 ) . compared to control rats , acrylamide treated rats exhibited a significant decrease in serum total testosterone and progesterone levels while serum estradiol was significantly increased ( p<0.05 ; table 1 ) . alpha - lipoic acid non - significantly increased serum total testosterone , progesterone and estradiol levels . administration of alpha - lipoic acid together with acrylamide significantly increased serum total testosterone level , non - significantly increased serum progesterone level and significantly increased serum estradiol level as compared to acrylamide treated rats . values are expressed as mean se the values with different letter within the same raw significantly differ at p < 0.05 in relation to control rats , acrylamide treated rats had greater level of mda in both testis and epididymis ( p<0.05 ) . acrylamide caused a reduction in enzymatic activities of gst , gpx and gr and level of gsh in both organs ( table 2 , 3 ) . alpha - lipoic acid treatment resulted in significant reduction in mda level with induction in enzymatic and non enzymatic antioxidant status in examined tissues ( p<0.05 ) . the rats treated with lipoic acid and acrylamide had a significant reduction in mda level and increased in gst , gpx and gr enzymatic activities and gsh content in testis and epididymis as compared to acrylamide treated rats ( table 2 , 3 ) . effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s testis . the values with different letter within the same raw significantly differ at p < 0.05 effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s epididymis . the values with different letter within the same raw significantly differ at p < 0.05 testicular sections of rats received lipoic acid had normal seminiferous tubules with complete spermatogenesis and normal interstitial connective tissue ( fig . other tubules showed coagulative necrosis and depletion of germinal epithelium with hyalinization of luminal contents ( fig . acrylamide treated rats exhibited histopathological alterations in the cauda epididymis as edema and moderate numbers of mononuclear cell infiltration of interstitial connective tissue beside the majority of epididymal ducts had no or low numbers of spermatozoa in their lumina ( fig . rats received acrylamide plus lipoic acid showed moderate enhancement in sperm density and epididymal structural integrity in caput ( e ) and cauda ( f ) epididymis . photomicrograph of rat testes stained with he : rats received lipoic acid ( 1% ) showing normal histoarchitecture of testes ( a x.160 ) . rats received acrylamide ( 0.05% ) exhibited vacuolar degeneration of the germinal epithelium and sertoli cells ( arrows ) ( b x.250 ) , sloughing of the germinal epithelium ( arrows ) with giant cell formations ( arrowheads ) in the lumen of seminiferous tubules ( c x.160 ) , depletion of germinal cells and hyalinization of the luminal contents ( arrows ) ( d x.160 ) and some seminiferous tubules loss of all germinal epithelium cells ( stars ) ( e x.160 ) . improvement of spermatogenesis in testes of rats treated with acrylamide + lipoic acid ( f x.160 ) . rats treated with acrylamide plus lipoic acid nearly normal structure and sperm density ( e & f ) . compared to control rats , acrylamide treated rats exhibited a significant decrease in serum total testosterone and progesterone levels while serum estradiol was significantly increased ( p<0.05 ; table 1 ) . alpha - lipoic acid non - significantly increased serum total testosterone , progesterone and estradiol levels . administration of alpha - lipoic acid together with acrylamide significantly increased serum total testosterone level , non - significantly increased serum progesterone level and significantly increased serum estradiol level as compared to acrylamide treated rats . values are expressed as mean se the values with different letter within the same raw significantly differ at p < 0.05 in relation to control rats , acrylamide treated rats had greater level of mda in both testis and epididymis ( p<0.05 ) . acrylamide caused a reduction in enzymatic activities of gst , gpx and gr and level of gsh in both organs ( table 2 , 3 ) . alpha - lipoic acid treatment resulted in significant reduction in mda level with induction in enzymatic and non enzymatic antioxidant status in examined tissues ( p<0.05 ) . the rats treated with lipoic acid and acrylamide had a significant reduction in mda level and increased in gst , gpx and gr enzymatic activities and gsh content in testis and epididymis as compared to acrylamide treated rats ( table 2 , 3 ) . effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s testis . the values with different letter within the same raw significantly differ at p < 0.05 effect of acrylamide and lipoic acid on antioxidants and oxidative stress of rat s epididymis . other tubules showed coagulative necrosis and depletion of germinal epithelium with hyalinization of luminal contents ( fig . the caput and cauda epididymis of rats received lipoic acid showed normal histological structure with normal sperm density ( fig . acrylamide treated rats exhibited histopathological alterations in the cauda epididymis as edema and moderate numbers of mononuclear cell infiltration of interstitial connective tissue beside the majority of epididymal ducts had no or low numbers of spermatozoa in their lumina ( fig . rats received acrylamide plus lipoic acid showed moderate enhancement in sperm density and epididymal structural integrity in caput ( e ) and cauda ( f ) epididymis . photomicrograph of rat testes stained with he : rats received lipoic acid ( 1% ) showing normal histoarchitecture of testes ( a x.160 ) . rats received acrylamide ( 0.05% ) exhibited vacuolar degeneration of the germinal epithelium and sertoli cells ( arrows ) ( b x.250 ) , sloughing of the germinal epithelium ( arrows ) with giant cell formations ( arrowheads ) in the lumen of seminiferous tubules ( c x.160 ) , depletion of germinal cells and hyalinization of the luminal contents ( arrows ) ( d x.160 ) and some seminiferous tubules loss of all germinal epithelium cells ( stars ) ( e x.160 ) . improvement of spermatogenesis in testes of rats treated with acrylamide + lipoic acid ( f x.160 ) . rats treated with acrylamide plus lipoic acid nearly normal structure and sperm density ( e & f ) . the present study clearly demonstrates that alpha - lipoic acid has marked protective effect against the development of acrylamide induced testicular oxidative stress through its antioxidant properties . ( 6 ) proved that level of testosterone hormone in rats was severely affected in different doses of acrylamide and this led to the spermatogenic effects and decreased the leydig cell viability . ( 7 ) indicated that testis is a target organ of acrylamide action as it caused severe damage in seminiferous tubules and caused decrease in plasma free and total testosterone in a dose dependent manner . ( 22 ) reported that the subchronic exposure of acrylamide could affect the normal development of sperm , cause changes of the activity of some enzymes in the testis and directly damage leydig cells and affects the endocrine function of the testis , as confirmed by histopathological changes . in addition , putative acrylamide - induced cytoskeletal dysfunction could inhibit the synthesis and/or transport of lh receptors to the cell membrane of leydig cells , thereby indirectly decreasing testosterone biosynthesis ( 25 ) . the decreased levels of estradiol and progesterone reported in the acr treated group may indicate that acr affects the ovarian follicles directly and/or indirectly through the effects on the pituitary gland and decrease its secretion of fsh which stimulates follicle growth and regulates the conversion of androstendione to estradiol ( 27 ) as confirmed by mannaa et al . ( 28 ) who reported that acrylamide induced significant decrease in the levels of estrogen and progesterone in rats . treatment with lipoic acid improved the changes in serum male sex hormones induced by acrylamide . ( 29 ) who demonstrated that lipoic acid sustained the levels of cholesterol and sex hormone binding globulin ( shbg ) to those seen in the control rats . our results revealed regarding oxidative stress and antioxidant status were in agreement with alturfan et al . ( 30 ) who mentioned that rats treated with acrylamide 40 mg / kg b.wt/day for 10 days exhibited an increased tissue mda level with decreased level of gsh . there was a significant decrease in thiol groups in lungs , kidney , brain , testis and liver homogenates in male rats treated with acrylamide in a dose dependent manner ( 31 ) . therefore , it reacts with gsh in a similar manner and forms glutathione s - conjugates , which is the initial step in the biotransformation of electrophiles into mercapturic acids ( 32 ) . in the present study , decreased gsh content and increased lipid peroxidation in tissues can be explained by the reaction of acr with gsh , which in turn causes the depletion of gsh and the enhancement of lipid peroxidation . ( 28 ) showed that acrylamide caused a significant decrease in total antioxidant and significant increase in lipid peroxidation in brain homogenate in female rats . the levels of oxidative stress indices and lipid peroxidation ( mda ) in the plasma of rats treated with acrylamide were significantly increased while total antioxidant activity was significantly decreased compared with their levels in the controls ( 5 ) . treatment of rats with la showed an increase in the activities of antioxidant enzymes and reduced glutathione level with a decrease in lipid peroxidation which might be attributed to the antioxidant and oxidative damage repairing ability of la ( 8) . ( 29 ) reported a decrease in the levels of tbars and protein carbonyl with normalized levels of endogenous antioxidants and protection of reproductive markers in the lipoic acid octylphenol treated rats compared to the control rats . ( 33 ) reported that co - administration of lipoic acid 20 mg / kg orally for 14 days together with bisphenol resulted in decreasing lipid peroxidation and increasing enzymatic and non - enzymatic antioxidants in rats . the current study confirms the results of other studies ( 34 ) who demonstrated that la regulated the gst activity in the hypothalamus and epididymal sperm and corrected their deficient thiol status by increasing the level of gsh . in addition , la can stimulate the recycling of tocopherol and ascorbic acid ( 35 ) and in combination with its scavenging ability , maintain levels of protein thiol and therefore modulated endogenous antioxidants in the hypothalamus and epididymal sperm . in conclusion , our results demonstrate for the first time that in acr induced oxidative stress , alpha - lipoic acid by inhibiting the changes in male sex hormones and balancing oxidant - antioxidant status protected the tissues and thus , supplementation of alpha - lipoic acid may be useful in individuals who are at risk to acr toxicity .

cloning , expression , and purification of recombinant proteins full - length varc ( pf08_0141 ) was pcr - amplified from 3d7 p. falciparum cdna using pfu polymerase and cloned into pet28a vector . deletion constructs for varc ( varc 1291 , 87392 , and 87291 ) were pcr - amplified using full - length varc as a template and cloned into pet28a vector . residues thr , thr , ser , ser , and ser in the varc 1291 construct were mutated to alanine by site - directed mutagenesis using the quikchange ii kit from stratagene to generate an n - terminal acidic cluster mutant of varc 1291 . all varc constructs were expressed in b834 cells and purified using ni - nitrilotriacetic acid affinity columns by virtue of their c - terminal hexahistidine tags . the best fractions from affinity chromatography were further purified by anion exchange chromatography on q - sepharose and then subjected to size exclusion on a superdex 75 column from amersham biosciences . two domains of kahrp ( k1a and k2a ) that interact with varc were pcr - amplified from p. falciparum 3d7 cdna using pfu polymerase and cloned into pet28a vector . these constructs were also expressed in b834 cells and purified on ni - nitrilotriacetic acid affinity columns . the nickel elutes were further purified by cation exchange chromatography on sp - sepharose and then subjected to gel filtration on a superdex 200 column . thin smears of mature stage p. falciparum were fixed in ice - cold methanol for 20 min . these were blocked with 5% bsa and then incubated with anti - varc antibody ( 1:1000 ) for 1 h. after washing with 1 pbst , the slides were incubated with anti - mouse antibodies ( 1:2000 ) conjugated to fluorescein isothiocyanate for 1 h , and treated with 0.1 m dapi for 5 min . slides were again washed extensively with pbst and mounted using antifade reagent ( bio - rad ) . the labeled parasites were visualized using a fluorescence microscope ( nikon ) at 40 magnification . falciparum laboratory strains ( 3d7 , fcr3-csa , and itg - icam ) were cultured in rpmi 1640 ( invitrogen ) supplemented with 0.5% albumax i ( invitrogen ) ( or 10% heat - inactivated human serum ) using o rbcs in an environment containing 5% o2 , 5% co2 , and 90% n2 . cultures were synchronized by using 5% sorbitol and 65% percoll using standard procedures . fcr3-csa and itg - icam cultures were panned on csa and icam-1 , respectively , to maintain their binding phenotypes . briefly , 10 g / ml csa ( or icam-1 ) were coated overnight on bacteriological petri plates at 37 c in a humidified chamber . purified trophozoites and schizonts were then incubated with bound csa for 1 h with intermittent shaking . the unbound parasites were removed by extensive washing with incomplete rpmi , and only bound parasites were cultured further . phosphorylation assays5 g of varc was phosphorylated with uninfected erythrocyte lysates ( 1 g of total protein ) in kinase buffer ( 20 mm tris - hcl , ph 8.0 , 2.5 mm mgcl2 , and 2.5 mm mncl2 , 1 mm sodium vanadate , and 0.5 mm sodium fluoride supplemented with 5 ci of [ -p]atp ) at 30 c for 1.5 h. erythrocyte lysates were prepared according to standard protocols . briefly , histopaque was used to purify erythrocytes from venous blood collected from healthy human volunteers . packed rbcs were lysed by adding an equal volume of hypotonic lysis solution ( 10 mm tris , ph 8.0 , 10 mm nacl ) . the membrane fraction was separated from the cytoplasmic fraction by centrifuging the lysed cells at 15,000 rpm in oakridge tubes ( ss34 rotor ) and collecting the supernatant ( cytoplasmic fraction ) . the erythrocyte ghosts were repeatedly washed with cold lysis solution until traces of hemoglobin were no longer visible . kinase reactions of uninfected rbc cytosol or membranes in the absence of recombinant protein , as relevant , were performed in parallel to act as negative controls . the phosphorylated samples were resolved by 12% sds - polyacrylamide gel electrophoresis and autoradiographed . inhibitors ( heparin ( sigma ) and chondroitin sulfate a ( csa ) , 4,5,6,7-tetrabromotriazole ( tbb ) , 2-dimethylamino-4,5,6,7-tetrabromo-1h - benzimidazole ( dmat ) , tetrabromocinnamic acid ( tbca ) , ( calbiochem ) ) and enhancers ( sigma ) were added at the desired final concentrations wherever needed . an in - gel kinase assay was also performed to confirm the identity of the kinase responsible for varc phosphorylation according to published protocols ( 18 ) . recombinant ckii ( 250 units ) and erythrocyte membranes ( 50 g of total protein ) were electrophoresed on sds - polyacrylamide ( 15% ) gels co - polymerized with 1 mg / ml varc 1291 for the assay . another gel containing 1 mg / ml bsa was used as a control . cultured 3d7 parasites were radiolabeled with 50 ci / ml s - protein labeling mix ( 12 h postinvasion rings ) or [ p]orthophosphate ( 2 h prior to sample collection ) . samples were collected at the specified time points as supernatants of parasite pellets lysed in a buffer containing 50 mm tris , ph 8.0 , 150 mm nacl , 5 mm edta , 1% triton x-100 , and 2% sds . pfemp1 was immunoprecipitated ( using varc antibodies ) from the above samples in nett buffer ( ph 8.1 ) using standard protocols , resolved on 6% polyacrylamide gels , dried , and autoradiographed . varc 1291 was phosphorylated using commercial casein kinase ii ( new england biolabs ) according to the manufacturer 's instructions . an identical reaction without atp was also done , and this was used as unphosphorylated varc sample in these experiments . phosphorylated and unphosphorylated varc were buffer - exchanged into 10 mm sodium phosphate buffer , ph 8.0 . far uv cd spectra were recorded on jasco j810 at 25 c in a quartz cuvette of 0.2-cm path length between wavelengths 190 and 250 nm , at a scan speed of 200 nm / min over three accumulations . the fluorescence measurements were recorded at room temperature in a perkinelmer ls 50b spectrometer with the excitation slit of 5 nm and emission slit of 10 nm at a scan speed of 900 nm / min . the intrinsic tryptophan fluorescence spectra of phosphorylated and unphosphorylated proteins were recorded from 310 to 500 nm with excitation at 280 nm . chymotrypsin fingerprinting a total of 100 g of varc 1291 was phosphorylated using casein kinase ii ( new england biolabs ) in a 100-l reaction . an identical reaction without atp was also performed , and this was used as unphosphorylated varc sample in the chymotrypsin digestion experiment . a kinase reaction of commercial ckii ( in the absence of varc protein ) was used as a control to negate bands corresponding to the enzyme itself . a total of 100 ng of chymotrypsin was added to the 100 g of varc and incubated at 37 c . after the indicated time points , 10 l of reaction mixture ( 10 g of varc ) was taken out from both phosphorylated and unphosphorylated tubes , and digestion was stopped by adding sds - polyacrylamide gel loading buffer . the samples were boiled , resolved by 15% sds - page and stained with coomassie brilliant blue r250 . a , phosphorylation of varc fl using erythrocyte cytosol ( cyto panel ) and membrane ( memb panel ) as the source of enzyme . 5 g of recombinant protein was phosphorylated using uninfected erythrocyte lysates ( 1 g of total protein each ) in kinase buffer ( 20 mm tris - hcl , ph 8.0 , 2.5 mm mgcl2 , 2.5 mm mncl2 , 1 mm sodium vanadate , 0.5 mm sodium fluoride supplemented with 5 ci of [ -p]atp ) at 30 c for 1.5 h. the phosphorylated samples were resolved by 12% sds - page and autoradiographed . varc fl is indicated by an arrow , and multiple bands above and below relate to phosphorylated erythrocyte proteins . b , autoradiographs of immunoprecipitated s / p - labeled pfemp1 resolved by sds - page at 16 , 22 , 32 , and 42 h postinvasion , as indicated . m , protein molecular weight marker ; e , irbcs ; c , uninfected rbcs . bands corresponding to full - length pfemp1 are marked by arrowheads in either panel . cultured 3d7 parasites were labeled using s - protein labeling mix ( s - labeled panel ) or [ p]orthophosphate ( p - labeled panel ) as detailed under samples were collected 16 , 22 , 32 , and 42 h postinvasion and immunoprecipitated using varc antibodies in nett buffer , resolved on 6% sds - page , and autoradiographed . a , phosphorylation of varc fl using erythrocyte cytosol ( cyto panel ) and membrane ( memb panel ) as the source of enzyme . 5 g of recombinant protein was phosphorylated using uninfected erythrocyte lysates ( 1 g of total protein each ) in kinase buffer ( 20 mm tris - hcl , ph 8.0 , 2.5 mm mgcl2 , 2.5 mm mncl2 , 1 mm sodium vanadate , 0.5 mm sodium fluoride supplemented with 5 ci of [ -p]atp ) at 30 c for 1.5 h. the phosphorylated samples were resolved by 12% sds - page and autoradiographed . varc fl is indicated by an arrow , and multiple bands above and below relate to phosphorylated erythrocyte proteins . b , autoradiographs of immunoprecipitated s / p - labeled pfemp1 resolved by sds - page at 16 , 22 , 32 , and 42 h postinvasion , as indicated . m , protein molecular weight marker ; e , irbcs ; c , uninfected rbcs . bands corresponding to full - length pfemp1 are marked by arrowheads in either panel . cultured 3d7 parasites were labeled using s - protein labeling mix ( s - labeled panel ) or [ p]orthophosphate ( p - labeled panel ) as detailed under experimental procedures . samples were collected 16 , 22 , 32 , and 42 h postinvasion and immunoprecipitated using varc antibodies in nett buffer , resolved on 6% sds - page , and autoradiographed . plate - based protein - protein interaction studies a total of 100 ng each of k1a , k2a , actin , and spectrin were coated on enzyme - linked immunosorbent assay plates overnight at 4 c . after blocking with 5% bsa , the coated ligands were allowed to interact with different amounts of varc fl ( commercial ckii phosphorylated versus unphosphorylated ) in 1 pbs ( + 2% bsa ) for 1 h at 37 c . the plates were then incubated with anti - varc antibodies ( 1:10,000 ) followed by anti - mouse hrpo ( 1:20,000 ) for 1 h each . the plates were developed using 1 mg / ml opd and h2o2 and read at 490 nm . static cytoadherence assays two microliters of csa ( 20 g / ml ) or icam-1 ( 25 g / ml ) in pbs were spotted on 60-mm bacteriological petri plates , allowed to adsorb for 2 h at 37 cin a humidified chamber , and used for binding assays with parasite cultures . pbs and bsa were spotted as negative controls for both csa and icam-1 binding , whereas csb and csc were also used in csa - binding assays . these plates were then blocked overnight with 1% bsa in pbs at 4 c . early to middle trophozoites were treated with 100 m of either of the ckii inhibitors for 30 min and added to the spotted plates in binding buffer ( fcr3-csa for csa and itg - icam for icam-1 , respectively ; 1.25 ml at 1% hematocrit , 3% parasitemia ) . the plates were rocked intermittently at 37 c for 1 h , and unbound erythrocytes were washed away with binding buffer . bound erythrocytes were fixed in 1% glutaraldehyde for 20 min , stained with 2% giemsa for 20 min , and scored using a nikon microscope with a10 objective . bound cells were counted from six randomly selected distinct fields in triplicate spots from three independent experiments . results were expressed as percentage binding in comparison with positive control ( no inhibitors added ) . figure 3.identification of the kinase responsible for varc phosphorylation . a , effect of various ckii inhibitors on varc phosphorylation . each of these lanes shows kinase reactions ( with or without inhibitors ) using erythrocyte membranes as the source of enzyme , loaded on a 12% sds - polyacrylamide gel , and autoradiographed . lane 1 , reaction minus varc ; lane 2 , reaction + varc ; lane 3 , as in lane 2 + heparin ( 0.5 g / ml ) ; lane 4 , as in lane 2 + tbb ( 2 m ) ; lane 5 , as in lane 2 + dmat ( 0.5 m ) ; lane 6 , as in lane 2 + tbca ( 0.5 m ) ; lane 7 , varc phosphorylation using rckii ( new england biolabs ) . b and c , effect of enhancers ( b , polylysine ; c , putrescine and spermine ) on varc phosphorylation . kinase reactions were performed with the respective concentration of enhancers , loaded on sds - page , and autoradiographed . -fold change was calculated considering band intensity of the reaction without any enhancer as 1 . the y axis depicts the -fold change in phosphorylation , whereas the x axis depicts the concentration of enhancer used . all experiments in b and c were performed three times in triplicate , and the data here are represented as an average of the same . d , in - gel kinase assay to confirm the identity of the kinase that phosphorylates varc . varc was co - polymerized ( 1 mg / ml ) in a 15% sds - polyacrylamide gel ; rckii and erythrocyte membranes were resolved on this gel . an in - gel kinase reaction , followed by autoradiography revealed a radioactive band of 45 kda in the erythrocyte membrane ( lane memb ) ; this corresponds to the size of recombinant ckii catalytic subunit ( lane rckii ) . identification of the kinase responsible for varc phosphorylation . a , effect of various ckii inhibitors on varc phosphorylation . each of these lanes shows kinase reactions ( with or without inhibitors ) using erythrocyte membranes as the source of enzyme , loaded on a 12% sds - polyacrylamide gel , and autoradiographed . lane 1 , reaction minus varc ; lane 2 , reaction + varc ; lane 3 , as in lane 2 + heparin ( 0.5 g / ml ) ; lane 4 , as in lane 2 + tbb ( 2 m ) ; lane 5 , as in lane 2 + dmat ( 0.5 m ) ; lane 6 , as in lane 2 + tbca ( 0.5 m ) ; lane 7 , varc phosphorylation using rckii ( new england biolabs ) . b and c , effect of enhancers ( b , polylysine ; c , putrescine and spermine ) on varc phosphorylation . kinase reactions were performed with the respective concentration of enhancers , loaded on sds - page , and autoradiographed . -fold change was calculated considering band intensity of the reaction without any enhancer as 1 . the y axis depicts the -fold change in phosphorylation , whereas the x axis depicts the concentration of enhancer used . all experiments in b and c were performed three times in triplicate , and the data here are represented as an average of the same . d , in - gel kinase assay to confirm the identity of the kinase that phosphorylates varc . varc was co - polymerized ( 1 mg / ml ) in a 15% sds - polyacrylamide gel ; rckii and erythrocyte membranes were resolved on this gel . an in - gel kinase reaction , followed by autoradiography revealed a radioactive band of 45 kda in the erythrocyte membrane ( lane memb ) ; this corresponds to the size of recombinant ckii catalytic subunit ( lane rckii ) . flow - based cytoadherence assays microslides were coated for 2 h at 37 c with icam-1 ( 25 g / ml ) or csa ( 20 g / ml ) in pbs and blocked overnight with 1% bsa in pbs at 4 c . parasitized rbcs ( 1% hematocrit and 3% parasitemia ) were treated for 30 min with a 100 m concentration of each of the ckii inhibitors . untreated parasite cultures were used as positive controls in these experiments to signify 100% binding . these irbc suspensions were then flowed over receptor - coated microslides for a total of 5 min , and then binding buffer was flowed over to remove unbound cells . the flow rate ( 0.18 ml / min ) yielded a wall shear stress of 0.05 pascals , which has been used widely to mimic wall shear stresses in the microvasculature . the number of stationary or rolling irbcs was counted in six random fields on the microslides from three independent experiments . results were expressed as percentage binding in comparison with positive control ( no inhibitors added ) . expression and characterization of recombinant varc full - length ( fl ) cytoplasmic c - terminal domain ( varc 1392 ) of pfemp1 ( pf08 _ 0141 ) was cloned and expressed in a bacterial overexpression system and purified to homogeneity ( fig . recombinant fl varc ( 45 kda ) is a dimer in solution , as shown by gel permeation chromatography ( fig . it migrates between bsa dimer ( 132 kda ) and bsa monomer ( 66 kda ) on an s200 superdex column . due to its flexible nature disorder prediction servers ( disopred ) predict the first 100 and the last 100 amino acids of fl varc to be highly unstable . 1a ; this construct was relatively more stable ) , and polyclonal serum was raised against purified varc 1291 in mice . immunofluorescence assays on cultured 3d7 parasites using these antibodies showed a ring fluorescence pattern characteristic of pfemp1 protein ( fig . phosphorylation of pfemp1 in vitro and in vivo since varc is exposed to the erythrocyte cytoplasm after being transported to the rbc membrane , we tested the ability of erythrocyte kinases to phosphorylate fl varc . uninfected rbc extracts ( cytosol and membrane ; 1 g of total protein each ) were used as the source of enzyme in in vitro kinase assays . the membrane fraction of rbcs ( fig . 2a , lane e , memb panel ) could phosphorylate varc significantly better than the cytoplasmic fraction ( fig . 2a , lane e , cyto panel ) in these kinase reactions , as indicated by the intensity of the band corresponding to recombinant varc . since the enzyme responsible for phosphorylation of varc was present in the erythrocyte membrane at a higher concentration , all in vitro phosphorylation reactions involving rbc extracts were hereafter performed using purified erythrocyte membranes . the phosphorylation state of pfemp1 was tested in cultured 3d7 parasites by radioactive labeling using [ p]orthophosphate , followed by immunoprecipitation of pfemp1 with varc antibodies . in order to detect expression of pfemp1 radioactive bands corresponding to the size of full - length pfemp1 could be observed 22 , 32 , and 42 h postinvasion ( fig . 2b , p - labeled panel ) , confirming the phosphorylation potential of pfemp1 in vivo . expression of pfemp1 was detectable at all four time points when samples were collected ( fig . identification of the kinase responsible for phosphorylation of varc a number of inhibitors were used in kinase reactions using erythrocyte membrane as the source of enzyme to identify the kinase responsible for varc phosphorylation . staurosporine , a generic ser / thr kinase inhibitor , and specific ckii inhibitors ( heparin ( 19 ) , tbb , dmat , and tbca ) were found to be effective against varc phosphorylation ( fig . also , various polyamines ( polylysine , putrescine , and spermine ) that act as specific enhancers of ckii activity ( 20 ) could increase the extent of phosphorylation of varc in kinase reactions by 2560% , characteristic of these molecules ( 20 ) ( fig . an in - gel kinase assay identified a protein band of 45 kda ( fig . 3d , lane memb ) from erythrocyte membranes to be responsible for the phosphorylation function ; this corresponds to the size of catalytic subunit of ckii ( fig . the control gel ( co - polymerized with bsa ) highlighted no radioactive bands ( data not shown ) , ruling out autophosphorylation of detected bands . identification of sites for ckii - mediated phosphorylation of varc bioinformatics - based analysis ( using phosphorylation and kinase prediction servers ) of varc suggested the presence of several ckii target sites with high scores in the protein . multiple sequence alignment ( clustalw ) of varcs from 3d7 was used to check which of these sites were conserved among pfemp1s ( fig . these analyses revealed thr , thr , ser , ser , ser , and thr as the most probable target residues for phosphorylation events . in order to verify these results and determine ckii target sites , two other deletion constructs ( residues 87291 and 87392 ) ( fig . phosphorylation of each of these constructs using erythrocyte membrane as the enzyme source in kinase reactions was performed , and the samples were resolved on sds - page . our results show that varc fl and varc 1291 could get heavily phosphorylated ( fig . 4b , lanes a and b ) , whereas varc 87291 showed no phosphorylation ( fig . , a faint radioactive band corresponding to the size of varc 87392 could be observed ( fig . these findings suggest that the ckii target sites are largely clustered in the first 87 residues of varc ( thr , thr , ser , ser , and ser ) , along with thr being a likely target . phosphorylation of a mutant varc 1291 , where thr , thr , ser , ser , and ser were changed to alanine by site - directed mutagenesis , showed significant reduction in its phosphorylation potential when tested in an in vitro kinase assay ( fig . figure 4.identification of ckii target sites on varc . a , multiple sequence alignment ( clustalw ) of varcs from 3d7 strain of p. falciparum . the gray boxes show conservation of probable target site residues thr , thr , ser , ser , and ser . b , phosphorylation of varc constructs ( 5 g ) with erythrocyte membrane extracts resolved on 12% sds - page and autoradiographed . a , varc full - length ; b , varc 1291 ; c , varc 87392 ; d , varc 87291 . c , phosphorylation of alanine site - directed mutant of varc 1291 at target sites thr , thr , ser , ser , and ser as compared with that of wild type varc 1291 . c , reaction minus varc ; 291 , reaction + varc 1291 ( wild type ) ; mut , reaction + mutant varc . a , multiple sequence alignment ( clustalw ) of varcs from 3d7 strain of p. falciparum . the gray boxes show conservation of probable target site residues thr , thr , ser , ser , and ser . b , phosphorylation of varc constructs ( 5 g ) with erythrocyte membrane extracts resolved on 12% sds - page and autoradiographed . a , varc full - length ; b , varc 1291 ; c , varc 87392 ; d , varc 87291 . c , phosphorylation of alanine site - directed mutant of varc 1291 at target sites thr , thr , ser , ser , and ser as compared with that of wild type varc 1291 . c , reaction minus varc ; 291 , reaction + varc 1291 ( wild type ) ; mut , reaction + mutant varc . cd spectroscopy studies were performed to study the changes in secondary structure of varc 1291 upon phosphorylation ( fig . characteristic spectra of a protein containing both sheets and random coils were obtained . comparison of cd spectra of unphosphorylated and phosphorylated varc ( using commercial ckii ) suggested that very small and subtle conformational changes occur in varc 1291 upon kinase treatment ( fig . 5a ) . the intrinsic tryptophan fluorescence of varc 1291 ( has four tryptophans ) in its phosphorylated and unphosphorylated states was also measured . fluorescence was enhanced and red shifted ( minor ) in the case of phosphorylated varc 1291 , suggesting solvent exposure of previously buried tryptophans in response to phosphorylation ( fig . mild proteolysis of phosphorylated and unphosphorylated varc 1291 using chymotrypsin produced several polymorphic bands ( fig . 5c ) , again suggesting the likelihood of minor structural changes . as shown in fig . 5c , bands 2 and 3 at the 5 min time point and the appearance of bands 1 , 4 , and 5 at various time points suggests that structural changes occur in varc due to phosphorylation . strength of varc interaction with p. falciparum - encoded proteins is altered by its phosphorylation k1a and k2a are domains of kahrp that strongly interact with varc ( 12 ) . the interaction of fl varc versus commercial ckii - phosphorylated fl varc was quantitatively measured with each of these kahrp domains in plate - based binding assays . phosphorylated fl varc bound significantly better to both k1a and k2a , as compared with its unphosphorylated form ( fig . 6 , a and b ) . although k2a interaction seemed to reach saturation at higher concentrations of phosphorylated varc , k1a binding to both forms of varc remained appreciably different even at higher concentrations ( fig . since pfemp1 is tethered to the rbc cytoskeleton via the binding of its cytoplasmic domain to actin and spectrin , we also studied the effect of fl varc phosphorylation on its binding to these proteins . the effect of varc phosphorylation on the interaction of either cytoskeleton proteins with varc was negligible , as depicted by the contiguous concentration - dependent binding curves ( fig . varc 1291 was phosphorylated with casein kinase ii and buffer - exchanged in 10 mm sodium phosphate buffer ( ph 8.0 ) . spectra were recorded on jasco j810 at 25 c in a quartz cuvette of 0.2-cm path length between wavelengths 190 and 250 nm , at a scan speed of 200 nm / min over three accumulations , after subtraction of buffer spectra . the spectrum indicated by a heavy line corresponds to unphosphorylated varc , whereas the spectrum indicated with a dotted line corresponds to phosphorylated varc . the intrinsic tryptophan fluorescence spectra of phosphorylated and unphosphorylated proteins were recorded from 310 to 500 nm with excitation wavelength at 280 nm . the spectrum indicated by a heavy line corresponds to unphosphorylated varc , whereas the spectrum indicated with a dotted line corresponds to phosphorylated varc . this digestion was performed for indicated times in identical sets for phosphorylated and unphosphorylated varc , as indicated under experimental procedures . lanes labeled u and p represent unphosphorylated and phosphorylated varc , respectively , followed by the indicated time point ( min ) . arrows 1 , 4 , and 5 indicate the appearance of new bands , whereas arrows 2 and 3 suggest domain rearrangement in response to phosphorylation . structural changes in varc upon phosphorylation . a , far uv cd spectra of phosphorylated and unphosphorylated varc . varc 1291 was phosphorylated with casein kinase ii and buffer - exchanged in 10 mm sodium phosphate buffer ( ph 8.0 ) . spectra were recorded on jasco j810 at 25 c in a quartz cuvette of 0.2-cm path length between wavelengths 190 and 250 nm , at a scan speed of 200 nm / min over three accumulations , after subtraction of buffer spectra . the spectrum indicated by a heavy line corresponds to unphosphorylated varc , whereas the spectrum indicated with a dotted line corresponds to phosphorylated varc . the intrinsic tryptophan fluorescence spectra of phosphorylated and unphosphorylated proteins were recorded from 310 to 500 nm with excitation wavelength at 280 nm . the spectrum indicated by a heavy line corresponds to unphosphorylated varc , whereas the spectrum indicated with a dotted line corresponds to phosphorylated varc . c , chymotryptic fingerprinting of phosphorylated varc . this digestion was performed for indicated times in identical sets for phosphorylated and unphosphorylated varc , as indicated under experimental procedures . lanes labeled u and p represent unphosphorylated and phosphorylated varc , respectively , followed by the indicated time point ( min ) . arrows 1 , 4 , and 5 indicate the appearance of new bands , whereas arrows 2 and 3 suggest domain rearrangement in response to phosphorylation . binding of different amounts of unphosphorylated and phosphorylated varc to k1a ( a ) , k2a ( b ) , actin ( c ) , and spectrin ( d ) is shown . 100 ng of individual proteins k1a , k2a , actin , and spectrin were coated on enzyme - linked immunosorbent assay plates and allowed to interact with different amounts of phosphorylated and unphosphorylated varc . the y axis represents binding ( measured as absorbance at 490 nm ) , whereas the x axis represents varc concentration ( unphosphorylated or phosphorylated , as indicated on the respective curves ) . each experiment was performed in triplicate , and the average of three experiments was plotted after deducting background signal from the negative control ( bsa ) . binding of different amounts of unphosphorylated and phosphorylated varc to k1a ( a ) , k2a ( b ) , actin ( c ) , and spectrin ( d ) is shown . 100 ng of individual proteins k1a , k2a , actin , and spectrin were coated on enzyme - linked immunosorbent assay plates and allowed to interact with different amounts of phosphorylated and unphosphorylated varc . the y axis represents binding ( measured as absorbance at 490 nm ) , whereas the x axis represents varc concentration ( unphosphorylated or phosphorylated , as indicated on the respective curves ) . each experiment was performed in triplicate , and the average of three experiments was plotted after deducting background signal from the negative control ( bsa ) . ckii inhibition affects cytoadherence in cultured parasites since phosphorylation of varc increases its affinity for kahrp domains and varc - kahrp interaction has been considered as an important parameter in cytoadherence , we tested the effect of a set of cell - permeable ckii inhibitors on cytoadhesion to soluble endothelial receptors . the binding reduction was scored as follows : 025% , low ; 2550% , moderate ; > 50% , high . the effect of these inhibitors on cytoadherence in static conditions was low to moderate ( fig . 7 , a c , granulated gray bars ) except in the case of tbb on itg - icam ( 55% reduction ; fig . 7a , icam-1 panel ) . however , under flow conditions , the decline in irbc binding to icam-1/csa in response to ckii inhibitors ranged from moderate to high ( fig . varcs are the cytoplasmic domains of the pfemp1 family of proteins , which are unique to p. falciparum . these domains are central to the phenomenon of cytoadherence , a major contributor to p. falciparum malaria - related deaths . varcs are likely to be involved in events downstream or upstream of host receptor engagement by the n termini of pfemp1 , making them important and interesting to work on . here , we have characterized a varc ( pf08_0141 ) as an example and assigned an important function to these domains . full - length recombinantly expressed varc is highly soluble and forms a dimer in solution , as determined by gel permeation chromatography ( fig . deletion constructs of this domain that are truncated at the amino ( residues 87291 and 87392 ) or carboxyl termini ( residues 1291 ) also run as dimers when subjected to size exclusion ( data not shown ) , implying that the minimal dimerization unit lies within the central portion of the protein . circular dichroism of full - length varc revealed that this protein is largely composed of -sheets and random coils ( data not shown ) . since varcs are cytoplasmic within the rbc , they are likely to serve as an important link between the extracellular cues and erythrocytic responses . protein phosphorylation constitutes a major mechanism by which cellular processes like metabolism , signal transduction , cell polarity , and cytoskeletal reorganization can be controlled ( 21 ) . the erythrocyte cytoskeleton is a meshwork of various proteins that interact with each other and regulate important cellular functions . many rbc membrane proteins are subjected to phosphorylation by the action of erythrocytic kinases ( 2225 ) , sometimes even by constitutive association ( 26 ) . during their growth within the red cells , malaria parasites export a number of their proteins to the rbc membrane ( e.g. pfemp1 and kahrp ) , which hereafter are treated by the cell as their own ( 27 ) . this penetration by plasmodium into the host system therefore imparts the parasite exported proteins the ability to interact with erythrocytic molecules ( e.g. kinases ) . keeping this in mind , we tested the phosphorylation potential of varc in vitro using erythrocyte extracts . we found that erythrocyte ghosts could efficiently phosphorylate recombinant full - length and 1291 varc , whereas the cytosolic kinases had little effect on varc ( fig . modification of a pseudomembrane protein by proximal erythrocytic membrane kinases may facilitate the parasites ' purpose to undergo phosphorylation . although expression of this protein can be seen in cultured parasites ( s - labeled ) as early as 16 h postinvasion , its phosphorylated form ( p - labeled ) is detectable only as the culture progresses to its mature stages ( 22 h postinvasion onward ) , when pfemp1 is exported to the erythrocyte surface ( it is widely believed that export of pfemp1 to rbc surface is effected in the 1620 h postinvasion window ( fig . the effect of tbb ( a ) , dmat ( b ) , and tbca ( c ) on binding of fcr3-csa to csa ( csa panels ) and itg - icam to icam-1 ( icam-1 panels ) . parasitized rbcs were treated for 30 min with a 100 m concentration of each of the ckii inhibitors , as indicated . irbc suspensions ( 1% hematocrit and 3% parasitemia ) were flowed over receptor - coated microslides for 5 min . the number of stationary or rolling irbcs was counted in six random fields on the microslides from three independent experiments . results were expressed as percentage bindingin comparison with untreated culture ( 100% binding ) . granulated gray bars represent data from static assays , whereas solid gray bars represent data from flow assays . the effect of tbb ( a ) , dmat ( b ) , and tbca ( c ) on binding of fcr3-csa to csa ( csa panels ) and itg - icam to icam-1 ( icam-1 panels ) . parasitized rbcs were treated for 30 min with a 100 m concentration of each of the ckii inhibitors , as indicated . irbc suspensions ( 1% hematocrit and 3% parasitemia ) were flowed over receptor - coated microslides for 5 min . the flow rate was set to 0.18 ml / min . the number of stationary or rolling irbcs was counted in six random fields on the microslides from three independent experiments . results were expressed as percentage bindingin comparison with untreated culture ( 100% binding ) . granulated gray bars represent data from static assays , whereas solid gray bars represent data from flow assays . we have identified ckii as the enzyme responsible for this post - translational modification by using specific inhibitors { heparin ( 19 ) , tbb , dmat , and tbca } and enhancers ( polyamines ) ( 20 ) in kinase assays ( fig . polyamines like spermine , putrescine , and polylysine are known to stimulate ckii by causing an aggregation of the substrate , thereby increasing its effective concentration ( 28 ) . an in - gel kinase assay for varc to identify the responsible enzyme ( in erythrocyte membranes ) highlights a band corresponding to the size of the catalytic subunit of ckii ( fig . 3d ) . this enzyme is a ubiquitously expressed ( in all tissues and organisms ) and probably constitutively active ser / thr kinase that is located in nearly all subcellular compartments ( 29 ) . the erythrocyte membrane has a distinct pool of ckii , which along with the cytosolic pool phosphorylates most of the cytoskeletal proteins , including spectrin , ankyrin , and adducin ( 23 , 26 , 30 ) . our bioinformatics analysis to identify the target residues for phosphorylation along with the kinasing data of n- and c - terminal deletion constructs suggests that most ckii target sites on varc exist in an n - terminal acidic cluster , typical of ckii recognition sites ( fig . 4 , a and b ) . a variant of varc 1291 , where the n - terminal acidic cluster has been mutated by alanine site - directed mutagenesis , shows significantly reduced phosphorylation capability in kinase assays using erythrocyte membranes ( fig . . another isolated residue of varc that is likely to undergo phosphorylation is thr , which lies on its c terminus . our results imply that varc harbors multiple phosphorylation sites for its modification within the rbc . ckii - mediated phosphorylation of various proteins is known to change their conformation upon modification ( 31 , 32 ) . our data suggest that varc undergoes subtle conformational alterations upon phosphorylation ( fig . phosphorylation of erythrocyte cytoskeletal proteins alters their binding affinity for each other , which has important implications in cellular function ( 23 , 26 , 30 ) . varc is known to tether pfemp1 to the rbc cytoskeleton by interacting with host proteins actin and spectrin and parasite encoded kahrp . here , we have shown that phosphorylation of varc has a considerable impact on its interaction with kahrp domains k1a and k2a , whereas the effect on resident host proteins ( actin and spectrin ) is negligible . our results depict that upon phosphorylation of varc , its binding to k1a was altered more dramatically than to k2a ( fig . since k1a interacts with the c terminus of varc , it is probable that thr phosphorylation confers a greater structural change in varc , as opposed to modification of the n terminus , which is known to bind k2a . although spectrin is also a substrate for ckii in vivo , we have observed that its phosphorylation does not have any effect on varc - spectrin association ( data not shown ) . therefore , a change in their binding strength for each other is likely to reflect on how well irbcs cytoadhere . we have studied cytoadhesion abilities of cultured parasites treated with commercially available , cell - permeable , and specific ckii inhibitors ( 33 ) . all three inhibitors used in this study reduce ckii mediated phosphorylation by competing for the enzyme 's atp binding pocket . the effect of these inhibitors on cytoadherence in static conditions was mostly insignificant ( fig . 7 , a ( csa panel ) , b , and c ) , with the exception of tbb on itg - icam ( 55% reduction ; fig . 7a , icam-1 panel ) . however , under flow conditions , the decline in irbc binding to icam-1/csa in response to ckii inhibitors was significant ( fig . 7 , a c ) . binding of pfemp1s to host receptors like csa and icam-1 is considered important in severe placental and cerebral malaria , respectively . our data show that ckii inhibition causes parasitized rbcs to cytoadhere more weakly to soluble receptors under flow conditions , as compared with static conditions . on the basis of these results , we propose the following . due to a decline in varc phosphorylation and hence weaker binding to kahrp , it is probable that pfemp1 is inadequately anchored to the erythrocyte cytoskeleton , impairing cytoadherence in circulation . on the other hand , binding of pfemp1s to their respective host endothelial receptors is not challenged in the absence of physiological shear stress ( i.e. static assays ) . due to the universal requirement of ckii in cellular functions , one could argue that the enzyme 's inhibition could be lethal to the parasite . however , the capability of dead parasites to cytoadhere equals that of live parasites.3 nonetheless , a possibility that ckii inhibition produces a more generic effect on irbc morphology to alter their cytoadherence properties can not be ruled out . due to polyamine biosynthesis or increased uptake of putrescine from the extracellular pool ( 34 , 35 ) , mature stage irbcs have significantly elevated levels of polyamines as compared with uninfected or ring stage parasites . this probably acts in favor of parasites so that they can utilize their resources to increase varc phosphorylation and cytoadhere better in order to evade host response . the presence of several polylysine stretches in kahrp may be a parasite 's evolutionary attempt to enhance varc phosphorylation and strengthen its roots . phosphorylation of major cytoskeletal proteins , such as ankyrin , band 4.1 , and band 4.9 , can weaken the rigidity of the cytoskeleton by reducing the binding affinity of these components ( 24 , 26 ) . this property of the erythrocytes is extremely important , since it regulates membrane deformability essential for passage through microvasculature . although the precise mechanisms leading to decreased deformability of irbcs are poorly understood , the contribution of parasite proteins to increased membrane rigidity is most likely attributable to their direct or indirect interactions with proteins of the rbc membrane skeleton or other exported parasite proteins ( 36 , 37 ) . since phosphorylation of varc increases its affinity for kahrp , it could be one of the factors for increased membrane rigidity of p. falciparum - infected rbcs . since cytoadhesion is a major mechanism by which p. falciparum implements its pathophysiology , drugs that potentially diminish cytoadherence are considered important agents in the fight against virulent malaria . this is the first report where small molecule ckii inhibitors have been assessed for their anti - cytoadherence activity by targeting intracellular events . this study suggests that molecules that block or reverse the interaction between varc and its binding partners have the ability to reduce cytoadherence and may therefore be effective against severe malaria . the ckii target site on varc presents itself as a potential new target for development of anti - cytoadherence agents . finally , therapeutics that act to suppress the molecular functions of conserved varcs are likely to have an edge over the ones that aim at abrogating the variable extracellular pfemp1-host receptor interactions .

plasmodium falciparum malaria is a major human health scourge and a key cause of mortality . its pathogenicity partly results from the phenomenon of cytoadherence mediated by the pfemp1 ( plasmodium falciparum erythrocyte membrane protein 1 ) family . extracellular domains of pfemp1s are variable and bind various host endothelial receptors , whereas their cytoplasmic domains ( varcs ) are relatively conserved . varcs affix pfemp1s in the human erythrocyte membrane by interacting with host cytoskeleton proteins and exported parasite proteins . here , we provide in vitro and in vivo evidence for pfemp1 phosphorylation ( on varc ) and propose an important function for this modification . specific inhibitors and enhancers have been used to identify erythrocytic casein kinase ii ( ckii ) as the enzyme responsible for varc modification activity . we have also delineated probable ckii target residues on varc , which mainly reside in an n - terminal acidic cluster . our data show that varc phosphorylation alters its binding to parasite encoded knob - associated histidine - rich protein ( kahrp ) . finally , we demonstrate reduced cytoadherence of infected rbcs to endothelial receptors like icam-1 and csa ( these contribute to cerebral and placental malaria , respectively ) in response to their ckii inhibition . collectively , this study furthers our understanding of varc function , underscores the importance of erythrocytic ckii in cytoadherence , and suggests a possible new target for anti - cytoadherence molecules .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION

residues thr , thr , ser , ser , and ser in the varc 1291 construct were mutated to alanine by site - directed mutagenesis using the quikchange ii kit from stratagene to generate an n - terminal acidic cluster mutant of varc 1291 . falciparum laboratory strains ( 3d7 , fcr3-csa , and itg - icam ) were cultured in rpmi 1640 ( invitrogen ) supplemented with 0.5% albumax i ( invitrogen ) ( or 10% heat - inactivated human serum ) using o rbcs in an environment containing 5% o2 , 5% co2 , and 90% n2 . phosphorylation assays5 g of varc was phosphorylated with uninfected erythrocyte lysates ( 1 g of total protein ) in kinase buffer ( 20 mm tris - hcl , ph 8.0 , 2.5 mm mgcl2 , and 2.5 mm mncl2 , 1 mm sodium vanadate , and 0.5 mm sodium fluoride supplemented with 5 ci of [ -p]atp ) at 30 c for 1.5 h. erythrocyte lysates were prepared according to standard protocols . inhibitors ( heparin ( sigma ) and chondroitin sulfate a ( csa ) , 4,5,6,7-tetrabromotriazole ( tbb ) , 2-dimethylamino-4,5,6,7-tetrabromo-1h - benzimidazole ( dmat ) , tetrabromocinnamic acid ( tbca ) , ( calbiochem ) ) and enhancers ( sigma ) were added at the desired final concentrations wherever needed . an in - gel kinase assay was also performed to confirm the identity of the kinase responsible for varc phosphorylation according to published protocols ( 18 ) . pfemp1 was immunoprecipitated ( using varc antibodies ) from the above samples in nett buffer ( ph 8.1 ) using standard protocols , resolved on 6% polyacrylamide gels , dried , and autoradiographed . varc 1291 was phosphorylated using commercial casein kinase ii ( new england biolabs ) according to the manufacturer 's instructions . chymotrypsin fingerprinting a total of 100 g of varc 1291 was phosphorylated using casein kinase ii ( new england biolabs ) in a 100-l reaction . a kinase reaction of commercial ckii ( in the absence of varc protein ) was used as a control to negate bands corresponding to the enzyme itself . after the indicated time points , 10 l of reaction mixture ( 10 g of varc ) was taken out from both phosphorylated and unphosphorylated tubes , and digestion was stopped by adding sds - polyacrylamide gel loading buffer . a , phosphorylation of varc fl using erythrocyte cytosol ( cyto panel ) and membrane ( memb panel ) as the source of enzyme . a , phosphorylation of varc fl using erythrocyte cytosol ( cyto panel ) and membrane ( memb panel ) as the source of enzyme . static cytoadherence assays two microliters of csa ( 20 g / ml ) or icam-1 ( 25 g / ml ) in pbs were spotted on 60-mm bacteriological petri plates , allowed to adsorb for 2 h at 37 cin a humidified chamber , and used for binding assays with parasite cultures . figure 3.identification of the kinase responsible for varc phosphorylation . a , effect of various ckii inhibitors on varc phosphorylation . each of these lanes shows kinase reactions ( with or without inhibitors ) using erythrocyte membranes as the source of enzyme , loaded on a 12% sds - polyacrylamide gel , and autoradiographed . b and c , effect of enhancers ( b , polylysine ; c , putrescine and spermine ) on varc phosphorylation . identification of the kinase responsible for varc phosphorylation . a , effect of various ckii inhibitors on varc phosphorylation . b and c , effect of enhancers ( b , polylysine ; c , putrescine and spermine ) on varc phosphorylation . flow - based cytoadherence assays microslides were coated for 2 h at 37 c with icam-1 ( 25 g / ml ) or csa ( 20 g / ml ) in pbs and blocked overnight with 1% bsa in pbs at 4 c . the flow rate ( 0.18 ml / min ) yielded a wall shear stress of 0.05 pascals , which has been used widely to mimic wall shear stresses in the microvasculature . phosphorylation of pfemp1 in vitro and in vivo since varc is exposed to the erythrocyte cytoplasm after being transported to the rbc membrane , we tested the ability of erythrocyte kinases to phosphorylate fl varc . since the enzyme responsible for phosphorylation of varc was present in the erythrocyte membrane at a higher concentration , all in vitro phosphorylation reactions involving rbc extracts were hereafter performed using purified erythrocyte membranes . identification of the kinase responsible for phosphorylation of varc a number of inhibitors were used in kinase reactions using erythrocyte membrane as the source of enzyme to identify the kinase responsible for varc phosphorylation . staurosporine , a generic ser / thr kinase inhibitor , and specific ckii inhibitors ( heparin ( 19 ) , tbb , dmat , and tbca ) were found to be effective against varc phosphorylation ( fig . identification of sites for ckii - mediated phosphorylation of varc bioinformatics - based analysis ( using phosphorylation and kinase prediction servers ) of varc suggested the presence of several ckii target sites with high scores in the protein . these analyses revealed thr , thr , ser , ser , ser , and thr as the most probable target residues for phosphorylation events . phosphorylation of each of these constructs using erythrocyte membrane as the enzyme source in kinase reactions was performed , and the samples were resolved on sds - page . our results show that varc fl and varc 1291 could get heavily phosphorylated ( fig . 4b , lanes a and b ) , whereas varc 87291 showed no phosphorylation ( fig . these findings suggest that the ckii target sites are largely clustered in the first 87 residues of varc ( thr , thr , ser , ser , and ser ) , along with thr being a likely target . phosphorylation of a mutant varc 1291 , where thr , thr , ser , ser , and ser were changed to alanine by site - directed mutagenesis , showed significant reduction in its phosphorylation potential when tested in an in vitro kinase assay ( fig . c , phosphorylation of alanine site - directed mutant of varc 1291 at target sites thr , thr , ser , ser , and ser as compared with that of wild type varc 1291 . c , phosphorylation of alanine site - directed mutant of varc 1291 at target sites thr , thr , ser , ser , and ser as compared with that of wild type varc 1291 . cd spectroscopy studies were performed to study the changes in secondary structure of varc 1291 upon phosphorylation ( fig . fluorescence was enhanced and red shifted ( minor ) in the case of phosphorylated varc 1291 , suggesting solvent exposure of previously buried tryptophans in response to phosphorylation ( fig . strength of varc interaction with p. falciparum - encoded proteins is altered by its phosphorylation k1a and k2a are domains of kahrp that strongly interact with varc ( 12 ) . although k2a interaction seemed to reach saturation at higher concentrations of phosphorylated varc , k1a binding to both forms of varc remained appreciably different even at higher concentrations ( fig . since pfemp1 is tethered to the rbc cytoskeleton via the binding of its cytoplasmic domain to actin and spectrin , we also studied the effect of fl varc phosphorylation on its binding to these proteins . the effect of varc phosphorylation on the interaction of either cytoskeleton proteins with varc was negligible , as depicted by the contiguous concentration - dependent binding curves ( fig . the spectrum indicated by a heavy line corresponds to unphosphorylated varc , whereas the spectrum indicated with a dotted line corresponds to phosphorylated varc . lanes labeled u and p represent unphosphorylated and phosphorylated varc , respectively , followed by the indicated time point ( min ) . arrows 1 , 4 , and 5 indicate the appearance of new bands , whereas arrows 2 and 3 suggest domain rearrangement in response to phosphorylation . lanes labeled u and p represent unphosphorylated and phosphorylated varc , respectively , followed by the indicated time point ( min ) . arrows 1 , 4 , and 5 indicate the appearance of new bands , whereas arrows 2 and 3 suggest domain rearrangement in response to phosphorylation . each experiment was performed in triplicate , and the average of three experiments was plotted after deducting background signal from the negative control ( bsa ) . each experiment was performed in triplicate , and the average of three experiments was plotted after deducting background signal from the negative control ( bsa ) . ckii inhibition affects cytoadherence in cultured parasites since phosphorylation of varc increases its affinity for kahrp domains and varc - kahrp interaction has been considered as an important parameter in cytoadherence , we tested the effect of a set of cell - permeable ckii inhibitors on cytoadhesion to soluble endothelial receptors . however , under flow conditions , the decline in irbc binding to icam-1/csa in response to ckii inhibitors ranged from moderate to high ( fig . varcs are the cytoplasmic domains of the pfemp1 family of proteins , which are unique to p. falciparum . these domains are central to the phenomenon of cytoadherence , a major contributor to p. falciparum malaria - related deaths . here , we have characterized a varc ( pf08_0141 ) as an example and assigned an important function to these domains . pfemp1 and kahrp ) , which hereafter are treated by the cell as their own ( 27 ) . keeping this in mind , we tested the phosphorylation potential of varc in vitro using erythrocyte extracts . we found that erythrocyte ghosts could efficiently phosphorylate recombinant full - length and 1291 varc , whereas the cytosolic kinases had little effect on varc ( fig . although expression of this protein can be seen in cultured parasites ( s - labeled ) as early as 16 h postinvasion , its phosphorylated form ( p - labeled ) is detectable only as the culture progresses to its mature stages ( 22 h postinvasion onward ) , when pfemp1 is exported to the erythrocyte surface ( it is widely believed that export of pfemp1 to rbc surface is effected in the 1620 h postinvasion window ( fig . the effect of tbb ( a ) , dmat ( b ) , and tbca ( c ) on binding of fcr3-csa to csa ( csa panels ) and itg - icam to icam-1 ( icam-1 panels ) . the effect of tbb ( a ) , dmat ( b ) , and tbca ( c ) on binding of fcr3-csa to csa ( csa panels ) and itg - icam to icam-1 ( icam-1 panels ) . we have identified ckii as the enzyme responsible for this post - translational modification by using specific inhibitors { heparin ( 19 ) , tbb , dmat , and tbca } and enhancers ( polyamines ) ( 20 ) in kinase assays ( fig . this enzyme is a ubiquitously expressed ( in all tissues and organisms ) and probably constitutively active ser / thr kinase that is located in nearly all subcellular compartments ( 29 ) . the erythrocyte membrane has a distinct pool of ckii , which along with the cytosolic pool phosphorylates most of the cytoskeletal proteins , including spectrin , ankyrin , and adducin ( 23 , 26 , 30 ) . our bioinformatics analysis to identify the target residues for phosphorylation along with the kinasing data of n- and c - terminal deletion constructs suggests that most ckii target sites on varc exist in an n - terminal acidic cluster , typical of ckii recognition sites ( fig . a variant of varc 1291 , where the n - terminal acidic cluster has been mutated by alanine site - directed mutagenesis , shows significantly reduced phosphorylation capability in kinase assays using erythrocyte membranes ( fig . another isolated residue of varc that is likely to undergo phosphorylation is thr , which lies on its c terminus . our data suggest that varc undergoes subtle conformational alterations upon phosphorylation ( fig . varc is known to tether pfemp1 to the rbc cytoskeleton by interacting with host proteins actin and spectrin and parasite encoded kahrp . here , we have shown that phosphorylation of varc has a considerable impact on its interaction with kahrp domains k1a and k2a , whereas the effect on resident host proteins ( actin and spectrin ) is negligible . our results depict that upon phosphorylation of varc , its binding to k1a was altered more dramatically than to k2a ( fig . since k1a interacts with the c terminus of varc , it is probable that thr phosphorylation confers a greater structural change in varc , as opposed to modification of the n terminus , which is known to bind k2a . although spectrin is also a substrate for ckii in vivo , we have observed that its phosphorylation does not have any effect on varc - spectrin association ( data not shown ) . however , under flow conditions , the decline in irbc binding to icam-1/csa in response to ckii inhibitors was significant ( fig . binding of pfemp1s to host receptors like csa and icam-1 is considered important in severe placental and cerebral malaria , respectively . our data show that ckii inhibition causes parasitized rbcs to cytoadhere more weakly to soluble receptors under flow conditions , as compared with static conditions . due to a decline in varc phosphorylation and hence weaker binding to kahrp , it is probable that pfemp1 is inadequately anchored to the erythrocyte cytoskeleton , impairing cytoadherence in circulation . on the other hand , binding of pfemp1s to their respective host endothelial receptors is not challenged in the absence of physiological shear stress ( i.e. due to the universal requirement of ckii in cellular functions , one could argue that the enzyme 's inhibition could be lethal to the parasite . although the precise mechanisms leading to decreased deformability of irbcs are poorly understood , the contribution of parasite proteins to increased membrane rigidity is most likely attributable to their direct or indirect interactions with proteins of the rbc membrane skeleton or other exported parasite proteins ( 36 , 37 ) . since cytoadhesion is a major mechanism by which p. falciparum implements its pathophysiology , drugs that potentially diminish cytoadherence are considered important agents in the fight against virulent malaria . this is the first report where small molecule ckii inhibitors have been assessed for their anti - cytoadherence activity by targeting intracellular events . the ckii target site on varc presents itself as a potential new target for development of anti - cytoadherence agents . finally , therapeutics that act to suppress the molecular functions of conserved varcs are likely to have an edge over the ones that aim at abrogating the variable extracellular pfemp1-host receptor interactions .

the colonization and growth of cancer metastases in the bone depends on a cooperative interaction of the cancer cells with the host cells in the bone microenvironment . this microenvironment includes the resident osteoblasts , osteoclasts , endothelial cells , bone - lining cells , stromal cells , hematopoietic stem cells , and transient cells such as macrophages , lymphocytes , neutrophils , and other blood cells . while cell - cell contacts are established between cancer cells and bone cells via adhesion molecules , a wider network of communication occurs through secreted cytokines and growth factors . these soluble molecules play a critical role in the normal bone remodeling process as well as in cancer cell colonization of the bone marrow . the interplay of the cancer cells with the cells of the bone marrow cavity has been described in terms of a vicious cycle . in brief , cytokines or growth factors secreted by invading cancer cells ( e.g. , parathyroid hormone - related protein , pthrp ) act to stimulate osteoblasts to produce more receptor activator of nuclear factor kappa - b ligand ( rankl ) and less osteoprotegerin ( opg ) , a decoy receptor for rankl . the rankl binds to rank on osteoclast precursors leading to differentiation and activation of osteoclasts . activated osteoclasts degrade bone matrix releasing growth factors such as transforming growth factor beta ( tgf- ) and insulin - like growth factor ( igf ) . this series of events provides an explanation of the osteolytic outcome of breast cancer metastasis in bone ; that is , an increase in osteoclast activation leads to excess bone breakdown and further stimulation of cancer cells . . however , by and large , the lesions do not heal . in our previous research , we found that metastatic breast cancer cells also inhibit the differentiation of osteoblasts , thereby diminishing bone formation . the combination of increased bone degradation and decreased bone rebuilding has a net outcome of bone loss . through cell culture studies when conditioned medium from metastatic human breast cancer cells , mda - mb-231 , was added to human osteoblasts ( hfob1.19 ) or murine ( mc3t3-e1 ) or primary osteoblasts , the osteoblasts increased their secretion of interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and monocyte chemoattractant protein-1 ( mcp-1 ) . under these conditions , the osteoblasts did not differentiate in culture ; that is , they did not produce characteristic osteoblast differentiation proteins such as alkaline phosphatase , osteocalcin , or bone sialoprotein [ 2 , 3 ] . these observations were followed with in vivo experiments . by using green fluorescent protein ( gfp ) expressing cancer cells in a xenograft model we saw that the cancer cells appeared throughout the bone but cleared quickly from the diaphysis . some cells localized in the ends of the femur where they developed into large colonies . for the most part , the cancer cells were associated with the endosteal surface of the bone marrow compartment . as part of an ex vivo study , we examined the cytokines produced by the bone cells in the presence of cancer cells . mice received intracardiac injections of mda - mb-231 cells and were sacrificed three weeks later . in this study , the marrow was removed from the femurs , the bones separated into diaphysis ( shaft ) and epiphyses ( ends ) , crushed and incubated in culture medium for 24 hr . species - specific antibodies were used to distinguish between host ( murine ) and cancer ( human ) cytokines . we found that murine il-6 , mcp-1 , macrophage inflammatory protein-2 ( mip-2 ) ( human il-8 ) , vascular endothelial growth factor ( vegf ) , and keratinocyte chemoattractant ( kc ) ( human growth - regulated oncogene - alpha , gro- ) were greater in ends of the bone compared to shafts and were increased in cancer - bearing mice . this ex vivo assay confirmed the in vitro findings that host cytokines in the bone microenvironment increase in the presence of cancer cells . these initial findings led us to investigate how the cytokine profile of the bone microenvironment changed over time following the appearance of cancer cells in the bone marrow . we designed an experiment to ask how cytokines changed over time in the femurs of mice inoculated with metastatic mda - mb-231 cells . concurrently , we wished to investigate whether or not the cytokine profile of the bone microenvironment differed when the mice were injected with the highly metastatic mda - mb-231 line or the metastasis - suppressed variant mda - mb-231brms1 which traffics to the bone but does not grow there . in order to examine the bone microenvironment in its entirety femurs were separated into shafts and ends , crushed and incubated for 24 hours in serum - free medium . an initial assay panel of 32 mouse cytokines revealed two cytokines , eotaxin and monkine induced by interferon gamma ( mig ) , in addition to il-6 , mcp-1 , mip-2 , kc , and vegf that merited further investigation . in the final experiment , athymic nude mice were injected in the left cardiac ventricle with either mda - mb-231 cells , mda - mb-231brms1 cells , or pbs . four days were chosen for sacrifice ( 3 , 11 , 19 , and 27 days after injection ) to represent early , middle , and late stage metastasis . culture supernatants from femoral shafts and ends were analyzed for mcp-1 , il-6 , kc , mip-2 , vegf , mig , and eotaxin . the human metastatic breast cancer cell line mda - mb-231 ( 231 ) and the metastasis - suppressed derivative mda - mb-231brms1 ( brms1 ) were obtained from danny welch , university of alabama , birmingham and cultured in dmem ( mediatech , herndon , va ) , 5% fetal bovine serum ( paa laboratories , etobicoke , on , canada ) , and 1x nonessential amino acids ( mediatech ) . antibiotics were not used to culture cells for a minimum of two weeks prior to injection . for intracardiac injection , cells were detached with trypsin - edta solution , centrifuged and washed twice with sterile phosphate - buffered saline ( pbs , hyclone , logan , utah ) . cells were resuspended at a concentration of 1.5 10 cells / ml in sterile pbs and held on ice until injection . six - week - old female athymic nude mice were obtained from charles river laboratories and were housed and handled in strict accordance with iacuc regulations ( penn state iacuc protocol 28631 ) . on the day of inoculation , mice were anesthetized with 120 mg / kg body weight of ketamine and 16 mg / kg of xylazine . when animals were completely anesthetized , 200 l of pbs or cancer cell suspension ( 3 10 cells ) were injected directly into the left ventricle of the heart . for the pilot experiment to screen for relevant cytokines , 3 mice were injected with either pbs , 231 , or brms1-expressing cells and kept for a period of 3 weeks before sacrifice . for the primary experiment , 8 mice were inoculated with either pbs , 231 , or brms1-expressing cells for each of the four time points . after recovery from the procedure , mice were returned to sterilized cages with air filters and observed daily for signs of illness or distress . on days 3 , 11 , 19 , and 27 after injection both femurs were removed from each mouse , cleaned of exterior tissue , and placed in pbs on ice prior to processing . femurs were examined by fluorescence stereomicroscopy ( 40x magnification ) with a nikon smz 1500 fluorescence stereoscope ( nikon instruments , inc . , melville , ny ) with gfp long bandpass fluorescence filter ( excitation = 488 nm ; emission = 515 nm , chroma technology corporation , rockingham , vt ) . images were captured using a nikon coolpix 8400 digital camera ( nikon instruments , inc . ) . proximal and distal ends of each femur were separated from the shaft of the bone . bone samples were crushed with a small glass pestle , and the fragments were cultured in 1 ml of mem ( mediatech ) for 24 hours . culture supernatants were then collected , centrifuged to remove cells and bone fragments , and frozen at 80c . to determine which cytokines might play an important role in the metastasis of cancer to bone , we first assayed bone culture supernatants of femurs of mice that had been injected with either pbs , 231 , or brms1-expressing cells 3 weeks prior to sacrifice . for this determination , we used a milliplex 32-plex mouse cytokine array ( millipore corporation , billerica , ma ) which allowed for the simultaneous quantitation of 32 mouse cytokines . after relevant cytokines were established for the main experiment , eotaxin , kc , mig , mip-2 , and vegf were assayed using a milliplex 5-plex mouse cytokine array . two other cytokines , il-6 and mcp-1 were assayed by standard sandwich elisa techniques as previously described . a statistical model was fit to allow for correlations within a mouse and within batches of mice using random effects in order to compare each cytokine across each group , that is , pbs , mda - mb-231 , and mda - mb-231brms1 . prior to statistical analyses , assumptions for linear statistics were verified and log10 transformations were used and rechecked to assure statistical validity of the analyses . for the shaft versus ends comparison , pg / ml values were analyzed with a two - way anova analysis with a bonferroni post hoc correction . to compare each cytokine among injection groups and over time , a two - way anova test with a fisher 's lsd post hoc analysis was used . shown in bar graphs is the mean standard error ( se ) . in previous experiments , we had access to a limited panel of cytokines available in a multiplex format . in preparation for this current study , we carried out a more extensive screen with a 32-plex cytokine array to identify more cytokines of interest . femurs were harvested from 9 mice ( 3 per group ) three weeks following intracardiac inoculation with mda - mb-231 cells , mda - mb-231brms1 cells , or pbs . two femurs from each mouse were incubated as described in the methods section and the culture supernatants were tested . the patterns of cytokines were similar in all three groups but generally highest in the culture supernatants from mda - mb-231 cells . of the 32 murine cytokines tested ( table 1 ) , nine were below the levels of detection , approximately 3.2 pg / ml . another four ( il-2 , il-17 , m - csf , and rantes ) were present in very small amounts , 10 pg / ml . thirteen ranged in concentrations from 10 to 100 pg / ml . six ( g - csf , il-6 , kc , mcp-1 , mig , and vegf ) were present at > 100 pg / ml . we had previously reported that il-6 , kc , mcp-1 , vegf , and mip-2 were secreted by osteoblasts and increased in the presence of breast cancer cells in vitro and in vivo . we choose to assay for these five cytokines plus mig which ranged in concentration from 1001000 pg / ml . we also selected eotaxin ( 1040 pg / ml ) because of its reported roles in angiogenesis in breast cancer metastasis patients and in multiple myeloma . the multiplex cytokine array for the remainder of the study included il-6 , kc , mcp-1 , vegf , mig , mip-2 , and eotaxin . at the conclusion of the study , it was found that mip-2 levels were negligible for most of the samples assayed and were not considered in further analyses . prior to crushing the femurs for incubation , they were examined with a fluorescence stereomicroscope . we detected gfp in some of the femurs of mice inoculated with mda - mb-231 or mda - mb - brms1 cells taken at various times ( figure 1 ) . however , the sensitivity of the microscope and the location of the cells combined with the thickness of the bone made it likely that not all metastatic cells were detected by microscopy . there were not enough femurs with gfp detectable colonies to be examined as a group for cytokines separate from the other femurs . for the most part , the gfp - expressing metastases appeared much larger in the femurs of mice inoculated with mda - mb-231 than those with mda - mb-231brms1 ( figure 1 ) . we first compared the levels of mcp-1 , il-6 , mig , kc , vegf , and eotaxin in the diaphysis ( shaft ) versus the epiphyses ( end ) of each bone . the epiphyseal end of the femur contains the metaphysis , the region of bone remodeling rich in cytokines and growth factors , in addition to the epiphyseal growth plate . in contrast , the function of the diaphysis is to provide support and is less metabolically active . as expected , the ends of long bone were a much richer source of cytokines than the shaft ( figure 2 ) . eotaxin was present in less than 10 pg / ml in the shaft supernatant but was 5 - 6- fold higher in the ends . little to no kc was found in the shaft but approximately 200 to 600 pg / ml were detected in the ends depending on the group of mice . vegf and il-6 were present in the shafts at about half of the concentration in the ends of the bone which was approximately 200 and 600 pg / ml , respectively . the cytokine found in the greatest concentration was mig registering 5002000 pg / ml for both shafts and ends . the cytokines from femurs of animals inoculated with mda - mb-231 cells , with mda - mb-231brms1 cells , or with pbs were compared at four times , day 3 , 11 , 19 , and 27 . the values presented ( figure 3 ) are the cytokine concentrations from the ends of the bone . at the earliest time ( day 3 ) , most of the cytokine concentrations were similar in all groups except for vegf . the femurs of mice inoculated with mda - mb-231 showed significantly greater amounts of vegf than the mice inoculated with mda - mb-231brms1 cells . however , neither group was different than pbs . at day 11 , il-6 was greater in the femurs of mice with mda - mb-231 than in the femurs of the pbs group . interestingly , at this time , the femurs of the mice inoculated with brms1-expressing cells had a greater concentration of vegf and mig than the mice with 231 cells . mig was significantly less in the animals injected with cancer cells than those injected with pbs . by day 27 , the differences among groups were most pronounced . mcp-1 , mig , eotaxin , and vegf were all significantly greater in the cancer - inoculated mice than in those inoculated with pbs . mice bearing mda - mb-231 showed less il-6 than those with pbs or mda - mb-231brms1 cells . no differences were apparent among the groups for kc at any of the times tested . one of the original objectives of this study was to examine the pattern of cytokine changes over time . we found that there was considerable variation from femur to femur even within the same animal . in the animals treated with pbs , there were increases and decreases over time in 5 cytokines tested ( figure 4 ) . since these animals did not harbor tumor cells , these differences likely reflect normal physiological variation over time . for the mice inoculated with mda - mb-231 cells , neither vegf nor eotaxin showed significant increases or decreases over the experimental time frame ( figure 4 ) . in contrast , mcp-1 , mig , and il-6 exhibited a significant decrease on day 19 when compared to day 3 . while il-6 and mig levels rose moderately on day 27 , the level of mcp-1 was substantially elevated . in animals injected with the metastasis suppressed variant , mda - mb-231brms1 , the expression pattern for mcp-1 , mig , and il-6 was similar to results obtained for the metastatic cells . interestingly , the brms1-expressing cells elicited a variable expression pattern for vegf and eotaxin that closely resembled the control pbs injection , suggesting that these two cytokines may be implicated in tumor cell colonization . previously , we have reported changes in the inflammatory cytokines il-6 , mcp-1 , vegf , kc , and mip-2 in the culture supernatants from femurs of athymic mice three weeks after intracardiac injection of mda - mb-231 cancer cells . we sought to verify and expand these findings to answer several key questions . how does the inclusion of the marrow affect the assay of cytokine expression in the presence of metastatic cancer ? does the cytokine profile of the bone microenvironment change over time after the introduction of cancer cells ? does the presence of metastasis - suppressed breast cancer cells elicit a bone cytokine profile that differs from the profile generated by metastatic cancer cells ? . cytokine analysis of bone culture supernatants with an expanded 32-plex array revealed the presence of several cytokines in addition to the five ( il-6 , mcp-1 , vegf , kc , and mip-2 ) previously reported . il-2 , il-17 , m - csf , and rantes were detected but only in small amounts ; due to cost constraint , we elected not to include them in the panel . mig and eotaxin were found to be expressed in the mouse femurs and appeared to vary with the presence of mda - mb-231 . mig is a target for rankl and as such is involved in osteoclast activation . eotaxin is believed to play a key role in angiogenesis . because osteolysis and tumor angiogenesis are intimately tied to cancer metastasis in bone , mig and eotaxin unlike the bone shaft , the ends of the long bones are areas of high bone turnover and are comprised of a specialized arrangement of osteoblasts , osteoclasts , stromal cells , hematopoietic cells , and endothelial cells . in order to examine the cytokine profile of the total bone microenvironment one obvious outcome of this study was that the cytokine concentrations in the ends of the bones were significantly higher than in the shaft . it has also been reported to be produced by osteoblasts . because the femurs are from athymic mice , the sources of mig in these experiments are likely osteoblasts and bone endothelial cells . eotaxin is also a product of t cells and endothelial cells , but not osteoblasts [ 14 , 15 ] ; thus , its likely origin is the bone vascular endothelium . we can not rule out that cytokines may also be due to transient monocytes , or to hematopoietic stem cells . using 8 mice per injection group per time point , we noticed a great deal of variation in cytokine levels from mouse to mouse within the same time and injection group . in many cases , two femurs from the same mouse yielded very different results . some of this variation may be due to imprecise sectioning of the ends and shaft of the bones . in addition , transient cells in the marrow such as monocytes or granulocytes and the general health of the animal independent of the presence of cancer metastasis could also account for this wide variation . in the case of animals injected with cancer cells , if the cancer cells had colonized another locus in the body , it is possible that the tumor may have had a more widespread effect on the cytokine levels in general . in order to examine the changes in mcp-1 il-6 , kc , mip-2 , vegf , mig , and eotaxin levels over time , we chose to sacrifice animals at 3 , 11 , 19 , and 27 days after injection of mda - mb-231 , mda - mb-231brms1 , or pbs . unfortunately , this experimental design did not allow us to sample the same animal over time . for this study , two cytokines , mip-2 and kc , were omitted from final analysis . mip-2 was not detected at all in a large number of the samples and kc showed no significant changes over time in any of the injection groups . at day 3 , 11 , and 19 , there were some statistically significant changes in mcp-1 , mig , vegf , eotaxin , and il-6 . since many of these changes also occurred in mice injected with pbs , the variation can likely be attributed to cyclic expression of cytokines in the bone , possibly due to age . originally , we postulated that if a particular cytokine was elevated early in the metastatic process , it could be acting as a chemoattractant for cancer cells or a catalyst for cancer cell colonization . however , there is insufficient evidence from this experiment to pinpoint such a cytokine from the seven cytokines assayed . the most striking results were observed at day 27 when levels of mcp-1 , mig , vegf , and eotaxin were significantly higher in mice injected with either breast cancer cell variant than in mice injected with pbs . il-6 , mcp-1 , vegf , and mig have all been implicated in osteoclastogenesis [ 8 , 16 ] . an increase in these molecules in the microenvironment in response to cancer cells correlates with increased osteoclast differentiation and activation and thus bone resorption . osteoblasts have been reported to display an inflammatory cytokine stress response to titanium in joint replacements and to bacteria in osteomyelitis . the same cytokine response occurs when breast cancer and likely other epithelial cells invade the marrow cavity . because several of these cytokines are also expressed by osteoblasts during their normal differentiation and during the bone remodeling process , it is easy to see how the introduction of cancer cells to the bone microenvironment can disrupt both of these important functions . additionally , vegf and eotaxin are known promoters of angiogenesis [ 11 , 19 ] and may be responsible for the vascularization of a newly formed metastatic tumor . in comparing the cytokine profiles of animals injected with metastatic mda - mb-231 to metastasis - suppressed mda - mb-231brms1 cells , we observed that at day 27 both cell types elicited significant elevations in mcp-1 , mig , vegf , and eotaxin levels . these data indicate that these four cytokines are not likely to be responsible for the inability of the brms1-expressing cells to colonize the bone . however , we were intrigued by the difference in cytokine expression patterns over time for vegf and eotaxin . while vegf and eotaxin levels remained unchanged in animals injected with 231 cells , the expression levels for pbs- and brms1-injected animals showed a similar pattern of significant variation over time ( i.e. , reduced expression levels at day 3 ) . one possible interpretation of these data is that higher sustained levels of vegf and eotaxin are enabling the metastatic cancer cells to colonize and thrive in the bone environment . it is interesting to note that mda - mb-231 and mda - mb-231brms1 themselves secrete il-6 , vegf , il-8 , and gro- ( the human homologues of mip-2 and kc , resp . ) . mcp-1 is made in small amounts and mig is reported to be absent from the 231 cancer cells . in this study , in addition , the cancer cells have been reported to express receptors to il-6 , mip-2 , kc , vegf , mcp-1 , and mig . the mrna for the receptor for eotaxin was not detected in mda - mb-231 cells . in a recent publication , mig was reported to be produced by bone marrow mesenchymal stem cells and enhanced the invasion and motility of mda - mb-231 cells . in the cross - species xenograft model for breast cancer utilized in this experiment thus the cytokine changes that occur in the microenvironment as a consequence of the cancer cells may also be responsible for the progression of the metastatic tumor . in summary , cytokines in the bone microenvironment the presence of cancer cells changes the normal levels of these cytokines which in turn disrupts the homeostatic balance in the bone . abnormal cytokine levels may also serve to fuel the propagation and further metastasis of breast cancer cells . whether these changes are limited to the immediate location of the cancer cells or are the result of a systemic effect has yet to be determined .

it is commonly accepted that cancer cells interact with host cells to create a microenvironment favoring malignant colonization . the complex bone microenvironment produces an ever changing array of cytokines and growth factors . in this study , we examined levels of mcp-1 , il-6 , kc , mip-2 , vegf , mig , and eotaxin in femurs of athymic nude mice inoculated via intracardiac injection with mda - mb-231gfp human metastatic breast cancer cells , mda - mb-231brms1gfp , a metastasis suppressed variant , or pbs . animals were euthanized ( day 3 , 11 , 19 , 27 after injection ) to examine femoral cytokine levels at various stages of cancer cell colonization . the epiphysis contained significantly more cytokines than the diaphysis except for mig which was similar throughout the bone . variation among femurs was evident within all groups . by day 27 , mcp-1 , mig , vegf and eotaxin levels were significantly greater in femurs of cancer cell - inoculated mice . these pro - osteoclastic and angiogenic cytokines may manipulate the bone microenvironment to enhance cancer cell colonization .

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

the colonization and growth of cancer metastases in the bone depends on a cooperative interaction of the cancer cells with the host cells in the bone microenvironment . this microenvironment includes the resident osteoblasts , osteoclasts , endothelial cells , bone - lining cells , stromal cells , hematopoietic stem cells , and transient cells such as macrophages , lymphocytes , neutrophils , and other blood cells . while cell - cell contacts are established between cancer cells and bone cells via adhesion molecules , a wider network of communication occurs through secreted cytokines and growth factors . these soluble molecules play a critical role in the normal bone remodeling process as well as in cancer cell colonization of the bone marrow . the interplay of the cancer cells with the cells of the bone marrow cavity has been described in terms of a vicious cycle . in brief , cytokines or growth factors secreted by invading cancer cells ( e.g. this series of events provides an explanation of the osteolytic outcome of breast cancer metastasis in bone ; that is , an increase in osteoclast activation leads to excess bone breakdown and further stimulation of cancer cells . in our previous research , we found that metastatic breast cancer cells also inhibit the differentiation of osteoblasts , thereby diminishing bone formation . through cell culture studies when conditioned medium from metastatic human breast cancer cells , mda - mb-231 , was added to human osteoblasts ( hfob1.19 ) or murine ( mc3t3-e1 ) or primary osteoblasts , the osteoblasts increased their secretion of interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and monocyte chemoattractant protein-1 ( mcp-1 ) . under these conditions , the osteoblasts did not differentiate in culture ; that is , they did not produce characteristic osteoblast differentiation proteins such as alkaline phosphatase , osteocalcin , or bone sialoprotein [ 2 , 3 ] . by using green fluorescent protein ( gfp ) expressing cancer cells in a xenograft model we saw that the cancer cells appeared throughout the bone but cleared quickly from the diaphysis . for the most part , the cancer cells were associated with the endosteal surface of the bone marrow compartment . as part of an ex vivo study , we examined the cytokines produced by the bone cells in the presence of cancer cells . mice received intracardiac injections of mda - mb-231 cells and were sacrificed three weeks later . in this study , the marrow was removed from the femurs , the bones separated into diaphysis ( shaft ) and epiphyses ( ends ) , crushed and incubated in culture medium for 24 hr . we found that murine il-6 , mcp-1 , macrophage inflammatory protein-2 ( mip-2 ) ( human il-8 ) , vascular endothelial growth factor ( vegf ) , and keratinocyte chemoattractant ( kc ) ( human growth - regulated oncogene - alpha , gro- ) were greater in ends of the bone compared to shafts and were increased in cancer - bearing mice . this ex vivo assay confirmed the in vitro findings that host cytokines in the bone microenvironment increase in the presence of cancer cells . these initial findings led us to investigate how the cytokine profile of the bone microenvironment changed over time following the appearance of cancer cells in the bone marrow . we designed an experiment to ask how cytokines changed over time in the femurs of mice inoculated with metastatic mda - mb-231 cells . concurrently , we wished to investigate whether or not the cytokine profile of the bone microenvironment differed when the mice were injected with the highly metastatic mda - mb-231 line or the metastasis - suppressed variant mda - mb-231brms1 which traffics to the bone but does not grow there . in order to examine the bone microenvironment in its entirety femurs were separated into shafts and ends , crushed and incubated for 24 hours in serum - free medium . an initial assay panel of 32 mouse cytokines revealed two cytokines , eotaxin and monkine induced by interferon gamma ( mig ) , in addition to il-6 , mcp-1 , mip-2 , kc , and vegf that merited further investigation . in the final experiment , athymic nude mice were injected in the left cardiac ventricle with either mda - mb-231 cells , mda - mb-231brms1 cells , or pbs . four days were chosen for sacrifice ( 3 , 11 , 19 , and 27 days after injection ) to represent early , middle , and late stage metastasis . culture supernatants from femoral shafts and ends were analyzed for mcp-1 , il-6 , kc , mip-2 , vegf , mig , and eotaxin . the human metastatic breast cancer cell line mda - mb-231 ( 231 ) and the metastasis - suppressed derivative mda - mb-231brms1 ( brms1 ) were obtained from danny welch , university of alabama , birmingham and cultured in dmem ( mediatech , herndon , va ) , 5% fetal bovine serum ( paa laboratories , etobicoke , on , canada ) , and 1x nonessential amino acids ( mediatech ) . six - week - old female athymic nude mice were obtained from charles river laboratories and were housed and handled in strict accordance with iacuc regulations ( penn state iacuc protocol 28631 ) . when animals were completely anesthetized , 200 l of pbs or cancer cell suspension ( 3 10 cells ) were injected directly into the left ventricle of the heart . for the primary experiment , 8 mice were inoculated with either pbs , 231 , or brms1-expressing cells for each of the four time points . on days 3 , 11 , 19 , and 27 after injection both femurs were removed from each mouse , cleaned of exterior tissue , and placed in pbs on ice prior to processing . proximal and distal ends of each femur were separated from the shaft of the bone . culture supernatants were then collected , centrifuged to remove cells and bone fragments , and frozen at 80c . to determine which cytokines might play an important role in the metastasis of cancer to bone , we first assayed bone culture supernatants of femurs of mice that had been injected with either pbs , 231 , or brms1-expressing cells 3 weeks prior to sacrifice . after relevant cytokines were established for the main experiment , eotaxin , kc , mig , mip-2 , and vegf were assayed using a milliplex 5-plex mouse cytokine array . a statistical model was fit to allow for correlations within a mouse and within batches of mice using random effects in order to compare each cytokine across each group , that is , pbs , mda - mb-231 , and mda - mb-231brms1 . to compare each cytokine among injection groups and over time , a two - way anova test with a fisher 's lsd post hoc analysis was used . in previous experiments , we had access to a limited panel of cytokines available in a multiplex format . in preparation for this current study , we carried out a more extensive screen with a 32-plex cytokine array to identify more cytokines of interest . femurs were harvested from 9 mice ( 3 per group ) three weeks following intracardiac inoculation with mda - mb-231 cells , mda - mb-231brms1 cells , or pbs . the patterns of cytokines were similar in all three groups but generally highest in the culture supernatants from mda - mb-231 cells . another four ( il-2 , il-17 , m - csf , and rantes ) were present in very small amounts , 10 pg / ml . six ( g - csf , il-6 , kc , mcp-1 , mig , and vegf ) were present at > 100 pg / ml . we had previously reported that il-6 , kc , mcp-1 , vegf , and mip-2 were secreted by osteoblasts and increased in the presence of breast cancer cells in vitro and in vivo . we choose to assay for these five cytokines plus mig which ranged in concentration from 1001000 pg / ml . the multiplex cytokine array for the remainder of the study included il-6 , kc , mcp-1 , vegf , mig , mip-2 , and eotaxin . at the conclusion of the study , it was found that mip-2 levels were negligible for most of the samples assayed and were not considered in further analyses . we detected gfp in some of the femurs of mice inoculated with mda - mb-231 or mda - mb - brms1 cells taken at various times ( figure 1 ) . however , the sensitivity of the microscope and the location of the cells combined with the thickness of the bone made it likely that not all metastatic cells were detected by microscopy . for the most part , the gfp - expressing metastases appeared much larger in the femurs of mice inoculated with mda - mb-231 than those with mda - mb-231brms1 ( figure 1 ) . we first compared the levels of mcp-1 , il-6 , mig , kc , vegf , and eotaxin in the diaphysis ( shaft ) versus the epiphyses ( end ) of each bone . the epiphyseal end of the femur contains the metaphysis , the region of bone remodeling rich in cytokines and growth factors , in addition to the epiphyseal growth plate . as expected , the ends of long bone were a much richer source of cytokines than the shaft ( figure 2 ) . vegf and il-6 were present in the shafts at about half of the concentration in the ends of the bone which was approximately 200 and 600 pg / ml , respectively . the cytokines from femurs of animals inoculated with mda - mb-231 cells , with mda - mb-231brms1 cells , or with pbs were compared at four times , day 3 , 11 , 19 , and 27 . at the earliest time ( day 3 ) , most of the cytokine concentrations were similar in all groups except for vegf . the femurs of mice inoculated with mda - mb-231 showed significantly greater amounts of vegf than the mice inoculated with mda - mb-231brms1 cells . at day 11 , il-6 was greater in the femurs of mice with mda - mb-231 than in the femurs of the pbs group . interestingly , at this time , the femurs of the mice inoculated with brms1-expressing cells had a greater concentration of vegf and mig than the mice with 231 cells . by day 27 , the differences among groups were most pronounced . mcp-1 , mig , eotaxin , and vegf were all significantly greater in the cancer - inoculated mice than in those inoculated with pbs . mice bearing mda - mb-231 showed less il-6 than those with pbs or mda - mb-231brms1 cells . one of the original objectives of this study was to examine the pattern of cytokine changes over time . since these animals did not harbor tumor cells , these differences likely reflect normal physiological variation over time . for the mice inoculated with mda - mb-231 cells , neither vegf nor eotaxin showed significant increases or decreases over the experimental time frame ( figure 4 ) . in contrast , mcp-1 , mig , and il-6 exhibited a significant decrease on day 19 when compared to day 3 . while il-6 and mig levels rose moderately on day 27 , the level of mcp-1 was substantially elevated . in animals injected with the metastasis suppressed variant , mda - mb-231brms1 , the expression pattern for mcp-1 , mig , and il-6 was similar to results obtained for the metastatic cells . interestingly , the brms1-expressing cells elicited a variable expression pattern for vegf and eotaxin that closely resembled the control pbs injection , suggesting that these two cytokines may be implicated in tumor cell colonization . previously , we have reported changes in the inflammatory cytokines il-6 , mcp-1 , vegf , kc , and mip-2 in the culture supernatants from femurs of athymic mice three weeks after intracardiac injection of mda - mb-231 cancer cells . does the cytokine profile of the bone microenvironment change over time after the introduction of cancer cells ? does the presence of metastasis - suppressed breast cancer cells elicit a bone cytokine profile that differs from the profile generated by metastatic cancer cells ? cytokine analysis of bone culture supernatants with an expanded 32-plex array revealed the presence of several cytokines in addition to the five ( il-6 , mcp-1 , vegf , kc , and mip-2 ) previously reported . il-2 , il-17 , m - csf , and rantes were detected but only in small amounts ; due to cost constraint , we elected not to include them in the panel . mig and eotaxin were found to be expressed in the mouse femurs and appeared to vary with the presence of mda - mb-231 . because osteolysis and tumor angiogenesis are intimately tied to cancer metastasis in bone , mig and eotaxin unlike the bone shaft , the ends of the long bones are areas of high bone turnover and are comprised of a specialized arrangement of osteoblasts , osteoclasts , stromal cells , hematopoietic cells , and endothelial cells . in order to examine the cytokine profile of the total bone microenvironment one obvious outcome of this study was that the cytokine concentrations in the ends of the bones were significantly higher than in the shaft . eotaxin is also a product of t cells and endothelial cells , but not osteoblasts [ 14 , 15 ] ; thus , its likely origin is the bone vascular endothelium . we can not rule out that cytokines may also be due to transient monocytes , or to hematopoietic stem cells . using 8 mice per injection group per time point , we noticed a great deal of variation in cytokine levels from mouse to mouse within the same time and injection group . in addition , transient cells in the marrow such as monocytes or granulocytes and the general health of the animal independent of the presence of cancer metastasis could also account for this wide variation . in the case of animals injected with cancer cells , if the cancer cells had colonized another locus in the body , it is possible that the tumor may have had a more widespread effect on the cytokine levels in general . in order to examine the changes in mcp-1 il-6 , kc , mip-2 , vegf , mig , and eotaxin levels over time , we chose to sacrifice animals at 3 , 11 , 19 , and 27 days after injection of mda - mb-231 , mda - mb-231brms1 , or pbs . for this study , two cytokines , mip-2 and kc , were omitted from final analysis . at day 3 , 11 , and 19 , there were some statistically significant changes in mcp-1 , mig , vegf , eotaxin , and il-6 . since many of these changes also occurred in mice injected with pbs , the variation can likely be attributed to cyclic expression of cytokines in the bone , possibly due to age . originally , we postulated that if a particular cytokine was elevated early in the metastatic process , it could be acting as a chemoattractant for cancer cells or a catalyst for cancer cell colonization . the most striking results were observed at day 27 when levels of mcp-1 , mig , vegf , and eotaxin were significantly higher in mice injected with either breast cancer cell variant than in mice injected with pbs . il-6 , mcp-1 , vegf , and mig have all been implicated in osteoclastogenesis [ 8 , 16 ] . an increase in these molecules in the microenvironment in response to cancer cells correlates with increased osteoclast differentiation and activation and thus bone resorption . because several of these cytokines are also expressed by osteoblasts during their normal differentiation and during the bone remodeling process , it is easy to see how the introduction of cancer cells to the bone microenvironment can disrupt both of these important functions . additionally , vegf and eotaxin are known promoters of angiogenesis [ 11 , 19 ] and may be responsible for the vascularization of a newly formed metastatic tumor . in comparing the cytokine profiles of animals injected with metastatic mda - mb-231 to metastasis - suppressed mda - mb-231brms1 cells , we observed that at day 27 both cell types elicited significant elevations in mcp-1 , mig , vegf , and eotaxin levels . these data indicate that these four cytokines are not likely to be responsible for the inability of the brms1-expressing cells to colonize the bone . however , we were intrigued by the difference in cytokine expression patterns over time for vegf and eotaxin . while vegf and eotaxin levels remained unchanged in animals injected with 231 cells , the expression levels for pbs- and brms1-injected animals showed a similar pattern of significant variation over time ( i.e. , reduced expression levels at day 3 ) . one possible interpretation of these data is that higher sustained levels of vegf and eotaxin are enabling the metastatic cancer cells to colonize and thrive in the bone environment . it is interesting to note that mda - mb-231 and mda - mb-231brms1 themselves secrete il-6 , vegf , il-8 , and gro- ( the human homologues of mip-2 and kc , resp . ) in this study , in addition , the cancer cells have been reported to express receptors to il-6 , mip-2 , kc , vegf , mcp-1 , and mig . in a recent publication , mig was reported to be produced by bone marrow mesenchymal stem cells and enhanced the invasion and motility of mda - mb-231 cells . in the cross - species xenograft model for breast cancer utilized in this experiment thus the cytokine changes that occur in the microenvironment as a consequence of the cancer cells may also be responsible for the progression of the metastatic tumor . in summary , cytokines in the bone microenvironment the presence of cancer cells changes the normal levels of these cytokines which in turn disrupts the homeostatic balance in the bone . abnormal cytokine levels may also serve to fuel the propagation and further metastasis of breast cancer cells .

a brain abscess is a focal intracerebral infection that leads to a life - threatening condition with severe neurological deficits . brain abscesses usually occur because of spreading from a contiguous focus of infection or hematogenous dissemination from a distant focus4,20,25 ) . in general , the brain is resistant to bacterial and fungal infection because of an abundant blood supply and an impermeable blood - brain barrier . however , conditions that compromise the integrity of the central nervous system such as head trauma and neurosurgery can also cause brain abscesses8,28,30 ) . in the past , however , the introduction of computed tomography ( ct ) in the early 1970s and the development of neurosurgical techniques along with the use of broad - spectrum antibiotics contributed to an improvement in patient prognoses6,24,26,34 ) . the purpose of this study was to review the data of 51 patients with brain abscesses admitted to a single tertiary referral hospital during a recent 10-year period and describe their clinical characteristics , outcome of treatment , and prognostic factors . this retrospective study included 51 patients with intracerebral abscesses treated with navigation - assisted abscess drainage at our institute between january 2004 and december 2013 . a brain abscess was defined as the presence of one or more lesions with a hypodense center and peripheral ring enhancement , after the injection of contrast media , on ct or magnetic resonance images ( mris ) . it was associated with at least one of the following characteristics : positive cultures of intracerebral materials , positive blood cultures , or histology of the intracerebral lesion suggesting a brain abscess . patients with subdural empyema and epidural abscesses , and those who underwent craniotomy for brain abscesses , were excluded . patient charts were reviewed for the following : age at diagnosis , sex , past history , symptom duration before diagnosis , level of consciousness [ assessed by the glasgow coma scale ( gcs ) ] , focal neurological status at admission , disease predisposition , various serum parameters [ complete blood count , erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) , and random blood sugar at admission ] , microorganisms identified on blood and abscess material cultures , antibiotic regimen , and duration of antibiotic treatment . the radiological parameters including the multiplicity of the lesion , abscess location , and initial and postoperative abscess volumes were also reviewed . abscess volume was calculated using the abc/2 formula , where a is the greatest abscess diameter measured by ct or mri , b is the diameter 90 to a , and c is the extent in the coronal direction or approximate number of slices within the abscess multiplied by the slice thickness ( fig . all patients underwent abscess aspiration under a neuronavigation system ( stealthstation , medtronic , minneapolis , mn , usa ) . the operative intervention was performed at the earliest time , if indicated , without preoperative antibiotics so as not to sterilize the cultures . patients who underwent delayed surgery were administered empirical antibiotics before the operation . during the operation , abscess material was maximally aspirated through a silicone catheter connected to soft collection bags placed in a dependent position and maintained for a minimum of 24 h. a repeat follow - up imaging study was performed routinely in all patients approximately 1 - 2 weeks postoperatively . brain abscess specimens obtained from the surgery were cultured for aerobic and anaerobic bacteria , mycobacteria , and fungi . antibiotic susceptibility tests were performed on the isolated microorganisms . immediately after the abscess drainage procedure , the choice of antibiotics for cases with sterile cultures was directed to the most likely organisms based on clinical history and the primary site of infection , if known . the referring physician was later consulted with regard to adjustments or additions to the antibiotics and maintenance of the agents . the outcome was assessed according to the glasgow outcome scale ( gos ) at the time of discharge . a favorable outcome ( gos 4 ) was defined as moderate disability ( having a disability , but being independent ) and good recovery . an unfavorable outcome ( gos < 4 ) was defined as death , persistent vegetative status , or severe disability ( conscious , but with disability ) . statistical analysis was performed using the statistical package for the social sciences software ( spss 11.5 , ibm corporation , armonk , ny , usa ) . univariate analyses ( chi - square test , fisher 's exact test , student 's t - test ) were performed to compare the variables between patients with a favorable outcome or with early response to antibiotics , and those with poor outcomes or with a delayed response to antibiotics . variables found to have p<0.1 in the univariate analyses were entered into a multivariate logistic regression model to identify independent predictors of neurological outcome and the duration of antibiotic use . there were 41 male and 10 female patients with a mean age of 53 years ( range : 14 - 81 years ) . headache , which occurred in 25 patients , was the most common symptom , while 3 patients complained of nausea and vomiting . motor weakness occurred in 20 patients , and fever was checked in 10 patients on admission . other symptoms and signs included speech disturbance ( 8 patients ) , visual disturbance ( 4 patients ) , impaired cognition ( 3 patients ) , seizures ( 2 patients ) , malaise ( 2 patients ) , and sensory changes ( 2 patients ) . clear or mildly disturbed consciousness ( gcs 13 ) was evident in 42 patients ( 82% ) and moderately or severely disturbed consciousness ( gcs < 13 ) was present in 9 patients ( 18% ) . the mean interval between symptom onset and admission was 8 days ( range : 0 - 45 days ) . a total of 24 patients had predisposing factors including pulmonary infection ( 7 patients ) , sinusitis ( 4 patients ) , dental infection ( 3 patients ) , prior trauma or neurosurgery ( 2 patients ) , otic infection ( 2 patients ) , endocarditis ( 2 patients ) , congenital heart disease ( 2 patients ) , skin infection ( 1 patient ) , and psoas abscess ( 1 patient ) . ten patients presented with known diabetes mellitus and 4 patients were in an immunocompromised state resulting from chemotherapy for cancer ( 2 patients ) , chronic renal failure ( 1 patient ) , and steroid therapy for addison 's disease ( 1 patient ) . of the 51 patients , leukocytosis [ white blood cell ( wbc ) 10000/l ] at admission was identified in 24 ( 47% ) patients . mean serum esr and crp levels at admission were 34.254.08 mm / h and 39.128.13 mg / l , respectively . an elevated crp level ( 10 mg / l ) was detected in 31 ( 61% ) patients . twenty - two ( 43% ) of the 51 patients were in a state of hyperglycemia ( blood glucose 140 mg / dl ) on admission . on brain images , a single lesion was observed in 44 ( 86% ) of the 51 patients and multiple lesions were observed in 7 patients ( 14% ) . the abscesses were most commonly found in the frontal lobe ( 30 patients , 59% ) , followed by the parietal lobe ( 12 patients , 24% ) , temporal lobe ( 8 patients , 16% ) , occipital lobe ( 5 patients , 10% ) , thalamus ( 2 patients , 4% ) , and pons ( 1 patient , 2% ) . the mean preoperative abscess volume was 19.112.00 cm and the residual volume of the abscesses after surgery was 7.030.97 cm ( table 2 ) . abscess pathogens were identified in 25 patients ( 49% ) during a culture study of the abscess material ( table 3 ) . a single organism was isolated from 21 patients and multiple organisms were isolated from 4 patients . of these isolated microorganisms , 21 ( 41% ) the most common aerobic bacteria were the streptococcus species ( 14 cases , 27% ) , followed by gemella morbillorum ( 4 cases , 8% ) , staphylococcus aureus ( 1 case , 2% ) , proteus mirabilis ( 1 case , 2% ) , and klebsiella pneumoniae ( 1 case , 2% ) . the most frequently isolated anaerobic bacteria belonged to the prevotella species ( 3 cases , 6% ) , followed by the peptostreptococcus species ( 2 cases , 4% ) , fusobacterium nucleatum ( 2 cases , 4% ) , and bacteroides intermedius ( 1 case , 2% ) . blood cultures were performed in 10 patients who presented with fever at admission and were positive in 2 patients ( 4% ) . in one patient , staphylococcus aureus was isolated from the blood , as well as from the abscess material . in the other patient , the abscess fluid culture was negative , and the blood culture showed streptococcus viridans growth . the mean duration between admission and surgery was 7.29 days ( range : 1 - 27 days ) . of the 51 patients , 6 patients ( 11.8% ) underwent surgery twice because a considerable portion of the abscess remained after the first intervention . intravenous administration of empirical antibiotics including vancomycin , ceftriaxone , and metronidazole were started after abscess aspiration until the pathogens were confirmed . in the 8 patients who were suspected to be in the cerebritis stage , the antibiotics were started before surgery . the most commonly used antibiotics included a combination of ceftriaxone and metronidazole ( 22 patients , 43% ) , sometimes with the addition of either vancomycin or gentamicin . the mean duration of intravenous antibiotic treatment for surviving patients was 42 days ( range : 23 - 66 days ) . none of the patients received oral antibiotics after they completed a course of parenteral antibiotic therapy . corticosteroids were selectively administered to patients with severely increased intracranial pressure ( 9 patients , 18% ) . during the course of treatment , 8 patients ( 16% ) died , with 3 ( 6% ) deaths being attributed to the abscess itself and 5 ( 10% ) deaths that may have resulted from the underlying pathology . in the latter cases , the cause of death was attributable to the aggravation of a pre - existing disease : 2 patients died of pulmonary infection - related sepsis , 1 patient with an atrial septum defect died from a pulmonary embolism , 1 patient with congestive heart failure died from a sudden heart attack , and 1 patient who had hepatoma died from bleeding of the original tumor . at discharge , a favorable outcome ( gos4 ) one of the patients with a thalamic abscess had a severe neurological deficit and was unable to live independently , and the other remained in a persistent vegetative state due to secondary ventriculitis caused by a rupture of the abscess . the results of comparison analyses between the patients with favorable and unfavorable outcomes are shown in table 4 . in the univariate analyses , admission variables associated with patient outcome included an initial gcs score < 13 ( p=0.003 ) , a high level of serum crp ( 10 mg / l , p=0.024 ) , and hyperglycemia ( 140 mg / dl , p=0.016 ) . among these , only a gcs score < 13 [ p=0.052 , 95% confidence interval ( ci ) 0.982 - 37.118 ] was likely to be an independent risk factor for poor patient outcome in the multivariate logistic regression analysis . to assess the clinical and radiological factors that were associated with the response to antibiotics , the patients were divided into two groups : 1 ) early responder group ( duration of antibiotics < 6 weeks ) comprising 25 patients , and 2 ) delayed responder group ( dead or using antibiotics for 6 weeks ) comprising 26 patients . the mean wbc count and plasma blood sugar level in the delayed responder group were significantly higher than in the early responder group ( 12639/l vs. 10116/l , p=0.045 ; 171.57 mg / dl vs. 122.84 mg / dl , p=0.003 , respectively ) ( table 5 ) . in the multivariate analyses , the independent risk factor for prolonged antibiotic use was hyperglycemia ( p=0.032 , 95% ci 1.002 - 1.040 ) . there was a positive correlation between the initial blood sugar level and the duration of antibiotic use ( r=0.471 and p=0.001 ) ( fig . there were 41 male and 10 female patients with a mean age of 53 years ( range : 14 - 81 years ) . headache , which occurred in 25 patients , was the most common symptom , while 3 patients complained of nausea and vomiting . motor weakness occurred in 20 patients , and fever was checked in 10 patients on admission . other symptoms and signs included speech disturbance ( 8 patients ) , visual disturbance ( 4 patients ) , impaired cognition ( 3 patients ) , seizures ( 2 patients ) , malaise ( 2 patients ) , and sensory changes ( 2 patients ) . clear or mildly disturbed consciousness ( gcs 13 ) was evident in 42 patients ( 82% ) and moderately or severely disturbed consciousness ( gcs < 13 ) was present in 9 patients ( 18% ) . the mean interval between symptom onset and admission was 8 days ( range : 0 - 45 days ) . a total of 24 patients had predisposing factors including pulmonary infection ( 7 patients ) , sinusitis ( 4 patients ) , dental infection ( 3 patients ) , prior trauma or neurosurgery ( 2 patients ) , otic infection ( 2 patients ) , endocarditis ( 2 patients ) , congenital heart disease ( 2 patients ) , skin infection ( 1 patient ) , and psoas abscess ( 1 patient ) . ten patients presented with known diabetes mellitus and 4 patients were in an immunocompromised state resulting from chemotherapy for cancer ( 2 patients ) , chronic renal failure ( 1 patient ) , and steroid therapy for addison 's disease ( 1 patient ) . of the 51 patients , leukocytosis [ white blood cell ( wbc ) 10000/l ] at admission was identified in 24 ( 47% ) patients . mean serum esr and crp levels at admission were 34.254.08 mm / h and 39.128.13 mg / l , respectively . an elevated crp level ( 10 mg / l ) was detected in 31 ( 61% ) patients . twenty - two ( 43% ) of the 51 patients were in a state of hyperglycemia ( blood glucose 140 mg / dl ) on admission . on brain images , a single lesion was observed in 44 ( 86% ) of the 51 patients and multiple lesions were observed in 7 patients ( 14% ) . the abscesses were most commonly found in the frontal lobe ( 30 patients , 59% ) , followed by the parietal lobe ( 12 patients , 24% ) , temporal lobe ( 8 patients , 16% ) , occipital lobe ( 5 patients , 10% ) , thalamus ( 2 patients , 4% ) , and pons ( 1 patient , 2% ) . the mean preoperative abscess volume was 19.112.00 cm and the residual volume of the abscesses after surgery was 7.030.97 cm ( table 2 ) . abscess pathogens were identified in 25 patients ( 49% ) during a culture study of the abscess material ( table 3 ) . a single organism was isolated from 21 patients and multiple organisms were isolated from 4 patients . of these isolated microorganisms , 21 ( 41% ) were aerobic and 8 ( 16% ) were anaerobic bacteria . the most common aerobic bacteria were the streptococcus species ( 14 cases , 27% ) , followed by gemella morbillorum ( 4 cases , 8% ) , staphylococcus aureus ( 1 case , 2% ) , proteus mirabilis ( 1 case , 2% ) , and klebsiella pneumoniae ( 1 case , 2% ) . the most frequently isolated anaerobic bacteria belonged to the prevotella species ( 3 cases , 6% ) , followed by the peptostreptococcus species ( 2 cases , 4% ) , fusobacterium nucleatum ( 2 cases , 4% ) , and bacteroides intermedius ( 1 case , 2% ) . blood cultures were performed in 10 patients who presented with fever at admission and were positive in 2 patients ( 4% ) . in one patient , staphylococcus aureus was isolated from the blood , as well as from the abscess material . in the other patient , the abscess fluid culture was negative , and the blood culture showed streptococcus viridans growth . the mean duration between admission and surgery was 7.29 days ( range : 1 - 27 days ) . of the 51 patients , 6 patients ( 11.8% ) underwent surgery twice because a considerable portion of the abscess remained after the first intervention . intravenous administration of empirical antibiotics including vancomycin , ceftriaxone , and metronidazole were started after abscess aspiration until the pathogens were confirmed . in the 8 patients who were suspected to be in the cerebritis stage , the antibiotics were started before surgery . the most commonly used antibiotics included a combination of ceftriaxone and metronidazole ( 22 patients , 43% ) , sometimes with the addition of either vancomycin or gentamicin . the mean duration of intravenous antibiotic treatment for surviving patients was 42 days ( range : 23 - 66 days ) . none of the patients received oral antibiotics after they completed a course of parenteral antibiotic therapy . corticosteroids were selectively administered to patients with severely increased intracranial pressure ( 9 patients , 18% ) . during the course of treatment , 8 patients ( 16% ) died , with 3 ( 6% ) deaths being attributed to the abscess itself and 5 ( 10% ) deaths that may have resulted from the underlying pathology . in the latter cases , the cause of death was attributable to the aggravation of a pre - existing disease : 2 patients died of pulmonary infection - related sepsis , 1 patient with an atrial septum defect died from a pulmonary embolism , 1 patient with congestive heart failure died from a sudden heart attack , and 1 patient who had hepatoma died from bleeding of the original tumor . at discharge , a favorable outcome ( gos4 ) one of the patients with a thalamic abscess had a severe neurological deficit and was unable to live independently , and the other remained in a persistent vegetative state due to secondary ventriculitis caused by a rupture of the abscess . the results of comparison analyses between the patients with favorable and unfavorable outcomes are shown in table 4 . in the univariate analyses , admission variables associated with patient outcome included an initial gcs score < 13 ( p=0.003 ) , a high level of serum crp ( 10 mg / l , p=0.024 ) , and hyperglycemia ( 140 mg / dl , p=0.016 ) . among these , only a gcs score < 13 [ p=0.052 , 95% confidence interval ( ci ) 0.982 - 37.118 ] was likely to be an independent risk factor for poor patient outcome in the multivariate logistic regression analysis . to assess the clinical and radiological factors that were associated with the response to antibiotics , the patients were divided into two groups : 1 ) early responder group ( duration of antibiotics < 6 weeks ) comprising 25 patients , and 2 ) delayed responder group ( dead or using antibiotics for 6 weeks ) comprising 26 patients . the mean wbc count and plasma blood sugar level in the delayed responder group were significantly higher than in the early responder group ( 12639/l vs. 10116/l , p=0.045 ; 171.57 mg / dl vs. 122.84 mg / dl , p=0.003 , respectively ) ( table 5 ) . in the multivariate analyses , the independent risk factor for prolonged antibiotic use was hyperglycemia ( p=0.032 , 95% ci 1.002 - 1.040 ) . there was a positive correlation between the initial blood sugar level and the duration of antibiotic use ( r=0.471 and p=0.001 ) ( fig . brain abscesses are considered serious and life threatening infections . the mortality rate associated with brain abscesses advances in radiographic scanning , the development of novel surgical techniques , and the availability of newer antibiotics have all helped decrease the mortality rate in patients with brain abscesses6,24,26,34 ) . recently , tseng and tseng31 ) have published the clinical outcomes of their patients with brain abscesses ( n=142 ) , of which 16.9% died during hospitalization . the deaths were caused by underlying systemic infection or the terminal stage of cancer and not by the abscess itself . similarly , in our series , the overall mortality rate was 16% ( 8 patients ) and the majority of these deaths were related to systemic infections , heart attacks , or aggravation of their pre - existing diseases . this finding suggests that , although the current therapeutic strategy for controlling these abscesses is highly effective , the decisive factor for patients ' survival still depends on their ability to recover from their medical comorbidities . several case series have reported headache , fever , and altered mental status as the most frequent presenting symptoms , while less commonly observed symptoms include nausea , vomiting , and focal neurological symptoms and signs15,18,25 ) . however , kao et al.15 ) reported that fever was not helpful diagnostically . in a study for patients with pneumococcal brain abscesses , fever was found in only 29% of patients , while 86% had focal deficits and 81% had headaches12 ) . in the present series , forty percent of patients had motor weakness , either hemiparesis or monoparesis , 18% had moderately or severely disturbed consciousness , and 16% had speech disturbances . overall , it seems that headache and focal neurological deficits including motor deficits and dysarthria are the most frequent presenting symptoms , with fever being common , but not invariable , since the systemic inflammatory response to an abscess may be minimal . chun et al.8 ) demonstrated that old age , male gender , altered sensorium on admission , and abscesses with a pulmonary source were associated with poor outcomes in patients with brain abscesses . other studies found that the mortality among treated patients correlated significantly with the initial neurological grade20,34 ) . seydoux and francioli30 ) also revealed that death and severe sequelae were related to the severity of neurological impairment on admission and to a shorter duration from symptom onset to admission . in the present study , patients with a low gcs score on admission had a greater likelihood of an unfavorable outcome than those with minimal mental disturbances when all potential confounds had been adjusted for in the multivariate analysis . direct spreading from an ear infection , sinusitis , or dental infection is the major cause for developing an abscess . this contiguous spreading of infection from adjacent sites has been reported in nearly half of all brain abscess cases in the past2,8,14 ) . however , in our series , only 13 ( 26% ) of the patients had a contiguous focus of infection as their predisposing cause ( 4 sinus , 3 dental , and 2 ear infections ) . similar to our findings , carpenter et al.6 ) also found that 24% of patients with brain abscesses had primary infections in their head and neck . aggressive and widespread therapy for the primary infections in the adjacent area is likely to lead to a corresponding decrease in the incidence of brain abscesses associated with these conditions . in addition to a documented focus of infection , patients with certain medical conditions have an increased risk of infection15,18 ) . many of these conditions , including diabetes and cancer , reduce the immune response in the patient . in this series , 14 ( 28% ) patients had a medical condition that might have increased their susceptibility to infection : diabetes mellitus in 10 patients and an immunocompromised state in 4 . this is in accord with other series , in which diabetes has been shown to be an important predisposing factor15,18 ) . kao et al.15 ) reported that patients with underlying diabetes and liver disease had a higher mortality . however , in the present study , we failed to find any apparent correlation between outcomes and the presence or type of predisposing factors . however , a lumbar puncture to obtain cerebrospinal fluid may be contraindicated in a patient with a brain abscess because raised intracranial pressure may lead to brainstem herniation and death20,30 ) . blood cultures are often helpful in some patients , especially those who did not undergo an operation , and in patients whose abscess fluid cultures were negative . in addition , if the abscess is thought to be the result of hematogenous spreading , a blood culture is indicated . the introduction , in the 1980s , and subsequent widespread acceptance of ct - guided stereotactic aspiration for brain abscesses , a minimally invasive procedure with low morbidity and mortality , has allowed rapid and effective surgical drainage of many brain abscess cases . in turn , this has enabled the culturing of pus and the identification of the organism(s ) responsible , with high rates of resolution and a good outcome . some studies have advocated nonsurgical treatments for patients who are poor candidates for surgery or for those with surgically inaccessible lesions3 ) . the major drawback to this approach is the potential toxicity involved in the prolonged administration of empirical antimicrobial therapy20 ) . even in regions with limited accessibility such as the brain stem or eloquent areas among patients with a high risk for surgery or with multiple brain abscesses , stereotactic aspiration combined with antibiotic treatment can be safely applied in most cases9 ) . in our center , we attempt to treat most of the lesions suspected to be brain abscesses on imaging and clinical manifestations by aspiration and drainage guided by navigation , except during the stage of cerebritis . this approach not only achieves a reduction of the mass effect , but also secures abscess materials for identifying the infecting pathogens , and thus for facilitating antibiotic selection16 ) . although the excision of abscesses may shorten the clinical course19 ) , no further improvement in the outcome has been observed16,34 ) . there has been no laboratory data of any value for outcome prediction in patients with brain abscesses . hyperglycemia is reportedly associated with a poor outcome in several systemic infectious and neurological disorders , such as sepsis , stroke , and severe head injury5,13,17,27 ) . intensive insulin therapy has been shown to prevent morbidity and reduce mortality in critically ill patients32,33 ) . elevated glucose in the blood has been associated with an increase in circulating cytokine concentrations and with an impaired ability to oppose infection11 ) . therefore , high blood glucose levels are regarded as an epiphenomenon of systemic disease severity and physical stress . on the contrary , there is evidence that hyperglycemia associated with brain pathology is a central phenomenon10 ) . implicated in the pathogenesis is an increase in sympathetic nervous system activity and a subsequent change in glucose metabolism through the hypothalamic - pituitary - adrenal axis , which is integral to the neuroendocrine stress response1 ) . a specific anatomical site responsible for governing glucose homeostasis has been presumed to exist21,22 ) . they suggested that the insular cortex influences autonomic function , especially the tone of the sympathetic nervous system , via its interconnections with the hypothalamus1,7,23 ) . in a recent study , schut et al.29 ) demonstrated that 69% of patients with bacterial meningitis have hyperglycemic blood glucose levels on admission . in addition , they showed an association between hyperglycemia on admission and patient outcomes . in the present study , we found that 43% of patients with brain abscess had an elevated glucose level ( 140 mg / dl ) at the initial presentation . although the exact mechanism remains uncertain , it can be assumed that central nervous system infection , including bacterial meningitis and brain abscesses may involve cortical and/or subcortical autonomic centers and affect the regulation of the autonomic nervous system , leading to hyperglycemia . our study showed that hyperglycemia is related to an unfavorable outcome in the univariate analysis ( p=0.016 ) , but this relationship do not remain robust in a multivariate analysis ( p=0.081 ) . interestingly , the patients who received prolonged antibiotic treatment ( 6 weeks ) had a significantly higher glucose level at admission compared to those who had a relatively short period of antibiotic therapy ( < 6 weeks , 171.57 mg / dl vs. 122.84 mg / dl , p=0.003 ) . in the multivariate analysis , elevated blood sugar on admission was an independent risk factor associated with prolonged antibiotic use ( p=0.032 , 95% ci=1.002 - 1.040 ) . finally , we found that the duration of antibiotic administration was directly correlated with the initial glucose level ( r=0.471 and p=0.001 ) . other laboratory parameters regarded as indicators for the severity of systemic infection , such as wbc count , esr , and crp , were of limited value for predicting the patients ' outcomes or the duration of antibiotic use in the statistical analysis . these findings suggest that hyperglycemia is likely the result of a central nervous system insult leading to disturbed blood - glucose regulation mechanisms , rather than the result of systemic insults ; thus , hyperglycemia may be a useful predictor of treatment outcome and the responsiveness of postoperative medical treatment in patients with brain abscesses . as with any observational study , there remains a possibility that unmeasured confounds influenced our findings . in addition , our study did not include patients who underwent a craniotomy with their abscess excision , which might have an independent effect on the outcome . we assessed a single random blood glucose level on admission to define hyperglycemia without measuring glycosylated hemoglobin ( hba1c ) . although we attempted to define the medical condition of the patients clearly , including pre - existing diabetes , by performing a critical review of the patients ' medical histories , it is likely that we also included patients with undiagnosed diabetes , which might have introduced an important bias in our analysis . in this retrospective study of 51 patients with brain abscesses , we found that the gcs score on admission was associated with the patients ' outcomes . the overall mortality rate was 16% ; however , the majority of the deaths were caused by patients ' pre - existing critical medical conditions and not by the abscesses themselves . in survivors , the response to a combination therapy of abscess drainage followed by parenteral antibiotic administration was excellent . an elevated blood glucose level on admission was associated with a prolonged use of antibiotics .

objectivethe purpose of this study was to describe the clinical characteristics , treatment outcomes , and prognostic factors in patients with brain abscesses treated in a single institute during a recent 10-year period.methodsfifty-one patients with brain abscesses who underwent navigation - assisted abscess aspiration with antibiotic treatment were included in this study . variable parameters were collected from the patients ' medical records and radiological data . a comparison was made between patients with favorable [ glasgow outcome scale ( gos ) 4 ] and unfavorable ( gos < 4 ) outcomes at discharge . additionally , we investigated the factors influencing the duration of antibiotic administration.resultsthe study included 41 male and 10 female patients with a mean age of 53 years . at admission , 42 patients ( 82% ) showed either clear or mildly disturbed consciousness ( gcs 13 ) and 24 patients ( 47% ) had predisposing factors . the offending microorganisms were identified in 25 patients ( 49% ) , and streptococcus species were the most commonly isolated bacteria ( 27% ) . the mean duration of antibiotic administration was 42 days . at discharge , 41 patients had a favorable outcome and 10 had an unfavorable outcome including 8 deaths . the decreased level of consciousness ( gcs < 13 ) on admission was likely associated with an unfavorable outcome ( p=0.052 ) , and initial hyperglycemia ( 140 mg / dl ) was an independent risk factor for prolonged antibiotic therapy ( p=0.032).conclusionwe found that the level of consciousness at admission was associated with treatment outcomes in patients with brain abscesses . furthermore , initial hyperglycemia was closely related to the long - term use of antibiotic agents .

INTRODUCTION MATERIALS AND METHODS RESULTS Patient demographics Laboratory and radiological findings Microbiological findings Treatments and outcomes Prognostic factors DISCUSSION CONCLUSION

the purpose of this study was to review the data of 51 patients with brain abscesses admitted to a single tertiary referral hospital during a recent 10-year period and describe their clinical characteristics , outcome of treatment , and prognostic factors . this retrospective study included 51 patients with intracerebral abscesses treated with navigation - assisted abscess drainage at our institute between january 2004 and december 2013 . patients with subdural empyema and epidural abscesses , and those who underwent craniotomy for brain abscesses , were excluded . patient charts were reviewed for the following : age at diagnosis , sex , past history , symptom duration before diagnosis , level of consciousness [ assessed by the glasgow coma scale ( gcs ) ] , focal neurological status at admission , disease predisposition , various serum parameters [ complete blood count , erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) , and random blood sugar at admission ] , microorganisms identified on blood and abscess material cultures , antibiotic regimen , and duration of antibiotic treatment . the radiological parameters including the multiplicity of the lesion , abscess location , and initial and postoperative abscess volumes were also reviewed . brain abscess specimens obtained from the surgery were cultured for aerobic and anaerobic bacteria , mycobacteria , and fungi . the outcome was assessed according to the glasgow outcome scale ( gos ) at the time of discharge . a favorable outcome ( gos 4 ) was defined as moderate disability ( having a disability , but being independent ) and good recovery . an unfavorable outcome ( gos < 4 ) was defined as death , persistent vegetative status , or severe disability ( conscious , but with disability ) . univariate analyses ( chi - square test , fisher 's exact test , student 's t - test ) were performed to compare the variables between patients with a favorable outcome or with early response to antibiotics , and those with poor outcomes or with a delayed response to antibiotics . variables found to have p<0.1 in the univariate analyses were entered into a multivariate logistic regression model to identify independent predictors of neurological outcome and the duration of antibiotic use . there were 41 male and 10 female patients with a mean age of 53 years ( range : 14 - 81 years ) . headache , which occurred in 25 patients , was the most common symptom , while 3 patients complained of nausea and vomiting . clear or mildly disturbed consciousness ( gcs 13 ) was evident in 42 patients ( 82% ) and moderately or severely disturbed consciousness ( gcs < 13 ) was present in 9 patients ( 18% ) . a total of 24 patients had predisposing factors including pulmonary infection ( 7 patients ) , sinusitis ( 4 patients ) , dental infection ( 3 patients ) , prior trauma or neurosurgery ( 2 patients ) , otic infection ( 2 patients ) , endocarditis ( 2 patients ) , congenital heart disease ( 2 patients ) , skin infection ( 1 patient ) , and psoas abscess ( 1 patient ) . of the 51 patients , leukocytosis [ white blood cell ( wbc ) 10000/l ] at admission was identified in 24 ( 47% ) patients . mean serum esr and crp levels at admission were 34.254.08 mm / h and 39.128.13 mg / l , respectively . twenty - two ( 43% ) of the 51 patients were in a state of hyperglycemia ( blood glucose 140 mg / dl ) on admission . the abscesses were most commonly found in the frontal lobe ( 30 patients , 59% ) , followed by the parietal lobe ( 12 patients , 24% ) , temporal lobe ( 8 patients , 16% ) , occipital lobe ( 5 patients , 10% ) , thalamus ( 2 patients , 4% ) , and pons ( 1 patient , 2% ) . abscess pathogens were identified in 25 patients ( 49% ) during a culture study of the abscess material ( table 3 ) . of these isolated microorganisms , 21 ( 41% ) the most common aerobic bacteria were the streptococcus species ( 14 cases , 27% ) , followed by gemella morbillorum ( 4 cases , 8% ) , staphylococcus aureus ( 1 case , 2% ) , proteus mirabilis ( 1 case , 2% ) , and klebsiella pneumoniae ( 1 case , 2% ) . the most frequently isolated anaerobic bacteria belonged to the prevotella species ( 3 cases , 6% ) , followed by the peptostreptococcus species ( 2 cases , 4% ) , fusobacterium nucleatum ( 2 cases , 4% ) , and bacteroides intermedius ( 1 case , 2% ) . blood cultures were performed in 10 patients who presented with fever at admission and were positive in 2 patients ( 4% ) . intravenous administration of empirical antibiotics including vancomycin , ceftriaxone , and metronidazole were started after abscess aspiration until the pathogens were confirmed . the most commonly used antibiotics included a combination of ceftriaxone and metronidazole ( 22 patients , 43% ) , sometimes with the addition of either vancomycin or gentamicin . the mean duration of intravenous antibiotic treatment for surviving patients was 42 days ( range : 23 - 66 days ) . during the course of treatment , 8 patients ( 16% ) died , with 3 ( 6% ) deaths being attributed to the abscess itself and 5 ( 10% ) deaths that may have resulted from the underlying pathology . in the latter cases , the cause of death was attributable to the aggravation of a pre - existing disease : 2 patients died of pulmonary infection - related sepsis , 1 patient with an atrial septum defect died from a pulmonary embolism , 1 patient with congestive heart failure died from a sudden heart attack , and 1 patient who had hepatoma died from bleeding of the original tumor . at discharge , a favorable outcome ( gos4 ) one of the patients with a thalamic abscess had a severe neurological deficit and was unable to live independently , and the other remained in a persistent vegetative state due to secondary ventriculitis caused by a rupture of the abscess . the results of comparison analyses between the patients with favorable and unfavorable outcomes are shown in table 4 . in the univariate analyses , admission variables associated with patient outcome included an initial gcs score < 13 ( p=0.003 ) , a high level of serum crp ( 10 mg / l , p=0.024 ) , and hyperglycemia ( 140 mg / dl , p=0.016 ) . among these , only a gcs score < 13 [ p=0.052 , 95% confidence interval ( ci ) 0.982 - 37.118 ] was likely to be an independent risk factor for poor patient outcome in the multivariate logistic regression analysis . to assess the clinical and radiological factors that were associated with the response to antibiotics , the patients were divided into two groups : 1 ) early responder group ( duration of antibiotics < 6 weeks ) comprising 25 patients , and 2 ) delayed responder group ( dead or using antibiotics for 6 weeks ) comprising 26 patients . the mean wbc count and plasma blood sugar level in the delayed responder group were significantly higher than in the early responder group ( 12639/l vs. 10116/l , p=0.045 ; 171.57 mg / dl vs. 122.84 mg / dl , p=0.003 , respectively ) ( table 5 ) . in the multivariate analyses , the independent risk factor for prolonged antibiotic use was hyperglycemia ( p=0.032 , 95% ci 1.002 - 1.040 ) . there was a positive correlation between the initial blood sugar level and the duration of antibiotic use ( r=0.471 and p=0.001 ) ( fig . there were 41 male and 10 female patients with a mean age of 53 years ( range : 14 - 81 years ) . headache , which occurred in 25 patients , was the most common symptom , while 3 patients complained of nausea and vomiting . clear or mildly disturbed consciousness ( gcs 13 ) was evident in 42 patients ( 82% ) and moderately or severely disturbed consciousness ( gcs < 13 ) was present in 9 patients ( 18% ) . a total of 24 patients had predisposing factors including pulmonary infection ( 7 patients ) , sinusitis ( 4 patients ) , dental infection ( 3 patients ) , prior trauma or neurosurgery ( 2 patients ) , otic infection ( 2 patients ) , endocarditis ( 2 patients ) , congenital heart disease ( 2 patients ) , skin infection ( 1 patient ) , and psoas abscess ( 1 patient ) . of the 51 patients , leukocytosis [ white blood cell ( wbc ) 10000/l ] at admission was identified in 24 ( 47% ) patients . an elevated crp level ( 10 mg / l ) was detected in 31 ( 61% ) patients . twenty - two ( 43% ) of the 51 patients were in a state of hyperglycemia ( blood glucose 140 mg / dl ) on admission . on brain images , a single lesion was observed in 44 ( 86% ) of the 51 patients and multiple lesions were observed in 7 patients ( 14% ) . the abscesses were most commonly found in the frontal lobe ( 30 patients , 59% ) , followed by the parietal lobe ( 12 patients , 24% ) , temporal lobe ( 8 patients , 16% ) , occipital lobe ( 5 patients , 10% ) , thalamus ( 2 patients , 4% ) , and pons ( 1 patient , 2% ) . abscess pathogens were identified in 25 patients ( 49% ) during a culture study of the abscess material ( table 3 ) . the most common aerobic bacteria were the streptococcus species ( 14 cases , 27% ) , followed by gemella morbillorum ( 4 cases , 8% ) , staphylococcus aureus ( 1 case , 2% ) , proteus mirabilis ( 1 case , 2% ) , and klebsiella pneumoniae ( 1 case , 2% ) . the most frequently isolated anaerobic bacteria belonged to the prevotella species ( 3 cases , 6% ) , followed by the peptostreptococcus species ( 2 cases , 4% ) , fusobacterium nucleatum ( 2 cases , 4% ) , and bacteroides intermedius ( 1 case , 2% ) . intravenous administration of empirical antibiotics including vancomycin , ceftriaxone , and metronidazole were started after abscess aspiration until the pathogens were confirmed . the most commonly used antibiotics included a combination of ceftriaxone and metronidazole ( 22 patients , 43% ) , sometimes with the addition of either vancomycin or gentamicin . the mean duration of intravenous antibiotic treatment for surviving patients was 42 days ( range : 23 - 66 days ) . during the course of treatment , 8 patients ( 16% ) died , with 3 ( 6% ) deaths being attributed to the abscess itself and 5 ( 10% ) deaths that may have resulted from the underlying pathology . in the latter cases , the cause of death was attributable to the aggravation of a pre - existing disease : 2 patients died of pulmonary infection - related sepsis , 1 patient with an atrial septum defect died from a pulmonary embolism , 1 patient with congestive heart failure died from a sudden heart attack , and 1 patient who had hepatoma died from bleeding of the original tumor . at discharge , a favorable outcome ( gos4 ) one of the patients with a thalamic abscess had a severe neurological deficit and was unable to live independently , and the other remained in a persistent vegetative state due to secondary ventriculitis caused by a rupture of the abscess . the results of comparison analyses between the patients with favorable and unfavorable outcomes are shown in table 4 . in the univariate analyses , admission variables associated with patient outcome included an initial gcs score < 13 ( p=0.003 ) , a high level of serum crp ( 10 mg / l , p=0.024 ) , and hyperglycemia ( 140 mg / dl , p=0.016 ) . among these , only a gcs score < 13 [ p=0.052 , 95% confidence interval ( ci ) 0.982 - 37.118 ] was likely to be an independent risk factor for poor patient outcome in the multivariate logistic regression analysis . to assess the clinical and radiological factors that were associated with the response to antibiotics , the patients were divided into two groups : 1 ) early responder group ( duration of antibiotics < 6 weeks ) comprising 25 patients , and 2 ) delayed responder group ( dead or using antibiotics for 6 weeks ) comprising 26 patients . the mean wbc count and plasma blood sugar level in the delayed responder group were significantly higher than in the early responder group ( 12639/l vs. 10116/l , p=0.045 ; 171.57 mg / dl vs. 122.84 mg / dl , p=0.003 , respectively ) ( table 5 ) . in the multivariate analyses , the independent risk factor for prolonged antibiotic use was hyperglycemia ( p=0.032 , 95% ci 1.002 - 1.040 ) . there was a positive correlation between the initial blood sugar level and the duration of antibiotic use ( r=0.471 and p=0.001 ) ( fig . the mortality rate associated with brain abscesses advances in radiographic scanning , the development of novel surgical techniques , and the availability of newer antibiotics have all helped decrease the mortality rate in patients with brain abscesses6,24,26,34 ) . recently , tseng and tseng31 ) have published the clinical outcomes of their patients with brain abscesses ( n=142 ) , of which 16.9% died during hospitalization . in a study for patients with pneumococcal brain abscesses , fever was found in only 29% of patients , while 86% had focal deficits and 81% had headaches12 ) . in the present series , forty percent of patients had motor weakness , either hemiparesis or monoparesis , 18% had moderately or severely disturbed consciousness , and 16% had speech disturbances . chun et al.8 ) demonstrated that old age , male gender , altered sensorium on admission , and abscesses with a pulmonary source were associated with poor outcomes in patients with brain abscesses . other studies found that the mortality among treated patients correlated significantly with the initial neurological grade20,34 ) . seydoux and francioli30 ) also revealed that death and severe sequelae were related to the severity of neurological impairment on admission and to a shorter duration from symptom onset to admission . in the present study , patients with a low gcs score on admission had a greater likelihood of an unfavorable outcome than those with minimal mental disturbances when all potential confounds had been adjusted for in the multivariate analysis . however , in our series , only 13 ( 26% ) of the patients had a contiguous focus of infection as their predisposing cause ( 4 sinus , 3 dental , and 2 ear infections ) . similar to our findings , carpenter et al.6 ) also found that 24% of patients with brain abscesses had primary infections in their head and neck . in this series , 14 ( 28% ) patients had a medical condition that might have increased their susceptibility to infection : diabetes mellitus in 10 patients and an immunocompromised state in 4 . however , in the present study , we failed to find any apparent correlation between outcomes and the presence or type of predisposing factors . however , a lumbar puncture to obtain cerebrospinal fluid may be contraindicated in a patient with a brain abscess because raised intracranial pressure may lead to brainstem herniation and death20,30 ) . the introduction , in the 1980s , and subsequent widespread acceptance of ct - guided stereotactic aspiration for brain abscesses , a minimally invasive procedure with low morbidity and mortality , has allowed rapid and effective surgical drainage of many brain abscess cases . even in regions with limited accessibility such as the brain stem or eloquent areas among patients with a high risk for surgery or with multiple brain abscesses , stereotactic aspiration combined with antibiotic treatment can be safely applied in most cases9 ) . in our center , we attempt to treat most of the lesions suspected to be brain abscesses on imaging and clinical manifestations by aspiration and drainage guided by navigation , except during the stage of cerebritis . there has been no laboratory data of any value for outcome prediction in patients with brain abscesses . hyperglycemia is reportedly associated with a poor outcome in several systemic infectious and neurological disorders , such as sepsis , stroke , and severe head injury5,13,17,27 ) . elevated glucose in the blood has been associated with an increase in circulating cytokine concentrations and with an impaired ability to oppose infection11 ) . in a recent study , schut et al.29 ) demonstrated that 69% of patients with bacterial meningitis have hyperglycemic blood glucose levels on admission . in the present study , we found that 43% of patients with brain abscess had an elevated glucose level ( 140 mg / dl ) at the initial presentation . our study showed that hyperglycemia is related to an unfavorable outcome in the univariate analysis ( p=0.016 ) , but this relationship do not remain robust in a multivariate analysis ( p=0.081 ) . interestingly , the patients who received prolonged antibiotic treatment ( 6 weeks ) had a significantly higher glucose level at admission compared to those who had a relatively short period of antibiotic therapy ( < 6 weeks , 171.57 mg / dl vs. 122.84 mg / dl , p=0.003 ) . in the multivariate analysis , elevated blood sugar on admission was an independent risk factor associated with prolonged antibiotic use ( p=0.032 , 95% ci=1.002 - 1.040 ) . finally , we found that the duration of antibiotic administration was directly correlated with the initial glucose level ( r=0.471 and p=0.001 ) . other laboratory parameters regarded as indicators for the severity of systemic infection , such as wbc count , esr , and crp , were of limited value for predicting the patients ' outcomes or the duration of antibiotic use in the statistical analysis . these findings suggest that hyperglycemia is likely the result of a central nervous system insult leading to disturbed blood - glucose regulation mechanisms , rather than the result of systemic insults ; thus , hyperglycemia may be a useful predictor of treatment outcome and the responsiveness of postoperative medical treatment in patients with brain abscesses . in addition , our study did not include patients who underwent a craniotomy with their abscess excision , which might have an independent effect on the outcome . although we attempted to define the medical condition of the patients clearly , including pre - existing diabetes , by performing a critical review of the patients ' medical histories , it is likely that we also included patients with undiagnosed diabetes , which might have introduced an important bias in our analysis . in this retrospective study of 51 patients with brain abscesses , we found that the gcs score on admission was associated with the patients ' outcomes . an elevated blood glucose level on admission was associated with a prolonged use of antibiotics .

orthodontic treatment is being performed more frequently in middle - aged patients , and middle - aged patients typically exhibit more dental restorations and a poorer periodontal status than adolescents and young adults . orthodontic bands are typically used for restored teeth in which direct bonding of the bracket is difficult . however , dental plaque may accumulate around the band margins1 as a result of the difficulty of shaping the bands to tooth surfaces . although surface conditioning techniques and adhesion materials have been developed for direct bracket bonding to the metal surface , the bond strength of the adhesive to gold alloy is less satisfactory than that of the adhesive to an enamel surface prepared with the classical acid etching method . adhesion is defined as " the maintained status of two materials at the interface by mechanical and chemical bonding forces . " acid etching of enamel is a good example of a method to enhance mechanical bond strength . bonding agents with desirable flowability infiltrate into fine irregularities formed at the enamel surface by acid etching , and efficient and strong adhesion is achieved with the micromechanical lock effect.2 however , acid etching can not be used with a metal surface . therefore , use of green stone3 and diamond burs for macromechanical retention , and aluminum oxide sandblasting4 for micromechanical retention , are widely applied in clinical situations . in addition to the methods used for enhanced mechanical retention , use of a metal primer for chemical bonding between the metal surface and adhesion material has been studied.5,6 the bond strength with these methods may not equal that with enamel etching , and there is controversy over the sustainability of the enhanced bond strength obtained by the application of metal primer.6 however , more and more orthodontists are using direct bonding to metal restorations because of improved oral hygiene , as well as convenience to the practitioner , compared with the conventional method using bands . recently , chemicophysical bonding using a silica coating and silanation ( silicoating),7,8 which can enhance mechanical and chemical bonds , has been introduced in the field of orthodontics . chemically cured resin is more fully polymerized than light - cured resin , particularly in the central area of the bracket , when a metal bracket is bonded to gold alloy.9 this suggests that the chemically cured resin may be superior in this circumstance . metal primer is popular among orthodontists because it can be applied easily after routine sandblasting using aluminum oxide . to date , there has been no study comparing the bond strength attained by different surface conditioning methods including sandblasting , application of metal primer , and silicoating , together with the use of chemically cured resin , for orthodontic bonding . the purpose of this study was to compare the shear bond strength ( sbs ) of three surface conditioning methods including aluminum oxide sandblasting only , aluminum oxide sandblasting and application of metal primer , and silicoating when a metal bracket was bonded to a gold alloy surface using chemically cured resin . the change in the sbs after thermocycling was also investigated to examine the influence of the stress caused by repetitive temperature changes , which is the primary cause of bond strength weakening in clinical practice . the adhesive remnant index ( plate - shaped specimens of type iii dental gold alloys used for casting ( goldenian c-48 ; shinhung , seoul , korea ) were placed at the bottom of a cuboidal silicon mold ( 2.4 2.4 1.9 cm ) . as the type iii dental gold alloy is primarily used for onlay , crown , and crown and bridge restorations in small areas , and assuming that the adherend was a posterior tooth restoration , the type iii gold alloy was considered appropriate for specimens in this investigation . the gold alloy used in the present study contained 48% gold , 38.2% silver , 4.0% palladium , and 9.8% microelements . orthodontic acrylic resin ( dentsply , york , pa , usa ) was poured into the silicon mold . after the resin blocks were completely cured , the mold was removed and the specimen was located on the upper surface of the block . 120 , 1,000 , 2,000 , and 3,000 sandpaper ( single - disc polisher ; gang hae industry , gwangju , korea ) under a spray of water , and then finely polished with a rouge for dental alloys ( hi - rouge ; high dental korea , seoul , korea ) and a cotton wheel ( dae - myung dental , seoul , korea ) to achieve a flat surface on which to bond the bracket to the specimen surface . a 5 5 mm adhesion area was delimited by applying scotch tape to the final polished gold alloy surface . the specimens were randomly divided into three groups of 70 samples each , and each group was treated with one of the three surface conditioning methods - aluminum oxide sandblasting ( as ) , aluminum oxide sandblasting plus metal primer ( mp ) , and silicoating ( sc ) . half of the specimens of each group underwent thermocycling - aluminum oxide sandblasting with thermocycling ( ast ) , aluminum oxide sandblasting plus metal primer with thermocycling ( mpt ) , and silicoating with thermocycling ( sct ) . finally , the gold alloy specimens were classified into six groups consisting of 35 samples per group ( table 1 ) . the surfaces of the specimens in the as , ast , mp , and mpt groups were roughened with 50 m aluminum oxide ( blasting medium ; dentaurum gmbh & co. kg , ispringen , germany ) by using an intraoral sandblaster ( microetcher ; danville materials inc . , san ramon , ca , usa ) at a distance of 1 cm and pressure of 3.6 kgf / cm for 10 s. afterward , the scotch tape was removed from the surfaces of the specimens , and the surfaces were rinsed and dried thoroughly with an air - water spray for 10 s each . a uniform coat of metal primer ( reliance orthodontic products , itasca , il , usa ) was painted onto the gold alloy specimens in the mp and mpt groups with a microbrush . the specimens were then allowed to dry for 30 s according to the manufacturer 's instructions . in the sc and sct groups , the specimens were sandblasted with 30 m silicon dioxide ( cojet sand ; 3 m espe , st . paul , mn , usa ) by using an intraoral sandblaster ( microetcher ) at a distance of 1 cm and pressure of 3.6 kgf / cm for 10 s. consequently , the gold alloy surfaces of the specimens became roughened and silica - coated . afterward , the scotch tape was removed from the specimens , and dry air was lightly applied for 5 s. silane ( espe sil ; 3 m espe ) agent was applied with a microbrush and evaporated for 5 min according to the manufacturer 's instructions . standard metal brackets for the upper central incisor ( standard edgewise bracket ; tomy inc . , tokyo , japan ) with a base area of approximately 14.22 mm were bonded to all specimens . the bonding adhesive used in this study was a chemically cured resin ( unite ; 3 m unitek , monrovia , ca , usa ) . the bracket base and gold alloy surfaces were coated with resin primer , and resin adhesive paste was applied to the bracket bases , according to the manufacturer 's instructions . the brackets were subsequently positioned and bonded to the gold alloy surfaces with constant pressure , and the excess resin extruded at the bracket - alloy interfaces was carefully removed with a dental explorer . specimens were kept at room temperature for 4 min , allowing chemical curing to take place . after this process daejeon , korea ) at 36.5 with 100% relative humidity for 24 h to achieve complete curing . each step in the procedure such as the fabrication of gold alloy blocks , surface conditioning , and bracket bonding was carried out by the same researcher for consistency and reproducibility of the experiment . the specimens in the ast , mpt , and sct groups underwent thermocycling based on the work of bishara et al.11 after storage in the water bath for 24 h. for thermocycling , 5 and 55 distilled water baths ( thermocycler ; gang hae industry ) were used . the samples were immersed in each bath for 20 s , which was considered one cycle of thermocycling , for 1,000 thermocycles . a universal testing machine with a 500 n load cell ( lloyd instruments ; ametek , berwyn , pa , usa ) was used to measure the sbs of each specimen . the acrylic resin blocks were fixed in the lower jig vice . the anterior / posterior and right / left positions of the acrylic resin blocks were adjusted so that the adhesive face between the bracket and the specimen was perpendicular to the crosshead ( figure 1 ) . the crosshead speed for the sbs measurement was 1 mm / min , and the maximum load at the moment of bracket debonding was measured . the sbs ( mpa ) of each specimen was measured by dividing this numerical value ( n ) by the base area ( mm ) of the bracket . the adhesion surface of each gold alloy specimen was photographed with a digital camera ( nikon d90 ; 105 mm micro lens , 1/125 , f25 , iso 200 , -0.3 ev ; nikon , tokyo , japan ) after measuring the sbs . using an image processing software ( photoshop cs5 extended 12.0 [ adobe systems inc . , san jose , ca , usa ] ) , each image was cropped , leaving only the 5 5 mm adhesion area , and a grid comprising horizontal and vertical lines at a width of 1 mm was superimposed on the image . the ari of the edited image observed on a monitor was used to evaluate the resin remnants . the shapiro - wilk test was performed to confirm that all six groups followed a normal distribution . two - way analysis of variance ( anova ) was used to assess the effect of the surface conditioning method and thermocycling process on the sbs . the kruskal - wallis h test was used to investigate the statistical significance of the ari among the different surface conditioning methods , and the mann - whitney u test was performed with bonferroni correction for multiple comparisons . the mann - whitney u test was used to evaluate the effect of thermocycling on the ari , and the correlation between the sbs and ari in each group was evaluated using spearman correlation analysis . plate - shaped specimens of type iii dental gold alloys used for casting ( goldenian c-48 ; shinhung , seoul , korea ) were placed at the bottom of a cuboidal silicon mold ( 2.4 2.4 1.9 cm ) . as the type iii dental gold alloy is primarily used for onlay , crown , and crown and bridge restorations in small areas , and assuming that the adherend was a posterior tooth restoration , the type iii gold alloy was considered appropriate for specimens in this investigation . the gold alloy used in the present study contained 48% gold , 38.2% silver , 4.0% palladium , and 9.8% microelements . orthodontic acrylic resin ( dentsply , york , pa , usa ) was poured into the silicon mold . after the resin blocks were completely cured , the mold was removed and the specimen was located on the upper surface of the block . 120 , 1,000 , 2,000 , and 3,000 sandpaper ( single - disc polisher ; gang hae industry , gwangju , korea ) under a spray of water , and then finely polished with a rouge for dental alloys ( hi - rouge ; high dental korea , seoul , korea ) and a cotton wheel ( dae - myung dental , seoul , korea ) to achieve a flat surface on which to bond the bracket to the specimen surface . a 5 5 mm adhesion area was delimited by applying scotch tape to the final polished gold alloy surface . the specimens were randomly divided into three groups of 70 samples each , and each group was treated with one of the three surface conditioning methods - aluminum oxide sandblasting ( as ) , aluminum oxide sandblasting plus metal primer ( mp ) , and silicoating ( sc ) . half of the specimens of each group underwent thermocycling - aluminum oxide sandblasting with thermocycling ( ast ) , aluminum oxide sandblasting plus metal primer with thermocycling ( mpt ) , and silicoating with thermocycling ( sct ) . finally , the gold alloy specimens were classified into six groups consisting of 35 samples per group ( table 1 ) . the surfaces of the specimens in the as , ast , mp , and mpt groups were roughened with 50 m aluminum oxide ( blasting medium ; dentaurum gmbh & co. kg , ispringen , germany ) by using an intraoral sandblaster ( microetcher ; danville materials inc . , san ramon , ca , usa ) at a distance of 1 cm and pressure of 3.6 kgf / cm for 10 s. afterward , the scotch tape was removed from the surfaces of the specimens , and the surfaces were rinsed and dried thoroughly with an air - water spray for 10 s each . a uniform coat of metal primer ( reliance orthodontic products , itasca , il , usa ) was painted onto the gold alloy specimens in the mp and mpt groups with a microbrush . the specimens were then allowed to dry for 30 s according to the manufacturer 's instructions . in the sc and sct groups , the specimens were sandblasted with 30 m silicon dioxide ( cojet sand ; 3 m espe , st . paul , mn , usa ) by using an intraoral sandblaster ( microetcher ) at a distance of 1 cm and pressure of 3.6 kgf / cm for 10 s. consequently , the gold alloy surfaces of the specimens became roughened and silica - coated . afterward , the scotch tape was removed from the specimens , and dry air was lightly applied for 5 s. silane ( espe sil ; 3 m espe ) agent was applied with a microbrush and evaporated for 5 min according to the manufacturer 's instructions . standard metal brackets for the upper central incisor ( standard edgewise bracket ; tomy inc . , tokyo , japan ) with a base area of approximately 14.22 mm were bonded to all specimens . the bonding adhesive used in this study was a chemically cured resin ( unite ; 3 m unitek , monrovia , ca , usa ) . the bracket base and gold alloy surfaces were coated with resin primer , and resin adhesive paste was applied to the bracket bases , according to the manufacturer 's instructions . the brackets were subsequently positioned and bonded to the gold alloy surfaces with constant pressure , and the excess resin extruded at the bracket - alloy interfaces was carefully removed with a dental explorer . specimens were kept at room temperature for 4 min , allowing chemical curing to take place . after this process , they were stored in a water bath ( jeio tech . , daejeon , korea ) at 36.5 with 100% relative humidity for 24 h to achieve complete curing . each step in the procedure such as the fabrication of gold alloy blocks , surface conditioning , and bracket bonding was carried out by the same researcher for consistency and reproducibility of the experiment . the specimens in the ast , mpt , and sct groups underwent thermocycling based on the work of bishara et al.11 after storage in the water bath for 24 h. for thermocycling , 5 and 55 distilled water baths ( thermocycler ; gang hae industry ) were used . the samples were immersed in each bath for 20 s , which was considered one cycle of thermocycling , for 1,000 thermocycles . a universal testing machine with a 500 n load cell ( lloyd instruments ; ametek , berwyn , pa , usa ) was used to measure the sbs of each specimen . the anterior / posterior and right / left positions of the acrylic resin blocks were adjusted so that the adhesive face between the bracket and the specimen was perpendicular to the crosshead ( figure 1 ) . the crosshead speed for the sbs measurement was 1 mm / min , and the maximum load at the moment of bracket debonding was measured . the sbs ( mpa ) of each specimen was measured by dividing this numerical value ( n ) by the base area ( mm ) of the bracket . the adhesion surface of each gold alloy specimen was photographed with a digital camera ( nikon d90 ; 105 mm micro lens , 1/125 , f25 , iso 200 , -0.3 ev ; nikon , tokyo , japan ) after measuring the sbs . using an image processing software ( photoshop cs5 extended 12.0 [ adobe systems inc . , san jose , ca , usa ] ) , each image was cropped , leaving only the 5 5 mm adhesion area , and a grid comprising horizontal and vertical lines at a width of 1 mm was superimposed on the image . the ari of the edited image observed on a monitor was used to evaluate the resin remnants . the shapiro - wilk test was performed to confirm that all six groups followed a normal distribution . two - way analysis of variance ( anova ) was used to assess the effect of the surface conditioning method and thermocycling process on the sbs . the kruskal - wallis h test was used to investigate the statistical significance of the ari among the different surface conditioning methods , and the mann - whitney u test was performed with bonferroni correction for multiple comparisons . the mann - whitney u test was used to evaluate the effect of thermocycling on the ari , and the correlation between the sbs and ari in each group was evaluated using spearman correlation analysis . the mean value and standard deviation of the sbs for each group , and the statistical significance of the sbs value among the groups , are shown in table 3 . the mean sbs values of the as , mp , and sc groups were 14.0 , 18.7 , and 21.9 mpa , respectively . sbs was significantly higher in the mp than as group ( p < 0.001 ) , and in the sc than mp group ( p < 0.001 ) ( table 3 ) . although the mean sbs values of the mpt and sct groups decreased after thermocycling , no significant difference in sbs was observed after thermocycling ( p = 0.133 ) ( table 3 ) . further , there was no interaction between the surface conditioning method and thermocycling process ( p = 0.340 ) . the most frequent ari score observed in each group was as follows : as and ast , 0 ; mp and mpt , 1 ; and sc and sct , 2 ( table 4 , figures 2 , 3 , and 4 ) . the results of the kruskal - wallis h test and the mann - whitney u test with bonferroni correction ( p < 0.0167 ) revealed significant differences in the ari among the three surface conditioning methods with and without application of the thermocycling process . the mann - whitney u test revealed no statistical differences in the ari between the as and ast groups ( p = 1.0 ) and sc and sct groups ( p = 0.194 ) , although the ari was significantly greater in the mp than mpt group ( table 4 ) . the sc and sct groups exhibited higher sbs values than the mp and mpt groups , respectively , and the mp and mpt groups showed higher sbs values than the as and ast groups , respectively ( table 3 ) . accordingly , the correlation between the sbs and ari was investigated , and a moderately positive correlation was observed between the sbs and ari ( spearman correlation coefficient = 0.637 , p < 0.05 ) . this study investigated bond strength of gold alloy treated with three different surface conditioning methods with or without thermocycling and directly bonded to metal brackets with chemically cured resin . the specimens treated with silica coating and silination yielded the highest sbs values , and the sbs values of each of the three surfaces were not significantly reduced by application of a thermocycling procedure . several studies7,12,13 have reported that sandblasting of metal with aluminum oxide effectively enhances micromechanical retention between the metal and adhesive resin . although the present study did not compare bond strengths before and after aluminum oxide sandblasting of the gold alloy , there was no significant change in sbs values of gold alloy treated with aluminum oxide sandblasting before and after thermocycling ; this result was similar to that of previous study.13 the effect on bond strength of chemical conditioning methods using metal primers has also been evaluated.6,8,14 various commercialized metal primers are available ; with the metal primer containing 4-meta ( methacryloxyethyl trimellitic anhydride ) , the acid anhydride group combines with the hydroxyl groups and oxygen atoms in the metal oxide layer , and the methacrylate group polymerizes with the composite resin.8,12 by this mechanism , the metal primer containing 4-meta increases the bond strength between the metal and the composite resin.8,12 in the present study , sufficient bond strength was maintained after thermocycling when the metal primer was used after sandblasting . this result corresponds with that of the investigation by matsumura et al.15 in which the samples were thermocycled after v - primer or metaltite application . however , lee et al.6 found that metal primers including v - primer , metaltite , and alloy primer significantly increased the bond strength of gold alloy , but that bond strength of the specimens treated with all three types of primer decreased after thermocycling , and concluded that metal primer had no advantage over aluminum oxide sandblasting in bond strength . this result is contrary to the result of the present study , and the reason appears to be the use of different resin types in each study . yoshida and atsuta16 reported that the effectiveness of the primers for noble metals may be influenced by the initiator system of the resin bonded to the metal . according to their study , when v - primer was applied to gold alloy , the tri - n - butylborane resin and benzoyl peroxide - amine resin exhibited higher bond strengths than the camphoroquinone - amine resin to gold alloy , with or without thermocycling.16 lee et al.6 used a light - cured resin containing camphoroquinone as the photoinitiator . there is no previous study evaluating whether the bond strength of gold alloy specimens treated with the metal primer containing 4-meta is maintained after thermocycling . if abrasive particles with a silicated surface are blasted onto a metal surface at high energies , kinetic energy is converted into thermal energy , and the high temperature formed by the impact of the abrasive particles results in silica being incorporated and embedded into the metal to a depth of 15 m.17 ats et al.18 reported that the silica particles were incorporated into a metal surface by blasting pressure when the metal surface was blasted with 30 m diameter aluminum oxide chemically modified with silica , thereby forming a fine roughened surface . watanabe et al.19 verified that a silica layer was formed on the surface of dental gold alloys by using electron microprobe analysis element mapping . when the silane is applied to the silica layer formed on the metal surface , silane and silica combine strongly by a dehydration reaction , separating the hydroxyl group.8 silanes have dual reactivity . non - hydrolyzable functional groups can combine with the resin composite monomers containing c = c double bonds , and the hydrolyzable alkoxy group can bond with hydroxyl group - rich inorganic substrates such as silica.18,20 silane for conditioning purposes is supplied as a liquid dissolved in a volatile solvent such as alcohol ; thus , application is straightforward . however , sufficient time is required for evaporation of the solvent , and for the chemical reaction between silica and silane to take place , to achieve adequate bond strength . therefore , an appropriate time interval is necessary for a successful silicoating procedure.21 manufacturers recommend 5 min of silanation before applying the adhesive for extraoral use , and 1 min for intraoral use . in this study , a time interval of 5 min after silane application was selected to avoid the effect of insufficient time for the evaporation of the solvent and chemical reaction . in clinical applications , 1 min after silane application may be adequate . because silicoating is achieved by incorporation of silica particles into the metal surface , this method is less affected by the gold alloy composition and oxide layer formation , compared with metal primer application after aluminum oxide sandblasting.8,22 in the present study , the silicoating group exhibited an sbs value of 20.73 2.46 ( mean standard deviation ) mpa after thermocycling , which represents a higher bond strength than that of the metal primer application after aluminum oxide sandblasting . this bond strength is comparable to that of a metal bracket bonded to an enamel surface.23 further , this sbs value is greater than the sbs value of 13.63 2.55 mpa reported in the study of jung et al.8 in which light - cured resin was used with silicoating . typically , the weaker the bond between the adherend and the adhesive , the fewer resin remnants will remain on the adherend surface21,24 ; this trend was also observed in the present study . more resin remnants were observed on the gold alloy specimens in the sc than mp group , and in the mp than as group . according to a study by jung et al.8 , the ari was higher with silicoating than aluminum oxide sandblasting ; however , there was no statistical significance . jung et al.8 regarded the cause of the result as the use of light - cured resin adhesive in their study , and reported that the insignificant difference in ari might have been produced by uncured resin remaining nonuniformly on the specimen surface . although bond strength was increased by treatment with a metal primer after aluminum oxide sandblasting , greater bond strength was attained with silicoating . based on this result , the success rate of direct bonding to gold alloy restorations or prostheses may be increased by application of silicoating to the surfaces of gold alloys . silicoating may increase bond strength with both light - cured resin8 and chemically cured resin . further , the brackets were bonded to flat surfaces of the gold alloy specimens in the present study . in practice , the surface of gold alloy restorations is not flat , so gap formation between the gold alloy surface and the bracket is inevitable and bond strengths are expected to be less than those obtained in this in vitro study . when a chemically cured resin is used to bond metal brackets to gold alloys treated with surface conditioning methods including aluminum oxide sand - blasting alone , metal primer application after aluminum oxide sandblasting , and silicoating : 1 .

objectivethe purpose of this study was to evaluate the effects of three different surface conditioning methods on the shear bond strength ( sbs ) of metal brackets bonded directly to gold alloy with chemically cured resin.methodstwo hundred ten type iii gold alloy specimens were randomly divided into six groups according to the combination of three different surface conditioning methods ( aluminum oxide sandblasting only , application of a metal primer after aluminum oxide sandblasting , silica coating and silanation ) and thermocycling ( with thermocycling , without thermocycling ) . after performing surface conditioning of specimens in accordance with each experimental condition , metal brackets were bonded to all specimens using a chemically cured resin . the sbs was measured at the moment of bracket debonding , and the resin remnants on the specimen surface were evaluated using the adhesive remnant index.resultsapplication of metal primer after aluminum oxide sandblasting yielded a higher bond strength than that with aluminum oxide sandblasting alone ( p < 0.001 ) , and silica coating and silanation yielded a higher bond strength than that with metal primer after aluminum oxide sandblasting ( p < 0.001 ) . there was no significant change in sbs after thermocycling in all groups.conclusionswith silica coating and silanation , clinically satisfactory bond strength can be attained when metal brackets are directly bonded to gold alloys using a chemically cured resin .

INTRODUCTION MATERIALS AND METHODS Fabrication of experimental specimens Bracket bonding Thermocycling Measurement of SBS ARI measurement Statistical analysis RESULTS DISCUSSION CONCLUSION

although surface conditioning techniques and adhesion materials have been developed for direct bracket bonding to the metal surface , the bond strength of the adhesive to gold alloy is less satisfactory than that of the adhesive to an enamel surface prepared with the classical acid etching method . bonding agents with desirable flowability infiltrate into fine irregularities formed at the enamel surface by acid etching , and efficient and strong adhesion is achieved with the micromechanical lock effect.2 however , acid etching can not be used with a metal surface . in addition to the methods used for enhanced mechanical retention , use of a metal primer for chemical bonding between the metal surface and adhesion material has been studied.5,6 the bond strength with these methods may not equal that with enamel etching , and there is controversy over the sustainability of the enhanced bond strength obtained by the application of metal primer.6 however , more and more orthodontists are using direct bonding to metal restorations because of improved oral hygiene , as well as convenience to the practitioner , compared with the conventional method using bands . recently , chemicophysical bonding using a silica coating and silanation ( silicoating),7,8 which can enhance mechanical and chemical bonds , has been introduced in the field of orthodontics . chemically cured resin is more fully polymerized than light - cured resin , particularly in the central area of the bracket , when a metal bracket is bonded to gold alloy.9 this suggests that the chemically cured resin may be superior in this circumstance . metal primer is popular among orthodontists because it can be applied easily after routine sandblasting using aluminum oxide . to date , there has been no study comparing the bond strength attained by different surface conditioning methods including sandblasting , application of metal primer , and silicoating , together with the use of chemically cured resin , for orthodontic bonding . the purpose of this study was to compare the shear bond strength ( sbs ) of three surface conditioning methods including aluminum oxide sandblasting only , aluminum oxide sandblasting and application of metal primer , and silicoating when a metal bracket was bonded to a gold alloy surface using chemically cured resin . the change in the sbs after thermocycling was also investigated to examine the influence of the stress caused by repetitive temperature changes , which is the primary cause of bond strength weakening in clinical practice . the adhesive remnant index ( plate - shaped specimens of type iii dental gold alloys used for casting ( goldenian c-48 ; shinhung , seoul , korea ) were placed at the bottom of a cuboidal silicon mold ( 2.4 2.4 1.9 cm ) . as the type iii dental gold alloy is primarily used for onlay , crown , and crown and bridge restorations in small areas , and assuming that the adherend was a posterior tooth restoration , the type iii gold alloy was considered appropriate for specimens in this investigation . after the resin blocks were completely cured , the mold was removed and the specimen was located on the upper surface of the block . 120 , 1,000 , 2,000 , and 3,000 sandpaper ( single - disc polisher ; gang hae industry , gwangju , korea ) under a spray of water , and then finely polished with a rouge for dental alloys ( hi - rouge ; high dental korea , seoul , korea ) and a cotton wheel ( dae - myung dental , seoul , korea ) to achieve a flat surface on which to bond the bracket to the specimen surface . the specimens were randomly divided into three groups of 70 samples each , and each group was treated with one of the three surface conditioning methods - aluminum oxide sandblasting ( as ) , aluminum oxide sandblasting plus metal primer ( mp ) , and silicoating ( sc ) . half of the specimens of each group underwent thermocycling - aluminum oxide sandblasting with thermocycling ( ast ) , aluminum oxide sandblasting plus metal primer with thermocycling ( mpt ) , and silicoating with thermocycling ( sct ) . finally , the gold alloy specimens were classified into six groups consisting of 35 samples per group ( table 1 ) . a uniform coat of metal primer ( reliance orthodontic products , itasca , il , usa ) was painted onto the gold alloy specimens in the mp and mpt groups with a microbrush . the specimens were then allowed to dry for 30 s according to the manufacturer 's instructions . afterward , the scotch tape was removed from the specimens , and dry air was lightly applied for 5 s. silane ( espe sil ; 3 m espe ) agent was applied with a microbrush and evaporated for 5 min according to the manufacturer 's instructions . , tokyo , japan ) with a base area of approximately 14.22 mm were bonded to all specimens . the bonding adhesive used in this study was a chemically cured resin ( unite ; 3 m unitek , monrovia , ca , usa ) . the bracket base and gold alloy surfaces were coated with resin primer , and resin adhesive paste was applied to the bracket bases , according to the manufacturer 's instructions . the brackets were subsequently positioned and bonded to the gold alloy surfaces with constant pressure , and the excess resin extruded at the bracket - alloy interfaces was carefully removed with a dental explorer . each step in the procedure such as the fabrication of gold alloy blocks , surface conditioning , and bracket bonding was carried out by the same researcher for consistency and reproducibility of the experiment . the specimens in the ast , mpt , and sct groups underwent thermocycling based on the work of bishara et al.11 after storage in the water bath for 24 h. for thermocycling , 5 and 55 distilled water baths ( thermocycler ; gang hae industry ) were used . the anterior / posterior and right / left positions of the acrylic resin blocks were adjusted so that the adhesive face between the bracket and the specimen was perpendicular to the crosshead ( figure 1 ) . the crosshead speed for the sbs measurement was 1 mm / min , and the maximum load at the moment of bracket debonding was measured . the adhesion surface of each gold alloy specimen was photographed with a digital camera ( nikon d90 ; 105 mm micro lens , 1/125 , f25 , iso 200 , -0.3 ev ; nikon , tokyo , japan ) after measuring the sbs . the ari of the edited image observed on a monitor was used to evaluate the resin remnants . two - way analysis of variance ( anova ) was used to assess the effect of the surface conditioning method and thermocycling process on the sbs . the kruskal - wallis h test was used to investigate the statistical significance of the ari among the different surface conditioning methods , and the mann - whitney u test was performed with bonferroni correction for multiple comparisons . the mann - whitney u test was used to evaluate the effect of thermocycling on the ari , and the correlation between the sbs and ari in each group was evaluated using spearman correlation analysis . plate - shaped specimens of type iii dental gold alloys used for casting ( goldenian c-48 ; shinhung , seoul , korea ) were placed at the bottom of a cuboidal silicon mold ( 2.4 2.4 1.9 cm ) . as the type iii dental gold alloy is primarily used for onlay , crown , and crown and bridge restorations in small areas , and assuming that the adherend was a posterior tooth restoration , the type iii gold alloy was considered appropriate for specimens in this investigation . after the resin blocks were completely cured , the mold was removed and the specimen was located on the upper surface of the block . 120 , 1,000 , 2,000 , and 3,000 sandpaper ( single - disc polisher ; gang hae industry , gwangju , korea ) under a spray of water , and then finely polished with a rouge for dental alloys ( hi - rouge ; high dental korea , seoul , korea ) and a cotton wheel ( dae - myung dental , seoul , korea ) to achieve a flat surface on which to bond the bracket to the specimen surface . the specimens were randomly divided into three groups of 70 samples each , and each group was treated with one of the three surface conditioning methods - aluminum oxide sandblasting ( as ) , aluminum oxide sandblasting plus metal primer ( mp ) , and silicoating ( sc ) . half of the specimens of each group underwent thermocycling - aluminum oxide sandblasting with thermocycling ( ast ) , aluminum oxide sandblasting plus metal primer with thermocycling ( mpt ) , and silicoating with thermocycling ( sct ) . finally , the gold alloy specimens were classified into six groups consisting of 35 samples per group ( table 1 ) . the surfaces of the specimens in the as , ast , mp , and mpt groups were roughened with 50 m aluminum oxide ( blasting medium ; dentaurum gmbh & co. kg , ispringen , germany ) by using an intraoral sandblaster ( microetcher ; danville materials inc . a uniform coat of metal primer ( reliance orthodontic products , itasca , il , usa ) was painted onto the gold alloy specimens in the mp and mpt groups with a microbrush . the specimens were then allowed to dry for 30 s according to the manufacturer 's instructions . afterward , the scotch tape was removed from the specimens , and dry air was lightly applied for 5 s. silane ( espe sil ; 3 m espe ) agent was applied with a microbrush and evaporated for 5 min according to the manufacturer 's instructions . , tokyo , japan ) with a base area of approximately 14.22 mm were bonded to all specimens . the bonding adhesive used in this study was a chemically cured resin ( unite ; 3 m unitek , monrovia , ca , usa ) . the bracket base and gold alloy surfaces were coated with resin primer , and resin adhesive paste was applied to the bracket bases , according to the manufacturer 's instructions . the brackets were subsequently positioned and bonded to the gold alloy surfaces with constant pressure , and the excess resin extruded at the bracket - alloy interfaces was carefully removed with a dental explorer . each step in the procedure such as the fabrication of gold alloy blocks , surface conditioning , and bracket bonding was carried out by the same researcher for consistency and reproducibility of the experiment . the specimens in the ast , mpt , and sct groups underwent thermocycling based on the work of bishara et al.11 after storage in the water bath for 24 h. for thermocycling , 5 and 55 distilled water baths ( thermocycler ; gang hae industry ) were used . the anterior / posterior and right / left positions of the acrylic resin blocks were adjusted so that the adhesive face between the bracket and the specimen was perpendicular to the crosshead ( figure 1 ) . the crosshead speed for the sbs measurement was 1 mm / min , and the maximum load at the moment of bracket debonding was measured . the sbs ( mpa ) of each specimen was measured by dividing this numerical value ( n ) by the base area ( mm ) of the bracket . the adhesion surface of each gold alloy specimen was photographed with a digital camera ( nikon d90 ; 105 mm micro lens , 1/125 , f25 , iso 200 , -0.3 ev ; nikon , tokyo , japan ) after measuring the sbs . , san jose , ca , usa ] ) , each image was cropped , leaving only the 5 5 mm adhesion area , and a grid comprising horizontal and vertical lines at a width of 1 mm was superimposed on the image . the ari of the edited image observed on a monitor was used to evaluate the resin remnants . two - way analysis of variance ( anova ) was used to assess the effect of the surface conditioning method and thermocycling process on the sbs . the kruskal - wallis h test was used to investigate the statistical significance of the ari among the different surface conditioning methods , and the mann - whitney u test was performed with bonferroni correction for multiple comparisons . the mann - whitney u test was used to evaluate the effect of thermocycling on the ari , and the correlation between the sbs and ari in each group was evaluated using spearman correlation analysis . the mean value and standard deviation of the sbs for each group , and the statistical significance of the sbs value among the groups , are shown in table 3 . sbs was significantly higher in the mp than as group ( p < 0.001 ) , and in the sc than mp group ( p < 0.001 ) ( table 3 ) . although the mean sbs values of the mpt and sct groups decreased after thermocycling , no significant difference in sbs was observed after thermocycling ( p = 0.133 ) ( table 3 ) . further , there was no interaction between the surface conditioning method and thermocycling process ( p = 0.340 ) . the results of the kruskal - wallis h test and the mann - whitney u test with bonferroni correction ( p < 0.0167 ) revealed significant differences in the ari among the three surface conditioning methods with and without application of the thermocycling process . accordingly , the correlation between the sbs and ari was investigated , and a moderately positive correlation was observed between the sbs and ari ( spearman correlation coefficient = 0.637 , p < 0.05 ) . this study investigated bond strength of gold alloy treated with three different surface conditioning methods with or without thermocycling and directly bonded to metal brackets with chemically cured resin . the specimens treated with silica coating and silination yielded the highest sbs values , and the sbs values of each of the three surfaces were not significantly reduced by application of a thermocycling procedure . several studies7,12,13 have reported that sandblasting of metal with aluminum oxide effectively enhances micromechanical retention between the metal and adhesive resin . although the present study did not compare bond strengths before and after aluminum oxide sandblasting of the gold alloy , there was no significant change in sbs values of gold alloy treated with aluminum oxide sandblasting before and after thermocycling ; this result was similar to that of previous study.13 the effect on bond strength of chemical conditioning methods using metal primers has also been evaluated.6,8,14 various commercialized metal primers are available ; with the metal primer containing 4-meta ( methacryloxyethyl trimellitic anhydride ) , the acid anhydride group combines with the hydroxyl groups and oxygen atoms in the metal oxide layer , and the methacrylate group polymerizes with the composite resin.8,12 by this mechanism , the metal primer containing 4-meta increases the bond strength between the metal and the composite resin.8,12 in the present study , sufficient bond strength was maintained after thermocycling when the metal primer was used after sandblasting . however , lee et al.6 found that metal primers including v - primer , metaltite , and alloy primer significantly increased the bond strength of gold alloy , but that bond strength of the specimens treated with all three types of primer decreased after thermocycling , and concluded that metal primer had no advantage over aluminum oxide sandblasting in bond strength . this result is contrary to the result of the present study , and the reason appears to be the use of different resin types in each study . yoshida and atsuta16 reported that the effectiveness of the primers for noble metals may be influenced by the initiator system of the resin bonded to the metal . according to their study , when v - primer was applied to gold alloy , the tri - n - butylborane resin and benzoyl peroxide - amine resin exhibited higher bond strengths than the camphoroquinone - amine resin to gold alloy , with or without thermocycling.16 lee et al.6 used a light - cured resin containing camphoroquinone as the photoinitiator . there is no previous study evaluating whether the bond strength of gold alloy specimens treated with the metal primer containing 4-meta is maintained after thermocycling . if abrasive particles with a silicated surface are blasted onto a metal surface at high energies , kinetic energy is converted into thermal energy , and the high temperature formed by the impact of the abrasive particles results in silica being incorporated and embedded into the metal to a depth of 15 m.17 ats et al.18 reported that the silica particles were incorporated into a metal surface by blasting pressure when the metal surface was blasted with 30 m diameter aluminum oxide chemically modified with silica , thereby forming a fine roughened surface . when the silane is applied to the silica layer formed on the metal surface , silane and silica combine strongly by a dehydration reaction , separating the hydroxyl group.8 silanes have dual reactivity . non - hydrolyzable functional groups can combine with the resin composite monomers containing c = c double bonds , and the hydrolyzable alkoxy group can bond with hydroxyl group - rich inorganic substrates such as silica.18,20 silane for conditioning purposes is supplied as a liquid dissolved in a volatile solvent such as alcohol ; thus , application is straightforward . because silicoating is achieved by incorporation of silica particles into the metal surface , this method is less affected by the gold alloy composition and oxide layer formation , compared with metal primer application after aluminum oxide sandblasting.8,22 in the present study , the silicoating group exhibited an sbs value of 20.73 2.46 ( mean standard deviation ) mpa after thermocycling , which represents a higher bond strength than that of the metal primer application after aluminum oxide sandblasting . this bond strength is comparable to that of a metal bracket bonded to an enamel surface.23 further , this sbs value is greater than the sbs value of 13.63 2.55 mpa reported in the study of jung et al.8 in which light - cured resin was used with silicoating . typically , the weaker the bond between the adherend and the adhesive , the fewer resin remnants will remain on the adherend surface21,24 ; this trend was also observed in the present study . more resin remnants were observed on the gold alloy specimens in the sc than mp group , and in the mp than as group . according to a study by jung et al.8 , the ari was higher with silicoating than aluminum oxide sandblasting ; however , there was no statistical significance . jung et al.8 regarded the cause of the result as the use of light - cured resin adhesive in their study , and reported that the insignificant difference in ari might have been produced by uncured resin remaining nonuniformly on the specimen surface . although bond strength was increased by treatment with a metal primer after aluminum oxide sandblasting , greater bond strength was attained with silicoating . based on this result , the success rate of direct bonding to gold alloy restorations or prostheses may be increased by application of silicoating to the surfaces of gold alloys . silicoating may increase bond strength with both light - cured resin8 and chemically cured resin . further , the brackets were bonded to flat surfaces of the gold alloy specimens in the present study . when a chemically cured resin is used to bond metal brackets to gold alloys treated with surface conditioning methods including aluminum oxide sand - blasting alone , metal primer application after aluminum oxide sandblasting , and silicoating : 1 .

parasites and animals - oncomelania hupensis hupensis snails infected with s. japonicum were purchased from the jiangsu institute of parasitic diseases , china . m. fortis in closed colonies , balb / c mice weighing 18 - 20 g each and new zealand white rabbits weighing 1 - 2 kg each were provided by the lab animal centre of central south university . the animal experiments were performed according to the protocols approved by the national animal care and use committee of china . serum preparation - blood samples from m. fortis and mice were collected using an eye - detachment technique , while the new zealand white rabbit blood samples were collected via heart puncture . the sera were separated by centrifugation and the sera of the rabbits and mice were then filtered ( millex gp filter unit , 0.22 m , millipore ) . schistosomula culture in vitro - cercariae were collected from infected o. hupensis snails as described previously ( salafsky et al . cercariae were washed four times with dulbecco s modified eagle s medium ( dmem ) ( gibco , usa ) containing 300 u / ml of penicillin g sodium and 300 g / ml of streptomycin . after centrifugation at 322 g for 10 min , schistosomula were transformed from cercariae by culturing in dmem containing 100 u / ml of penicillin g sodium and 100 g / ml of streptomycin at 37c , 5% co 2 with 25% new zealand white rabbit sera for 24 - 48 h. schistosomula were next cultured in 48-well cell plates with 100 20 schistosomula per well at 37c and 5% co 2 . the viability of the incubated schistosomula was assessed directly with light microscopy ( olympus co , tokyo , japan ) . dead schistosomula showed a loss of motility , an increase in opacity and occasionally gross morphological changes in shape or obvious lesions on the tegument . the viability assessment was ensured by methylene blue ( mb ) uptake testing after observation with the unstained schistosomula considered dead and the stained ones alive ( gold & flescher 2000 ) . screening of anti - schistosomula - associated protein - to obtain an effective schistosomula protein from m. fortis serum , molecular sieve chromatography was performed with a sephadex g50 column . chromatographic separations were performed by high - performance liquid chromatograph ( hplc ) ( waters company , usa ) . to confirm the anti - schistosome activity of hplc separations further , eluted peaks were pooled , desalted and lyophilised after the separations were gradient - eluted with 50 - 80% acetonitrile containing 0.1% trifluoroacetic acid . affinity chromatography - pre - swollen blue sepharose ff was mixed thoroughly and was poured into a chromatography column ( 3 cm i.d . 10 cm ) , which was then equilibrated thoroughly with solution a ( 0.05 m kh 2 po 4 , ph 7.0 ) . m. fortis serum was then diluted 1:1 ( v / v ) with solution a and was dialysed overnight against phosphate buffered saline ( pbs ) using dalton membranes ( 8,000 - 14,000 ) . after centrifugation at 8,050 g for 10 min , the supernatant was applied to the blue sepharose ff affinity chromatography column ( 4.8 cm 40 cm ) ( pharmacia ) at a flow rate of 2 ml / min . the column was eluted with solution a , followed by solution b ( 0.05 m kh 2 po 4 , 1.5 m kcl , ph 7.0 ) . the elution peak was pooled and dialysed overnight at 4c against pbs using dalton membranes ( 8,000 - 14,000 ) . finally , sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) analysis was conducted to ensure the purity of the m. fortis- albumin ( mf -albumin ) . anti - schistosomula experiment of mf - albumin in vitro - the inhibitory effects of mf- albumin on schistosomula were detected in vitro . after the live schistosomula were counted , samples corresponding to 1.25 m of mf- albumin were added and then incubated at 37c . m. fortis serum ( 40% , final concentration ) was used as the positive control and mice serum albumin was used as the negative control . schistosomula were cultivated for up to 96 h and the residual live schistosomula were counted and the mortality rate of schistosomula was calculated . the death rate of schistosomula was evaluated by the following formula : death rate % = ( total number of schistosomula - live number of schistosomula)/total number of schistosomula 100 . preparation of the conditioned medium - full - length mf -albumin genes ( genbank accession ay885264 ) were amplified from the total rna of m. fortis livers by reverse transcription polymerase chain reaction ( pcr ) using the following pcr primers : 5-tattggatccaccatgaggggcttgtttcgccgaga-3 and 5-tattctcgagagcaacaagttttggaccctctag-3 for mf -albumin and 5-tattggatccaccatgaggggtgtgtttcgccgag-3 and 5-tattctcgagagcaacaagttttggaccctctag-3 for mouse serum albumin ( ms -albumin ) . the generated dna fragment was cloned into plasmid pcdna3.1 ( + ) and was transferred into the escherichia coli strain dh5a . the recombinant plasmid ( pcdna3.1-albumin ) was sequenced and transiently transfected into 293 t cells using lipofectamine 2000 ( invitrogen , usa ) . twelve hours after dna transfection , the cells were cultured with 2 ml of serum - free dmem . supernatant ( conditioned medium ) from each pool was collected 36 h later and was tested at a concentration of 50% for its anti - schistosomula potential . protective experiment in mice with mf - albumin - thirty - six - week - old male balb / c mice were infected with 40 s. japonicum cercariae percutaneously and were randomly distributed into three groups containing 10 mice each : one blank pbs group , one ms -albumin control group and one mf -albumin test group . on the first day , the blank group was administered 0.3 ml of pbs , the control group was administered 0.3 ml of ms -albumin and the test group with 0.3 ml mf -albumin , all via intravenous injection . on the third and sixth days after the first injection s. japonicum adult worms in the hepatic and portomesenteric veins were recovered and counted . the number of eggs in the liver was estimated after the tissue was digested with 4% koh . the percentage changes in worm and egg burden were evaluated by the following formula : % change = ( mean number in infected controls - mean number in infected , treated mice)/mean number in infected control 100 . isolation of digestive tract excretions from schistosomula and adult worms - after schistosomula were harvested and washed three times with distilled water , they were placed in distilled water at room temperature for 20 min until the water became turbid with dark granular material from the parasites digestive tracts . these excretions were collected , adjusted to a ph of 4.9 with 0.1 m citrate and then centrifuged at 8,050 g for 10 min , after which the supernatant was collected , lyophilised , sealed and kept at -70c until further use ( chappell & dresden 1986 ) . immediately before use , the powder was dissolved with pbs and the protein content was measured using bovine serum albumin as a standard ( lowry et al . 1951 ) . on - site detection of mf - albumin in schistosomula - labelling of protein was performed with fluorescein isothiocyanate ( fitc ) according to a conventional protocol . fitc- mf- albumin was added to the schistosomula cultures and was incubated for 24 h and 48 h in vitro . fitc - labelled ms -albumin , fluorescein ( 30 g / ml ) and schistosomula without treatment were used as controls . to ascertain the site of fluorescence , schistosomula were collected after 24 h and 48 h of incubation , washed with 0.01 m pbs ( ph 7.0 ) three times and then were observed under an olympus fluorescence microscope . albumin digestion with digestive tract excretions in vitro - samples of 5 g/l mf- albumin and digestive tract excretions were incubated for 0 h , 1 h , 2 h , 4 h , 8 h , 16 h and 24 h , respectively , at 37c in citrate saline buffer solution , ph 4.0 , in the presence of 20 mm cysteine . after digestion , the reaction was terminated by 100 l of 0.5 m tris buffer ( ph 6.8 , 10% sds ) . twenty microlitres of digestion products and 10 l of loading buffer were run on 10% sds - page . statistical analysis - the differences between groups are presented as means standard deviation by one - way anova , using spss statistical software version 13.0 . schistosomula culture in vitro - the tails of cercariae were detached in dmem medium containing 25% fresh new zealand white rabbit serum for 24 - 48 h and the transformation rate remained greater than 90% with a less than 10% blank mortality rate . under light microscopy , the live schistosomula were very vivacious with semitransparent bodies , crawling unhindered on the bottom of culture plate , whereas the dead schistosomula showed ankylosis and immobility , increased opacity and obvious lesions on the tegument and they did not display mb staining ( fig . 1 ) . fig . 1 : micrographic images of schistosomula : dead schistosomula showed an increased opacity and obvious lesions in the tegumental outer membrane . the live schistosomula took up the dye and stained into blue - black with orbicular tegumental outer membrane , whereas the dead ones have not taken up the dye and unstained . a : schistosomula cultured in dulbecco s modified eagle s medium ( dmem ) without methylene blue ; b : live ( stained ) and dead ( unstained ) schistosomula after introduction of methylene blue into the dmem . identification of the protein associated with the inhibitory effects on schistosomula - the protein components of m. fortis serum were isolated by means of molecular sieve chromatography using a sephadex g50 column and hplc and the protein that induced resistance to schistosomula was acquired . the amino - terminal end was identified as dahkseiahr by protein sequencing and blast showed that it had 85% homology with mouse albumin and was named mf -albumin . at a protein concentration of 1 mg / ml , it induced a schistosomula mortality rate of 36.9% ( = 3.8594 , p < 0.05 ) in vitro , significantly greater than that of the negative control . albumin purification - blue sepharose ff was used to purify m. fortis and ms -albumin . there were two main peaks in the blue sepharose ff affinity chromatography ( data not shown ) . thus , it was accumulated and dialysed thoroughly with distilled water and then was placed in a -80c ultra - low temperature freezer for at least 4 h. the purity of the albumin was detected by sds - page and the result showed a single monomeric protein migration at approximately 66 kda ( fig the purified mf serum albumin was subjected to n - terminal amino acid sequencing for further verification . 2 : sodium dodecyl sulfate polyacrylamide gel electrophoresis of microtus fortis -albumin purified with blue sepharose ff affinity chromatography . lane m : broad range protein molecular weight marker ; 1 : m. fortis serum albumin purified . inhibitory effects of mf - albumin on schistosomula in vitro - to confirm the anti - schistosomula effects of mf -albumin , mf -albumin was added to cultured schistosomula at final concentrations of 0.31 , 0.62 , 1.25 , 2.5 , 5.0 , 10.0 and 20.0 mg / ml , with ms - albumin as a negative control and m. fortis serum as a positive control . in addition to the morphological changes of the dead schistosomula previously described , we also observed that there were one or two large blebs on the bodies of schistosomula during the period of mf -albumin treatment . the same change was observed in the positive control group , but no change was seen in the negative control group . most of the schistosomula in the negative control group were vivacious with semitransparent bodies and we observed them to be crawling on the bottom of the cell plate ( fig . the results of the morphological observations and schistosomula death rate data showed that mf -albumin had obvious inhibitory activity on schistosomula in vitro . our results showed that when the concentration of mf -albumin was 1.25 mg / ml , the death rate of schistosomula was 46.2% ( = 8.4703 , p this diagram showed that when the target protein concentration was equal or greater than 1.25 mg / ml , significant difference of schistosomula mortality rate was observed when compared with negative control and the dead rate of schistosomula raised markedly along with the rise of albumin concentration ( asterisk means p < 0.05 , compare with negative control ) . 3 : micrographic images of schistosomula cultured in 96 h : dead schistosomula showed an increased opacity and obvious lesions in the tegumental outer membrane . sometimes the blebs appeared in their tegumental outer membrane ( arrowed ) . a : schistosomula treated with 5 mg / ml microtus fortis ( mf ) -albumin ; b : schistosomula treated with 10 mg / ml mf -albumin ; c : schistosomula treated with represented 40% m. fortis serum ; d : schistosomula treated with 10 mg / ml mouse serum albumin . effects of conditioned media on schistosomula - to verify further the anti - schistosome effects of mf -albumin , conditioned medium containing mf -albumin or ms -albumin was added to schistosomula culture at a concentration of 50% . we observed and calculated the death rate of schistosomula at 96 h. our results showed the average schistosomula - killing rate of pcdna3.1/ mf -albumin conditioned medium was 38.7% , which was significantly higher than that of the negative control ( fig . fig . 5 : inhibitory effect of pcdna3.1/ microtus fortis conditioned medium on schistosomula after 96 h of incubation . a : schistosomula cultured with control pcdna3.1/mouse serum albumin ( ms -albumin ) conditioned medium ; b : schistosomula cultured with pcdna3.1/ m. fortis -albumin ( mf -albumin ) conditioned medium ; c : schistosomula cultured with pcdna3.1/amp conditioned medium ( negative control ) ; d : schistosomula cultured with 20% m. fortis sera ( positive control ) ( more dead schistosomula were observed in mf - albumin group than the negative control group in 96 h ) ; e : histogram of schistosomula mortality rate after incubation with conditioned medium ( p < 0.05 compared with negative control ) . results represented mean standard deviation for triplicates . anti - schistosome effects of mf - albumin in vivo - balb / c mice were randomly divided into three groups and were infected with s. japonicum cercariae . the resulting worm burden and number of lepg in balb / c mice treated with mf -albumin and ms -albumin are presented in table i. balb / c mice administered mf -albumin ( 15 mg / animal ) showed a significant worm burden reduction of 43.5% ( p < 0.05 ) compared with ms -albumin administration . moreover , we also observed a highly significant reduction in lepg in the mf -albumin treated group , which had a 48.1% ( p < 0.05 ) egg reduction . additionally , there were no differences in the numbers of egg granuloma observed between the controls ( data not shown ) . all of these results showed that mf -albumin possessed significant inhibitory activity on s. japonicum in vivo . tablethe anti - schistosome effect of microtus fortis ( mf ) -albumin on schistosoma japonicum cercariae infection in balb / c micegroupsworm burden ( mean se)worms reduction rate ( % ) lepg ( mean se)egg reduction rate ( % ) ppbs24.9 4.8 - 59.543 6.887- 6.256- > 0.05 mf -albumin13.9 2.643.530.900 3.29848.1 < 0.05worm burden rate and egg reduction rate were calculated relative to phosphate buffered saline ( pbs ) control . worm burden and liver eggs per gram ( lepg ) between pbs and mouse serum albumin ( worm burden and lepg of mf -albumin group were statistically different from those of pbs and ms -albumin groups ( p < 0.05 ) . worm burden rate and egg reduction rate were calculated relative to phosphate buffered saline ( pbs ) control . worm burden and liver eggs per gram ( lepg ) between pbs and mouse serum albumin ( worm burden and lepg of mf -albumin group were statistically different from those of pbs and ms -albumin groups ( p < 0.05 ) . detection of the deposit site of mf - albumin in schistosomula - schistosomula culture was incubated with fitc - labelled proteins or other control proteins . 6 , clearly showed that a fluorescent enrichment effect could be found in the gut lumen of schistosomula after incubation with fitc- mf -albumin for 24 h. after 48 h of incubation , the fluorescent enrichment effect became even stronger in the gut of schistosomula by fluorescence microscopy . however , there was no distinct fluorescence enrichment in schistosomula that were treated with the fitc - labelled ms -albumin , fluorescence and the blank control . fig . 6 : on - site of microtus fortis -albumin ( mf -albumin ) in schistosomula ( 24 h and 48 h post - treated ) . a1 : treated with fluorescein isothiocyanate ( fitc)-labelled mf -albumin after 24 h incubation ; a2 : treated with fitc - labelled mf -albumin after 48 h incubation ; b1 : treated with fitc - labelled mouse serum albumin ( ms -albumin ) after 24 h incubation ; b2 : treated with fitc - labelled ms -albumin after 48 h incubation ; c1 : fluorescein control after 24 h incubation ; c2 : fluorescein control after 48 h incubation ; d1 : blank control after 24 h incubation ; d2 : blank control after 48 h incubation . digestion of mf - albumin by digestive tract excretions from schistosomula - digestive tract excretions obtained from schistosomula were used to evaluate the ability of the digestion processes of m. fortis and mice albumin . the results showed that evident digestion could be observed when ms -albumin was incubated with digestive tract excretions of schistosomula for only 2 h , but no evident digestion of mf -albumin was detected until co - incubation for 24 h ( fig . fig . 7 : digestion of albumins by digestive tract excretions from schistosomula and adult worms . albumins microtus fortis and mice were incubated with digestive tract excretions from schistosomula and adult worms at 37c for 0 h , 1 h , 2 h , 4 h , 8 h , 16 h and 24 h , respectively . then the products were collected and tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis . a : mouse serum albumin ( ms -albumin ) treated with distilled water ; b : ms -albumin treated with digestive tract excretions of schistosomula ; c : m. fortis -albumin ( mf -albumin ) treated with distilled water ; d : mf -albumin treated with digestive tract excretions of schistosomula . it is well known that m. fortis possesses natural resistance to s. japonicum and this characteristic could be steadily inherited . in addition , there can be variations in host resistance and parasite infectivity within a species or across different species ( schwarzenbach et al . however , the degree of resistance , which can be a plastic response to environmental variation in the natural population , is not well understood ( nagel et al . some studies have shown that environmental variation might increase ischaemia - modified albumin concentrations , which are a form of human serum albumin ( hsa ) ( bar - or et al . in addition , there has also been evidence of possible genetic variants of hsa , particularly structural changes at the n - terminus , which abolish transition metal binding ( sheat et al . therefore , we hypothesised that a genetic difference in albumin between m. fortis and mice would result in functional disparity between them , giving mf -albumin stronger anti - schistosome potential . laboratory studies have shown that m. fortis sera possessed anti - schistosome activity in vitro and in vivo . in the present investigation , we showed that extract of albumin from m. forti s sera exhibited anti - schistosomal activity both in vitro and in vivo . it is well known that serum albumin is the most abundant protein in the circulatory system and it possesses many important physiological functions that contribute to colloid osmotic blood pressure and aid in the transport , distribution and metabolism of many endogenous and exogenous substances . in addition , it was reported that albumin might play an important role in innate immunity ( fassi et al . giles and czuprynski ( 2003 ) showed that the albumin from several mammalian species had obvious inhibitory activity against blastomyces dermatitidis , while analbuminaemic rat serum failed to inhibit b. dermatitidis growth in vivo . ( 1992 ) indicated that maleylated hsa inhibited human immunodeficiency virus-1 infection and syncytia formation via binding to target cells with high affinity in vitro . recently , a series of studies suggested that fluid resuscitation with albumin was associated with higher mortality in patients ( schierhout & roberts 1998 , vincent et al all of these data indicated that , apart from the basic functions , albumin possesses an immunological effect related to the innate immunity of living things . m. fortis is a non - permissive host , the serum of which has been found to possess innate resistance to s. japonicum in vitro and in vivo . in this study , we isolated and purified m. fortis albumin . the inhibitory activity of the albumin to schistosomula was examined in vitro and in vivo . schistosomula culture treated with 1.25 mg / ml of mf -albumin caused a significantly higher schistosomula death rate when compared to the negative controls . the mortality rate of schistosomula approached 100% when the mf -albumin concentration was increased to 10 mg / ml . mf -albumin was administered to mice infected with cercariae by intravenous injection through the tail vein . after perfusion of the mice 42 d after infection , we found that mice injected with mf- albumin had 43.5% worm burden reduction and 48.1% lepg reduction compared with the control group . all of the results showed that mf -albumin possessed significant inhibitory activity against schistosomula . until now , the possible mechanism of mf -albumin anti - schistosomula had been unclear . it is well known that the definitive hosts of s. japonicum are widespread , infecting more than 40 mammalian species . however , m. fortis was shown to be the only non - permissive host of schistosome among mammalians in china ( li et al . when infected , the growth and development of s. japonicum are hindered and no adult worms and eggs could be found in both wild and laboratory - bred m. fortis ( he et al . fitc - labelled mf -albumin accumulated in the gut lumen of schistosomula after incubation for 24 h and 48 h. thus , mf -albumin must be transported intact and be metabolised slowly by schistosomula . we speculated that the anti - schistosomal activity would be accompanied by a series of proteins involved in signal transduction and protein trafficking ; these proteins might activate or strengthen the immune system and induce anti - schistosome effects during different stages of the schistosome life cycle in the vertebrate host . recent experimental evidence has shown that hormonal - like proteins can regulate a variety of cellular and physiological functions of schistosome in establishment , growth and reproduction , such as adrenal steroid hormones and testosterone ( barrabes et al . 2002 ) . in combination with the results of fitc - labelled mf -albumin accumulated in the intestines of schistosomula , it is possible that mf -albumin could influence the microenvironment or be indigested by the digestive tract excretions of schistosomula . thus , we presumed mf -albumin might play an important role in resistance to schistosome , by acting as a kind of a stressor . to explore further possible mechanisms of mf -albumin against s. japonicum , we collected digestive tract excretions of schistosomula to test their digestion differences from m. fortis and mice serum albumin in vitro . the results showed that mf -albumin was more difficult to be digested by the digestive tract excretions of schistosomula , compared with ms -albumin . when infected , the growth and development of s. japonicum were hindered and no adult worms or eggs could be found in either wild or laboratory - bred m. fortis ( he et al . therefore , we presumed that mf -albumin might be an important factor of the anti - schistosomula effects of m. fortis during s. japonicum development . in combination with the results of fitc - labelled mf -albumin accumulated in the intestines of s. japonicum , it also provided insight that digestive tract excretions could be used for rapid immunodiagnosis , even during early stages of infection . it is worthwhile to point out that when we studied the inhibitory effects of mf -albumin on schistosomula in vitro , we observed some large blebs appearing in the tegumental outer membrane . the appearance of these blebs was reported and was suggested to be the result of the action of complement ( mclaren et al . however , in our in vitro experimental system , there was no complement or the complement was inactivated . thus , we suggest that the presence of mf -albumin created an environment that was not suitable for the survival of schistosomula because of the obvious morphological changes to the tegument of the schistosomula . in summary , we screened proteins of m. fortis serum by molecular sieve chromatography , hplc and blue sepharose ff and identified mf -albumin as having anti - schistosomula effects , both in vitro and in vivo . we preliminarily explored the possible mechanism of mf -albumin anti - schistosomula and concluded that mf -albumin was an effective protein against schistosome .

schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease . experimental and epidemiological evidence strongly suggests that microtus fortis ( mf ) is a naturally resistant vertebrate host of schistosoma japonicum . in the present study , we found that mf serum albumin ( mf -albumin ) and the conditioned medium of pcdna3.1- mf -albumin caused 46.2% and 38.7% schistosomula death rates in 96 h , respectively , which were significantly higher than that of the negative control ( p < 0.05 ) . we also found that mice injected with mf -albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram ( p < 0.05 ) in comparison to the control animals . to characterise the mechanisms involved in clearance , schistosomula were incubated with fluorescein isothiocyanate - labelled mf -albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation . next , digestive tract excretions from schistosomula were collected and the sensitivity of mf -albumin to digestive tract excretions was evaluated . the results indicated that schistosomula digestive tract excretions showed indigestibility of mf -albumin . the death of schistosomula could be partially attributed to the lack of digestion of mf -albumin by digestive tract excretions during the development of the schistosomula stage . therefore , these data indicate the potential of mf -albumin as one of the major selective forces for schistosomiasis .

MATERIALS AND METHODS RESULTS DISCUSSION

after centrifugation at 322 g for 10 min , schistosomula were transformed from cercariae by culturing in dmem containing 100 u / ml of penicillin g sodium and 100 g / ml of streptomycin at 37c , 5% co 2 with 25% new zealand white rabbit sera for 24 - 48 h. schistosomula were next cultured in 48-well cell plates with 100 20 schistosomula per well at 37c and 5% co 2 . finally , sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) analysis was conducted to ensure the purity of the m. fortis- albumin ( mf -albumin ) . m. fortis serum ( 40% , final concentration ) was used as the positive control and mice serum albumin was used as the negative control . schistosomula were cultivated for up to 96 h and the residual live schistosomula were counted and the mortality rate of schistosomula was calculated . the death rate of schistosomula was evaluated by the following formula : death rate % = ( total number of schistosomula - live number of schistosomula)/total number of schistosomula 100 . preparation of the conditioned medium - full - length mf -albumin genes ( genbank accession ay885264 ) were amplified from the total rna of m. fortis livers by reverse transcription polymerase chain reaction ( pcr ) using the following pcr primers : 5-tattggatccaccatgaggggcttgtttcgccgaga-3 and 5-tattctcgagagcaacaagttttggaccctctag-3 for mf -albumin and 5-tattggatccaccatgaggggtgtgtttcgccgag-3 and 5-tattctcgagagcaacaagttttggaccctctag-3 for mouse serum albumin ( ms -albumin ) . on the first day , the blank group was administered 0.3 ml of pbs , the control group was administered 0.3 ml of ms -albumin and the test group with 0.3 ml mf -albumin , all via intravenous injection . isolation of digestive tract excretions from schistosomula and adult worms - after schistosomula were harvested and washed three times with distilled water , they were placed in distilled water at room temperature for 20 min until the water became turbid with dark granular material from the parasites digestive tracts . on - site detection of mf - albumin in schistosomula - labelling of protein was performed with fluorescein isothiocyanate ( fitc ) according to a conventional protocol . fitc- mf- albumin was added to the schistosomula cultures and was incubated for 24 h and 48 h in vitro . to ascertain the site of fluorescence , schistosomula were collected after 24 h and 48 h of incubation , washed with 0.01 m pbs ( ph 7.0 ) three times and then were observed under an olympus fluorescence microscope . albumin digestion with digestive tract excretions in vitro - samples of 5 g/l mf- albumin and digestive tract excretions were incubated for 0 h , 1 h , 2 h , 4 h , 8 h , 16 h and 24 h , respectively , at 37c in citrate saline buffer solution , ph 4.0 , in the presence of 20 mm cysteine . schistosomula culture in vitro - the tails of cercariae were detached in dmem medium containing 25% fresh new zealand white rabbit serum for 24 - 48 h and the transformation rate remained greater than 90% with a less than 10% blank mortality rate . at a protein concentration of 1 mg / ml , it induced a schistosomula mortality rate of 36.9% ( = 3.8594 , p < 0.05 ) in vitro , significantly greater than that of the negative control . thus , it was accumulated and dialysed thoroughly with distilled water and then was placed in a -80c ultra - low temperature freezer for at least 4 h. the purity of the albumin was detected by sds - page and the result showed a single monomeric protein migration at approximately 66 kda ( fig the purified mf serum albumin was subjected to n - terminal amino acid sequencing for further verification . inhibitory effects of mf - albumin on schistosomula in vitro - to confirm the anti - schistosomula effects of mf -albumin , mf -albumin was added to cultured schistosomula at final concentrations of 0.31 , 0.62 , 1.25 , 2.5 , 5.0 , 10.0 and 20.0 mg / ml , with ms - albumin as a negative control and m. fortis serum as a positive control . in addition to the morphological changes of the dead schistosomula previously described , we also observed that there were one or two large blebs on the bodies of schistosomula during the period of mf -albumin treatment . the same change was observed in the positive control group , but no change was seen in the negative control group . most of the schistosomula in the negative control group were vivacious with semitransparent bodies and we observed them to be crawling on the bottom of the cell plate ( fig . the results of the morphological observations and schistosomula death rate data showed that mf -albumin had obvious inhibitory activity on schistosomula in vitro . our results showed that when the concentration of mf -albumin was 1.25 mg / ml , the death rate of schistosomula was 46.2% ( = 8.4703 , p this diagram showed that when the target protein concentration was equal or greater than 1.25 mg / ml , significant difference of schistosomula mortality rate was observed when compared with negative control and the dead rate of schistosomula raised markedly along with the rise of albumin concentration ( asterisk means p < 0.05 , compare with negative control ) . 3 : micrographic images of schistosomula cultured in 96 h : dead schistosomula showed an increased opacity and obvious lesions in the tegumental outer membrane . a : schistosomula treated with 5 mg / ml microtus fortis ( mf ) -albumin ; b : schistosomula treated with 10 mg / ml mf -albumin ; c : schistosomula treated with represented 40% m. fortis serum ; d : schistosomula treated with 10 mg / ml mouse serum albumin . effects of conditioned media on schistosomula - to verify further the anti - schistosome effects of mf -albumin , conditioned medium containing mf -albumin or ms -albumin was added to schistosomula culture at a concentration of 50% . we observed and calculated the death rate of schistosomula at 96 h. our results showed the average schistosomula - killing rate of pcdna3.1/ mf -albumin conditioned medium was 38.7% , which was significantly higher than that of the negative control ( fig . 5 : inhibitory effect of pcdna3.1/ microtus fortis conditioned medium on schistosomula after 96 h of incubation . a : schistosomula cultured with control pcdna3.1/mouse serum albumin ( ms -albumin ) conditioned medium ; b : schistosomula cultured with pcdna3.1/ m. fortis -albumin ( mf -albumin ) conditioned medium ; c : schistosomula cultured with pcdna3.1/amp conditioned medium ( negative control ) ; d : schistosomula cultured with 20% m. fortis sera ( positive control ) ( more dead schistosomula were observed in mf - albumin group than the negative control group in 96 h ) ; e : histogram of schistosomula mortality rate after incubation with conditioned medium ( p < 0.05 compared with negative control ) . the resulting worm burden and number of lepg in balb / c mice treated with mf -albumin and ms -albumin are presented in table i. balb / c mice administered mf -albumin ( 15 mg / animal ) showed a significant worm burden reduction of 43.5% ( p < 0.05 ) compared with ms -albumin administration . moreover , we also observed a highly significant reduction in lepg in the mf -albumin treated group , which had a 48.1% ( p < 0.05 ) egg reduction . all of these results showed that mf -albumin possessed significant inhibitory activity on s. japonicum in vivo . tablethe anti - schistosome effect of microtus fortis ( mf ) -albumin on schistosoma japonicum cercariae infection in balb / c micegroupsworm burden ( mean se)worms reduction rate ( % ) lepg ( mean se)egg reduction rate ( % ) ppbs24.9 4.8 - 59.543 6.887- 6.256- > 0.05 mf -albumin13.9 2.643.530.900 3.29848.1 < 0.05worm burden rate and egg reduction rate were calculated relative to phosphate buffered saline ( pbs ) control . worm burden and liver eggs per gram ( lepg ) between pbs and mouse serum albumin ( worm burden and lepg of mf -albumin group were statistically different from those of pbs and ms -albumin groups ( p < 0.05 ) . worm burden and liver eggs per gram ( lepg ) between pbs and mouse serum albumin ( worm burden and lepg of mf -albumin group were statistically different from those of pbs and ms -albumin groups ( p < 0.05 ) . detection of the deposit site of mf - albumin in schistosomula - schistosomula culture was incubated with fitc - labelled proteins or other control proteins . 6 , clearly showed that a fluorescent enrichment effect could be found in the gut lumen of schistosomula after incubation with fitc- mf -albumin for 24 h. after 48 h of incubation , the fluorescent enrichment effect became even stronger in the gut of schistosomula by fluorescence microscopy . 6 : on - site of microtus fortis -albumin ( mf -albumin ) in schistosomula ( 24 h and 48 h post - treated ) . a1 : treated with fluorescein isothiocyanate ( fitc)-labelled mf -albumin after 24 h incubation ; a2 : treated with fitc - labelled mf -albumin after 48 h incubation ; b1 : treated with fitc - labelled mouse serum albumin ( ms -albumin ) after 24 h incubation ; b2 : treated with fitc - labelled ms -albumin after 48 h incubation ; c1 : fluorescein control after 24 h incubation ; c2 : fluorescein control after 48 h incubation ; d1 : blank control after 24 h incubation ; d2 : blank control after 48 h incubation . digestion of mf - albumin by digestive tract excretions from schistosomula - digestive tract excretions obtained from schistosomula were used to evaluate the ability of the digestion processes of m. fortis and mice albumin . the results showed that evident digestion could be observed when ms -albumin was incubated with digestive tract excretions of schistosomula for only 2 h , but no evident digestion of mf -albumin was detected until co - incubation for 24 h ( fig . 7 : digestion of albumins by digestive tract excretions from schistosomula and adult worms . albumins microtus fortis and mice were incubated with digestive tract excretions from schistosomula and adult worms at 37c for 0 h , 1 h , 2 h , 4 h , 8 h , 16 h and 24 h , respectively . a : mouse serum albumin ( ms -albumin ) treated with distilled water ; b : ms -albumin treated with digestive tract excretions of schistosomula ; c : m. fortis -albumin ( mf -albumin ) treated with distilled water ; d : mf -albumin treated with digestive tract excretions of schistosomula . some studies have shown that environmental variation might increase ischaemia - modified albumin concentrations , which are a form of human serum albumin ( hsa ) ( bar - or et al . therefore , we hypothesised that a genetic difference in albumin between m. fortis and mice would result in functional disparity between them , giving mf -albumin stronger anti - schistosome potential . in the present investigation , we showed that extract of albumin from m. forti s sera exhibited anti - schistosomal activity both in vitro and in vivo . it is well known that serum albumin is the most abundant protein in the circulatory system and it possesses many important physiological functions that contribute to colloid osmotic blood pressure and aid in the transport , distribution and metabolism of many endogenous and exogenous substances . recently , a series of studies suggested that fluid resuscitation with albumin was associated with higher mortality in patients ( schierhout & roberts 1998 , vincent et al all of these data indicated that , apart from the basic functions , albumin possesses an immunological effect related to the innate immunity of living things . schistosomula culture treated with 1.25 mg / ml of mf -albumin caused a significantly higher schistosomula death rate when compared to the negative controls . the mortality rate of schistosomula approached 100% when the mf -albumin concentration was increased to 10 mg / ml . after perfusion of the mice 42 d after infection , we found that mice injected with mf- albumin had 43.5% worm burden reduction and 48.1% lepg reduction compared with the control group . all of the results showed that mf -albumin possessed significant inhibitory activity against schistosomula . when infected , the growth and development of s. japonicum are hindered and no adult worms and eggs could be found in both wild and laboratory - bred m. fortis ( he et al . fitc - labelled mf -albumin accumulated in the gut lumen of schistosomula after incubation for 24 h and 48 h. thus , mf -albumin must be transported intact and be metabolised slowly by schistosomula . we speculated that the anti - schistosomal activity would be accompanied by a series of proteins involved in signal transduction and protein trafficking ; these proteins might activate or strengthen the immune system and induce anti - schistosome effects during different stages of the schistosome life cycle in the vertebrate host . in combination with the results of fitc - labelled mf -albumin accumulated in the intestines of schistosomula , it is possible that mf -albumin could influence the microenvironment or be indigested by the digestive tract excretions of schistosomula . to explore further possible mechanisms of mf -albumin against s. japonicum , we collected digestive tract excretions of schistosomula to test their digestion differences from m. fortis and mice serum albumin in vitro . the results showed that mf -albumin was more difficult to be digested by the digestive tract excretions of schistosomula , compared with ms -albumin . when infected , the growth and development of s. japonicum were hindered and no adult worms or eggs could be found in either wild or laboratory - bred m. fortis ( he et al . therefore , we presumed that mf -albumin might be an important factor of the anti - schistosomula effects of m. fortis during s. japonicum development . in combination with the results of fitc - labelled mf -albumin accumulated in the intestines of s. japonicum , it also provided insight that digestive tract excretions could be used for rapid immunodiagnosis , even during early stages of infection . it is worthwhile to point out that when we studied the inhibitory effects of mf -albumin on schistosomula in vitro , we observed some large blebs appearing in the tegumental outer membrane . thus , we suggest that the presence of mf -albumin created an environment that was not suitable for the survival of schistosomula because of the obvious morphological changes to the tegument of the schistosomula . in summary , we screened proteins of m. fortis serum by molecular sieve chromatography , hplc and blue sepharose ff and identified mf -albumin as having anti - schistosomula effects , both in vitro and in vivo . we preliminarily explored the possible mechanism of mf -albumin anti - schistosomula and concluded that mf -albumin was an effective protein against schistosome .

the earliest host - cell response to viral infection is activation of the type - i interferon ( ifn/ ) system ( see fig . 1 ) , which ultimately leads to the establishment of antiviral responses in infected / neighboring cells and contributes to the shaping of an effective adaptive response [ 55 , 70 ] . initiation of the ifn response follows recognition of pathogen associated molecular patterns ( pamps ) by germline - encoded pattern - recognition proteins ( prps ) ( e.g. toll - like receptors [ tlrs ] and retinoic acid - inducible gene i [ rigi]-like receptors [ rlrs ] ) [ 55 , 70 ] . rna virus pamps include double stranded rna ( dsrna ) and uncapped rna with a 5 triphosphate , which are presented by genomic rna or products of transcription / replication ; these variously stimulate the cytoplasmic rlrs rig - i and melanoma differentiation - associated protein 5 ( mda5 ) ( which principally recognize pamps generated within infected cells ) and certain of the transmembrane tlrs ( which detect extracellularly - derived pamps ) [ 6 , 22 , 55 , 70 ] . rlr signaling proceeds through the adapter protein mitochondrial antiviral - signaling protein ( mavs ) , ultimately activating the kinases , tank - binding kinase 1 ( tbk1 ) and i - kappa b kinase ( ikk ) ( fig . 1 ) ; an initially distinct signaling pathway from the double stranded rna receptor tlr3 also converges at tbk1/ikk . subsequent phosphorylation / dimerisation of ifn regulatory factor 3 ( irf3 ) in most cell types ( and irf7 in certain immune cells or ifn - primed cells ) precedes nuclear localization leading to ifn/ expression . nuclear factor - kappa b ( nfkb ) is also activated to form part of the ifn enhanceosome ( transcription factor complex ) , as well as activating the expression of other pro - inflamatory cytokines . secreted ifn/ subsequently binds to the type - i ifn receptor ( ifnar ) to activate the transcription factors signal transducers and activators of transcription 1 and 2 ( stat1 and 2 ) ( fig . stat1/2 heterodimerization via reciprocal phosphotyrosine - sh2 interactions precedes nuclear translocation and formation with irf9 of the ifn - stimulated gene factor 3 ( isgf3 ) , which upregulates several hundred ifn - stimulated gene ( isg ) products , including antiviral ( e.g. protein kinase r [ pkr ] , tetherin , myxoma virus resistance [ mx ] , promyelocytic leukemia [ pml ] ) and immunostimulatory ( e.g. major histocompatibility complex [ mhc ] ) proteins , to coordinate a potent antiviral response [ 55 , 63 ] . experiments using genetically modified virus and/or infection of dendritic cells to minimize viral mechanisms that inhibit immune signaling ( see below and fig . 1 ) , have indicated that rabv can induce ifn through pathways dependent on rig - i , mda5 and mavs , but independent of tlr signaling , consistent with increased pathogenicity in mavs - deficient mice [ 16 , 22 ] . since pathogenicity is also enhanced in ifnar mice , this indicates that rabv can induce type i - ifns which are at least partially effective in slowing the onset and/or reducing the severity of symptoms in vivo . nevertheless , rabv remains a potent killer , indicative of efficient mechanisms to counteract the ifn response . in common with many other viruses , rabv expresses proteins that can directly inhibit ifn signaling pathways . collectively termed viral ifn antagonists , which encompass a remarkable array of proteins ( with hundreds of examples in the literature ) , variously reported to target all stages of the ifn response through diverse mechanisms [ 44 , 55 , 70 ] . these mechanisms can be broadly categorized as : ( i ) general inhibition of host gene expression , ( ii ) sequestration / masking of pamps and ( iii ) sequestration / modification of ifn signaling components or isg products . more generally , mechanisms may be considered as virus - targeted ( where antagonists affect other viral components to minimize pamp production or exposure to prps ) or host - targeted ( where antagonists , often autonomously , form inhibitory interactions with cellular factors ) . many ifn antagonists are multifunctional with roles in the basic viral life cycle as well as accessory roles in ifn antagonism , which often encompass multiple mechanisms targeting different stages of the response [ 55 , 70 ] . this is particularly important to rna viruses where limited genome size excludes the expression of dedicated antagonists , resulting in the evolution of accessory ifn - antagonist functions within conserved structural proteins and/or alternative products encoded by conserved genes ( e.g. [ 1 , 11 , 55 , 66 , 70 , 72 ] ) . this has made analysis of the significance of ifn antagonism in infection / pathogenicity challenging due to potential off - target effects in mutagenic studies ; however , several recent studies of rabv and other viruses indicate critical roles in disease ( see below ) . rabv p , m and n proteins have ifn antagonist functions that encompass many archetypal features of this class of protein . p protein is the best defined , with in vitro infection and protein expression studies , indicative of several mechanisms , mediated through a large interactome incorporating host innate immune factors , nuclear trafficking receptors and elements of the cytoskeleton ( reviewed in [ 11 , 31 , 49 , 60 ] ; , summarized below and in figs . 1 and 2 ) . notably , p protein forms many other interactions with host proteins not obviously associated with immunity , indicative of roles for p as a major hub at the virus - host interface ( e.g. [ 17 , 32 , 51 , 56 ] ) . this is perhaps consistent with a high degree of evolutionary flexibility in this non - enzymatic component of the replication machinery , which is encoded by a p gene in all mononegaviruses and is essential to replication , but shows little to no sequence conservation across the mnv order or even between genera of the same family . the first indications of a role for p protein in immune evasion came from the conzelmann laboratory , through analysis of rabv in which p protein expression was downregulated by translocation of the p gene within the genome . although the precise molecular interactions involved in tbk1 inhibition remain unresolved [ 8 , 58 ] , it was recently shown that , p proteins of certain wild - type street strains , but not those of representative fixed laboratory strains , have an additional function to inhibit ikk , involving physical interaction with this adapter molecule . subsequently , the conzelmann and blondel groups identified an interaction of p protein with stat1 and stat2 that inhibits their nuclear translocation and transactivation in response to type - i and type - ii ifns ( [ 9 , 71 ] , fig . this interaction is conserved among lyssaviruses and notably is strongly dependent on ifn - activation ( phospholylation of stats ) , possibly reflecting a mechanism to ensure that p protein is diverted from replication / transcription function only when required [ 9 , 75 ] . recently , p protein was also shown to target stat3 , enabling inhibition of il6-dependent signaling , indicative of immune antagonistic roles beyond ifns . other than the full length p protein ( p or p1 ) , which interacts with l protein in the rdrp complex , rabv p gene expresses four n - terminal truncated isoforms ( p2-p5 ) from four extra in - frame start codons via a ribosomal leaky scanning mechanism . p2-p5 lack the n - terminal l - binding region , suggestive of alternative functions ( reviewed in ) , including specialized roles in ifn antagonism . indeed , analysis of rabv in which isoform expression is modulated using an internal ribosomal entry site ( ires ) has suggested that , while p1 is required for efficient replication , p2 has more important roles in suppressing ifn signaling . protein expression studies have also identified a number of apparently isoform - specific mechanisms of ifn antagonism , suggestive of a multifaceted strategy ( fig . the mechanisms involved in inhibition of ifn signaling by rabv p proteins are shown . all of the mechanisms require a physical interaction of p proteins ( p1 , p2 and p3 ) with stats . mechanisms directly implicated in pathogenicity are indicated by an asterisk . ) ; this might be required to effect an efficient generalized shutdown of the potent antiviral ifn system or to enable targeting of specific elements / pathways of the ifn response that may be more important during different stages of infection and/or in different cell types . p1/p2 exploit the cellular trafficking machinery to undergo active nuclear export via an n - terminal nuclear export sequence ( nes ) , thereby effecting strong nuclear exclusion of p1/p2-associated stats ( [ 9 , 23 , 33 , 52 , 71 , 75 , 76 ] , fig . n - terminal truncation inactivates the nes , activates a nuclear localization sequence ( nls ) and induces association with microtubules [ 7 , 47 , 50 ] ; together with several additional trafficking sequences [ 45 , 46 , 49,50,51,52 ] , this results in p3 distribution to the cytoskeleton and nucleus , enabling cytoskeletal arrest of p3-associated stat1 and a potential intra - nuclear blockade of stat1-dna interaction ( [ 47 , 72 ] , fig . 2 ) . p protein may also target functions of isg products , since p1 and p3 interact with the pml protein . although the significance of this interaction is unclear , expression of certain pml isoforms can inhibit infection , suggestive of antiviral functions toward rabv [ 4 , 5 ] . specific mechanisms of antagonism of ifn signaling by rabv p proteins . the mechanisms involved in inhibition of ifn signaling by rabv p proteins are shown . all of the mechanisms require a physical interaction of p proteins ( p1 , p2 and p3 ) with stats . m protein immune antagonistic activity involves interaction with a novel isoform of nfkb rela protein , relap43 , which was initially identified in a screen for m protein interactors and shown to be a positive regulator of nfkb signaling ( , fig . m protein interaction inhibited expression of type i ifn , proinflammatory cytokines and antiviral genes irf1 and hiap , consistent with important roles for m - relap43 in immune evasion . recent data indicate that rabv n protein acts as an ifn antagonist by suppressing ifn induction , although this uses a mechanism distinct from that of p protein , acting upstream of tbk1/irf3 at the level of rig - i ( , fig . 1 ) . notably , this effect was only observed in the context of rabv infection , where the pathogenic rabv strain nishigahara induced lower irf3 activation and expression of type - i ifn / chemokines than the attenuated nishigahara - derivative strain ni - ce . mutagenic analysis demonstrated that this is due to substitutions in the ni - ce n gene affecting two n protein residues . since single expression of n protein from a plasmid did not inhibit responses to the dsrna analogue polyi : c or infection by a heterologous virus , this appears to involve a virus - targeted mechanism , potentially due to n protein function in encapsidating the rabv genome / replication products and/or forming cage - like structures around cytoplasmic virus factories ( negri bodies ) [ 30 , 42 ] , thereby inhibiting pamp - prp recognition . comparative analyses of wild - type street strains and fixed laboratory / attenuated vaccine strains have included the demonstration that the cvs - b2c laboratory strain induces greater inflammatory responses in mice ( including elevated type - i ifn - related gene and pro - inflamatory cytokine expression ) than the street strain of bat origin ( shbrv ) , indicative of greater immuno - evasive function in the latter . similarly , relap3 targeting has been reported to be observed for m protein from wild - type lyssaviruses but not from laboratory / vaccine strains , with analogous results reported for ikk targeting by p protein . while these data indicate a correlation between ifn antagonist function and pathogenesis , some other findings argue against such a simple relationship , including the report that the cvs - b2c laboratory strain harbors greater capacity to antagonize ifn signaling than a wild - type dog rabv strain drv , which reverse genetic analysis indicated to be due to differences in the ifn - antagonist - encoding p gene of the respective strains . this is perhaps to be expected , since the attenuated phenotypes of different strains , particularly fixed strains adapted to laboratory animal models and/or cell culture , are likely to have multigenic origin ( e.g. ) ; indeed , previous data suggested that g protein is responsible for the attenuated phenotype of cvs - b2c strain via a mechanism distinct from p protein - mediated immune evasion . similarly , the behavior of street strains derived from host species , such as bats and dogs , may differ significantly when transferred to laboratory animal models . in this respect , it is possible that the p protein - dependent difference observed between drv and cvs - b2c in murine neuronal cells relates to some extent to the adaptation of the latter to rodents since the parental cvs strain was serially passaged in mouse brains . thus , more direct evidence for key roles of ifn - antagonism in disease has come from reverse genetics studies , which have enabled direct comparison of the pathogenicity of viruses that are homogeneous except for altered expression of , or sequences within , specific ifn - antagonists . suppression of p protein expression in the sad rabv strain using transcription and translation - based approaches has been shown to cause attenuation in vivo , correlating with reduced capacity to inhibit ifn induction and ifn signaling ( [ 8 , 9 , 36 , 58 ] and see above ) . the fact that the p - deficient viruses showed more virulent phenotype in ifnar - deficient mice is consistent with an important role of ifn antagonism in the viral pathogenesis ; however , approaches using altered expression of the entire p protein are complicated by potential off - target effects due to p protein s function in genome transcription / replication . furthermore , such approaches can not assess the contribution of particular mechanisms of ifn antagonism . directed mutagenesis of rabv has enabled such analyses , where deletions within a 10-residue central region of p protein and substitutions in the hydrophobic w - hole of the p protein c - terminal domain have been shown to specifically impair antagonism of ifn induction and stat1/2 signaling , respectively [ 58 , 76 ] . importantly , defects in replication / transcription function or growth in ifn - incompetent cells were not apparent for viruses carrying either the deletions or the substitutions , but neurovirulence in mice was reduced in both cases , indicating significant roles for antagonism of both ifn induction and signaling in disease . reverse genetics studies using the nishigahara / ni - ce viruses ( see above ) have also revealed roles in virulence for p protein nuclear trafficking and consequent inhibition of stat1 trafficking / signaling [ 23 , 37,38,39 ] . nishigahara is lethal in mice following intracerebral ( ic ) or intramuscular ( i m ) inoculation , indicative of neurovirulence and neuroinvasiveness , respectively , while the derivative ni - ce strain is non - lethal by either route [ 67 , 78 ] . although attenuation of ni - ce is multigenic , a significant restoration of pathogenicity in the recombinant ce(nip ) strain , in which the nishigahara p gene is expressed in the ni - ce genetic background , identified p protein as a pathogenic determinant by both inoculation routes [ 23 , 67 , 78 ] . this correlated with altered viral sensitivity to ifn in neuronal and muscle cell lines , suggesting that circumvention of ifn responses by p protein is important not only to propagation in neurons , but also in muscle cells en route to peripheral nerves [ 23 , 78 ] . analysis of p protein function in neuronal cells indicated that ni - ce p protein is not defective for suppression of ifn induction or binding to stat1 , but can not prevent stat1 nuclear accumulation / signaling due to mutations in the p protein nes that impair nuclear export of p - stat1 complexes . notably , in muscle cells , ni - ce p protein displayed an additional defect in antagonizing ifn induction , suggestive of differences in ifn responses of these cell types . this is consistent with the idea that the diverse ifn - antagonistic mechanisms identified for p protein might have developed to enable immune evasion in different cell - types . the reverse genetics approach using nishigahara / ni - ce chimeric viruses has also highlighted the importance of n protein - dependent inhibition of rig - i signaling in the pathogenicity : the expression of the nishigahara n gene in the ni - ce background [ generating the ce(nin ) strain ] resulted in increased suppression of viral ifn induction and pathogenicity in mice , with decreased ifn responses and increased viral spread observed in the brain [ 38 , 39 ] . rabv not only impacts on innate immune signaling within infected cells , but also has developed mechanisms to suppress broader adaptive inflammatory responses . in the cns , despite high levels of viral replication in naturally infected humans and animals , a lack of significant infiltration by inflammatory immune cells is a hallmark of pathogenic infection . several lines of evidence indicate key roles in pathogenesis [ 2 , 74 ] . . demonstrated that infection of mouse brain with the pathogenic rabv strain shbrv , only modestly induces expression of chemokines , such as mcp-1 ( ccl2 ) , ip-10 ( cxcl10 ) and rantes ( ccl5 ) , that are critical for leukocyte recruitment to sites of inflammation , while the less pathogenic laboratory strain cvs - b2c induced expression more strongly . cytokines including proinflammatory il-6 , and cytokine receptors , were also up - regulated in cvs - b2c - infected brain . these data indicated a correlation between the ability of rabv to suppress inflammatory responses in the cns and pathogenicity , suggesting that rabv s mechanisms to maintain low levels of chemokines and cytokines and/or their cognate receptors in the infected cns ( including the mechanisms described above ) , suppress inflammation and virus clearance , resulting in a lethal outcome . similar approaches have provided insights into the mechanisms by which rabv regulates inflammatory cell infiltration of the cns , highlighting a role for the blood - brain barrier ( bbb ) . in particular , infection with the attenuated cvs - f3 strain increased bbb permeability , which was accompanied by high - levels of expression of several chemokines including ccl2 , ccl5 and cxcl10 in the cerebellum as well as infiltration of cd4 t cells and cd19 b cells into this region . in contrast , infection with pathogenic shbrv maintained bbb integrity and prevented infiltration of immune effectors . these data strongly indicated that enhancement of bbb permeability is important for recruitment of immune effectors into the cns and subsequent clearance of attenuated rabv , while pathogenic strains harbor mechanisms to maintain the bbb . the key role of bbb integrity in the outcome of viral infection was also supported by the finding that in plsjl mice , which show moderate resistance to shbrv infection , reduction of bbb permeability through treatment with a steroid hormone increased the mortality rate of infected mice , while enhancement of bbb permeability by immunization with myelin basic protein resulted in decreased mortality . it was also reported that , in mouse brain infected with cvs - f3 strain , virus - specific antibody produced in situ after recruitment of b cells into the brain plays a critical role in virus clearance , highlighting the role of the humoral response in the cns . several studies have begun to delineate the mechanisms by which attenuated rabv infection results in loss of bbb integrity . phares et al . reported that in cvs - f3-infected mouse , cd4 t cells , but not cd8 t cells or b cells , play an important role in enhancement of bbb permeability . the study suggested that ifn- released by cd4 t cells that accumulate in the neurovasculature stimulates neurovascular endothelial cells to produce peroxynitrite ( onoo ) , which directly triggers enhancement of bbb permeability . a recent study demonstrated that in mouse brain infected with attenuated cvs - b2c strain , cxcl10 produced by infected neurons promotes infiltration of cxcr3 cd4 t cells , which can differentiate into ifn--producing th1 cells as well as th17 cells expressing il-17 , an important contributor to disruption of the bbb . the identification of mechanisms by which attenuated rabv infection results in recruitment of immune effectors via enhanced bbb permeability has provided insights into the strategies used by pathogenic rabv to inhibit inflammatory responses in the cns . specifically , it is thought that pathogenic rabv minimizes expression of cxcl10 in neurons , thereby preventing infiltration of cxcr3 cd4 t cells ( fig . 3afig . ( a ) infection of neurons by pathogenic but not attenuated rabv suppresses induction of cxcl10 expression , reducing cxcl10-dependent infiltration of cxcr3 cd4 + t cells , thereby preventing transition to il17-producing th17 cells that promote bbb permeability and immune cell infiltration . ( b ) rabv infection induces expression of b7-h1 and other immunosuppressive molecules on infected neurons ( and potentially non - infected glia ) , which induce the apoptosis of infiltrating t cells . ) ; this hypothesis is based on the observations that infection of mice by pathogenic shbrv only modestly induces expression of cxcl10 in the mouse brains and that infection of cultured neuroblastoma with nishigahara induces significantly lower levels of cxcl10 than infection with the attenuated ni - ce . thus , reduction of cxcl10 expression in infected neurons may be a general strategy used by pathogenic rabv strains to suppress inflammatory responses in the cns . ( a ) infection of neurons by pathogenic but not attenuated rabv suppresses induction of cxcl10 expression , reducing cxcl10-dependent infiltration of cxcr3 cd4 + t cells , thereby preventing transition to il17-producing th17 cells that promote bbb permeability and immune cell infiltration . ( b ) rabv infection induces expression of b7-h1 and other immunosuppressive molecules on infected neurons ( and potentially non - infected glia ) , which induce the apoptosis of infiltrating t cells . interestingly , studies by other groups have suggested that rabv may have evolved additional , distinct strategies to suppress inflammatory t - cell responses in the cns . in contrast to the model above , it was reported that infection both by pathogenic cvs strain and by attenuated pv strain led to infiltration of cd3 t cells into the cns of infected mice after i m inoculation . however , by seven days post - inocultion , apoptosis of infiltrating t cells and the resultant disappearance of t cells from the cns were observed in mice infected with cvs , but not with pv . these findings are particularly intriguing as rabv generally does not infect lymphocytes , raising the question of how pathogenic rabv induces t - cell apoptosis . the most likely scenario appears to relate to the finding that pathogenic rabv infection induces the expression of immunosuppressive molecules , such as b7-h1 , hla - g and fasl , on neurons , which can trigger apoptosis of activated t cells , both in mouse cns and in cultured neurons [ 3 , 27 , 28 , 41 ] . this indicates that pathogenic rabv can hijack host immunosuppressive mechanisms to destroy infiltrating t cells ( fig . this hypothesis is directly supported by the finding that a pathogenic cvs strain caused significantly milder symptoms in b7-h1 knockout mice than in wild - type mice . notably , it was also found that the percentages of cd3 t cells and cd8 t cells in the total t cell infiltrate in the cns of b7-h1 knockout mice were higher than those in the cns of the wild - type mice and that the proportion of apoptotic cd8 t cells was reduced in the cns of the b7-h1 knockout mice . these findings indicate that the b7-h1-mediated apoptosis of t cells infiltrating the cns fulfills a key role not only in suppression of the inflammatory response , but also in the pathogenesis of rabv . while the findings above are suggestive of a sophisticated strategy for immune evasion by rabv , there is a discrepancy that needs to be resolved : expression of the immunosuppressive b7-h1 and hla - g , both known isg products , is induced by ifn [ 14 , 27 , 28 ] , but ifn production and signaling is suppressed in neurons infected by pathogenic rabv via the function of n , m and p proteins ( see above ) . however , even pathogenic rabv infection induces modest but detectable level of ifn in the brain and in cultured neurons [ 37 , 74 ] . ifn might be sufficient to induce b7-h1 and hla - g expression and to cause destruction of infiltrating t cells . although it has been speculated that non - infected glia cells play a role in the destruction of t cells in the cns , this remains to be proven experimentally . current knowledge indicates that rabv effects a potent and multipronged strategy to disable host immunity at many levels , with a growing body of evidence from experiments using genetically modified viruses / hosts indicating critical roles in disease . in ifn - mediated innate immunity , the capacity of p protein to interact with and effect mislocalization of stats , and that of p and n proteins to inhibit rlr - driven ifn induction , have been demonstrated to impact on pathogenicity in animals . in adaptive immunity / inflammation of the cns , rabv has a clear capacity to suppress host responses , with two mechanisms suggested in in vivo models , specifically blockage of immune effector infiltration via maintenance of bbb integrity , and induction of t - cell apoptosis ( fig . firstly , the roles in pathogenicity of a number of ifn - antagonist mechanisms of p protein identified in vitro , such as targeting of pml , stat3 , and microtubules remain unresolved and await research using reverse genetics approaches / animal infection models similar to those that indicated roles in disease for nes - driven p protein nuclear export . secondly , there are apparently conflicting reports with respect to the roles of ifn signaling / isg expression in infection ( where antiviral and proviral roles have been identified ) , as well as the contribution of altered bbb permeability and t - cell apoptosis in the cns to rabv suppression of t - cell immunity [ 28 , 62 ] . although such observations may relate in part to differences in experimental design / models used , it seems likely that lyssaviruses have evolved diverse mechanisms to effect immune subversion , as indicated by in vitro studies of ifn - antagonism , and that certain mechanisms may predominate in different settings due to factors , such as the viral strains / species examined , cell type infected , stage of infection , infectious dose , etc . for example , a pathogenic strain that only moderately suppresses the expression of ifn and/or cxcl10 in neurons might have evolved mechanisms to induce apoptosis of t cells . thirdly , in spite of clear evidence that suppression of inflammation in the cns is important to disease , the molecular mechanisms by which the virus achieves this remain undefined , although involvement of p , m or n protein in suppressing signaling during cytokine / chemokine production seems likely . future research using genetically modified viruses / hosts should help to delineate these issues , but what is clear at present is that mutations impacting viral evasion of immunity can potently impact disease , indicating that such mutants are potentially useful for the generation of attenuated vaccine strains . in particular , novel mutations affecting immune evasion may be combined with mutations affecting other genes / mechanisms [ 15 , 24 , 38 , 67,68,69 ] to achieve high levels of safety while retaining largely native structure and replication to generate self - adjuventing vaccines ; such advances should greatly enhance deliverability , including providing the potential means to achieve single - dose oral vaccination , thereby overcoming current limitations of multiple - dose / needle injection regimes , as well as providing potent protection in diverse host species . importantly , such vaccines should not simply be weakened , but should have the potential to induce stronger immune responses that could generate protection more efficiently . this promise is yet to be realized , however , as a recent study using a p protein - mutated rabv strain unable to prevent ifn induction could induce protection in orally - vaccinated foxes , but gave no apparent improvement in protection compared with the wild - type strain . the effectiveness of such mutations in attenuating strains optimized for immunogenicity , or of alternative strategies targeting stat antagonism by p protein , or rig - i - antagonism by n protein , awaits further research . finally , another clear corollary of the above considerations is that the mechanisms underlying immune evasion may provide targets for therapeutics for rabies . several molecular interfaces have been shown to be critical to viral inhibition of immune signaling and virulence ( e.g. p protein - stat1 , p - protein - exportin interactions ) . others have been defined as important in vitro but not confirmed in vivo ( see above ) , and some involve interactions as yet undefined at the molecular level ( e.g. n protein - mediated inhibition of rig - i ) . in most cases , however , progress has been made toward elucidating the sites involved by mapping important domains / sequences [ 5 , 38 , 76 ] . the ultimate outcome of such research would be molecular and structural definition of the interfaces , providing knowledge that might guide the development of inhibitors capable of preventing viral evasion of innate immunity . until the mechanisms underlying viral modulation of bbb permeability and t - cell apoptosis are resolved , potential pathways to therapeutic targeting of these processes are less clear . however , since viral targeting of innate signaling is probably linked to effects on adaptive immunity , the above mentioned therapeutics may have a dual effect on both arms of the response . it is also possible that targeting the host to enhance adaptive responses , rather than targeting viral counter - measures might be useful ; indeed , mouse studies indicated that strategies to directly alter bbb permeability can improve outcomes of infection , providing proof - of - principal to assess such possibilities towards antiviral therapies .

rabies is a zoonotic disease caused by the lyssavirus rabies virus ( rabv ) that can infect most mammals , including humans , where it has a case - fatality rate of almost 100% . although preventable by vaccination , rabies causes c. 59,000 human fatalities every year worldwide . thus , there exists an urgent need to establish an effective therapy and/or improve dissemination of vaccines for humans and animals . these outcomes require greater understanding of the mechanisms of rabv pathogenesis to identify new molecular targets for the development of therapeutics and/or live vaccines with high levels of safety . importantly , a number of studies in recent years have indicated that rabv specifically suppresses host immunity through diverse mechanisms and that this is a key process in pathogenicity . here , we review current understanding of immune modulation by rabv , with an emphasis on its significance to pathogenicity and the potential exploitation of this knowledge to develop new vaccines and antivirals .

THE IFN RESPONSE AND ITS INDUCTION BY RABV VIRAL COUNTERMEASURES: IFN ANTAGONISTS LYSSAVIRUS IFN ANTAGONISTS ROLES OF IFN ANTAGONISM IN PATHOGENICITY EVASION OF INFLAMMATORY RESPONSES IN THE CNS PERSPECTIVES

the earliest host - cell response to viral infection is activation of the type - i interferon ( ifn/ ) system ( see fig . 1 ) , which ultimately leads to the establishment of antiviral responses in infected / neighboring cells and contributes to the shaping of an effective adaptive response [ 55 , 70 ] . initiation of the ifn response follows recognition of pathogen associated molecular patterns ( pamps ) by germline - encoded pattern - recognition proteins ( prps ) ( e.g. rna virus pamps include double stranded rna ( dsrna ) and uncapped rna with a 5 triphosphate , which are presented by genomic rna or products of transcription / replication ; these variously stimulate the cytoplasmic rlrs rig - i and melanoma differentiation - associated protein 5 ( mda5 ) ( which principally recognize pamps generated within infected cells ) and certain of the transmembrane tlrs ( which detect extracellularly - derived pamps ) [ 6 , 22 , 55 , 70 ] . nuclear factor - kappa b ( nfkb ) is also activated to form part of the ifn enhanceosome ( transcription factor complex ) , as well as activating the expression of other pro - inflamatory cytokines . stat1/2 heterodimerization via reciprocal phosphotyrosine - sh2 interactions precedes nuclear translocation and formation with irf9 of the ifn - stimulated gene factor 3 ( isgf3 ) , which upregulates several hundred ifn - stimulated gene ( isg ) products , including antiviral ( e.g. 1 ) , have indicated that rabv can induce ifn through pathways dependent on rig - i , mda5 and mavs , but independent of tlr signaling , consistent with increased pathogenicity in mavs - deficient mice [ 16 , 22 ] . since pathogenicity is also enhanced in ifnar mice , this indicates that rabv can induce type i - ifns which are at least partially effective in slowing the onset and/or reducing the severity of symptoms in vivo . in common with many other viruses , rabv expresses proteins that can directly inhibit ifn signaling pathways . collectively termed viral ifn antagonists , which encompass a remarkable array of proteins ( with hundreds of examples in the literature ) , variously reported to target all stages of the ifn response through diverse mechanisms [ 44 , 55 , 70 ] . many ifn antagonists are multifunctional with roles in the basic viral life cycle as well as accessory roles in ifn antagonism , which often encompass multiple mechanisms targeting different stages of the response [ 55 , 70 ] . this is particularly important to rna viruses where limited genome size excludes the expression of dedicated antagonists , resulting in the evolution of accessory ifn - antagonist functions within conserved structural proteins and/or alternative products encoded by conserved genes ( e.g. this has made analysis of the significance of ifn antagonism in infection / pathogenicity challenging due to potential off - target effects in mutagenic studies ; however , several recent studies of rabv and other viruses indicate critical roles in disease ( see below ) . p protein is the best defined , with in vitro infection and protein expression studies , indicative of several mechanisms , mediated through a large interactome incorporating host innate immune factors , nuclear trafficking receptors and elements of the cytoskeleton ( reviewed in [ 11 , 31 , 49 , 60 ] ; , summarized below and in figs . this is perhaps consistent with a high degree of evolutionary flexibility in this non - enzymatic component of the replication machinery , which is encoded by a p gene in all mononegaviruses and is essential to replication , but shows little to no sequence conservation across the mnv order or even between genera of the same family . the first indications of a role for p protein in immune evasion came from the conzelmann laboratory , through analysis of rabv in which p protein expression was downregulated by translocation of the p gene within the genome . recently , p protein was also shown to target stat3 , enabling inhibition of il6-dependent signaling , indicative of immune antagonistic roles beyond ifns . p2-p5 lack the n - terminal l - binding region , suggestive of alternative functions ( reviewed in ) , including specialized roles in ifn antagonism . indeed , analysis of rabv in which isoform expression is modulated using an internal ribosomal entry site ( ires ) has suggested that , while p1 is required for efficient replication , p2 has more important roles in suppressing ifn signaling . protein expression studies have also identified a number of apparently isoform - specific mechanisms of ifn antagonism , suggestive of a multifaceted strategy ( fig . the mechanisms involved in inhibition of ifn signaling by rabv p proteins are shown . all of the mechanisms require a physical interaction of p proteins ( p1 , p2 and p3 ) with stats . mechanisms directly implicated in pathogenicity are indicated by an asterisk . ) ; this might be required to effect an efficient generalized shutdown of the potent antiviral ifn system or to enable targeting of specific elements / pathways of the ifn response that may be more important during different stages of infection and/or in different cell types . although the significance of this interaction is unclear , expression of certain pml isoforms can inhibit infection , suggestive of antiviral functions toward rabv [ 4 , 5 ] . specific mechanisms of antagonism of ifn signaling by rabv p proteins . the mechanisms involved in inhibition of ifn signaling by rabv p proteins are shown . all of the mechanisms require a physical interaction of p proteins ( p1 , p2 and p3 ) with stats . recent data indicate that rabv n protein acts as an ifn antagonist by suppressing ifn induction , although this uses a mechanism distinct from that of p protein , acting upstream of tbk1/irf3 at the level of rig - i ( , fig . notably , this effect was only observed in the context of rabv infection , where the pathogenic rabv strain nishigahara induced lower irf3 activation and expression of type - i ifn / chemokines than the attenuated nishigahara - derivative strain ni - ce . mutagenic analysis demonstrated that this is due to substitutions in the ni - ce n gene affecting two n protein residues . similarly , relap3 targeting has been reported to be observed for m protein from wild - type lyssaviruses but not from laboratory / vaccine strains , with analogous results reported for ikk targeting by p protein . while these data indicate a correlation between ifn antagonist function and pathogenesis , some other findings argue against such a simple relationship , including the report that the cvs - b2c laboratory strain harbors greater capacity to antagonize ifn signaling than a wild - type dog rabv strain drv , which reverse genetic analysis indicated to be due to differences in the ifn - antagonist - encoding p gene of the respective strains . ; indeed , previous data suggested that g protein is responsible for the attenuated phenotype of cvs - b2c strain via a mechanism distinct from p protein - mediated immune evasion . in this respect , it is possible that the p protein - dependent difference observed between drv and cvs - b2c in murine neuronal cells relates to some extent to the adaptation of the latter to rodents since the parental cvs strain was serially passaged in mouse brains . thus , more direct evidence for key roles of ifn - antagonism in disease has come from reverse genetics studies , which have enabled direct comparison of the pathogenicity of viruses that are homogeneous except for altered expression of , or sequences within , specific ifn - antagonists . the fact that the p - deficient viruses showed more virulent phenotype in ifnar - deficient mice is consistent with an important role of ifn antagonism in the viral pathogenesis ; however , approaches using altered expression of the entire p protein are complicated by potential off - target effects due to p protein s function in genome transcription / replication . furthermore , such approaches can not assess the contribution of particular mechanisms of ifn antagonism . directed mutagenesis of rabv has enabled such analyses , where deletions within a 10-residue central region of p protein and substitutions in the hydrophobic w - hole of the p protein c - terminal domain have been shown to specifically impair antagonism of ifn induction and stat1/2 signaling , respectively [ 58 , 76 ] . importantly , defects in replication / transcription function or growth in ifn - incompetent cells were not apparent for viruses carrying either the deletions or the substitutions , but neurovirulence in mice was reduced in both cases , indicating significant roles for antagonism of both ifn induction and signaling in disease . although attenuation of ni - ce is multigenic , a significant restoration of pathogenicity in the recombinant ce(nip ) strain , in which the nishigahara p gene is expressed in the ni - ce genetic background , identified p protein as a pathogenic determinant by both inoculation routes [ 23 , 67 , 78 ] . analysis of p protein function in neuronal cells indicated that ni - ce p protein is not defective for suppression of ifn induction or binding to stat1 , but can not prevent stat1 nuclear accumulation / signaling due to mutations in the p protein nes that impair nuclear export of p - stat1 complexes . this is consistent with the idea that the diverse ifn - antagonistic mechanisms identified for p protein might have developed to enable immune evasion in different cell - types . the reverse genetics approach using nishigahara / ni - ce chimeric viruses has also highlighted the importance of n protein - dependent inhibition of rig - i signaling in the pathogenicity : the expression of the nishigahara n gene in the ni - ce background [ generating the ce(nin ) strain ] resulted in increased suppression of viral ifn induction and pathogenicity in mice , with decreased ifn responses and increased viral spread observed in the brain [ 38 , 39 ] . in the cns , despite high levels of viral replication in naturally infected humans and animals , a lack of significant infiltration by inflammatory immune cells is a hallmark of pathogenic infection . these data indicated a correlation between the ability of rabv to suppress inflammatory responses in the cns and pathogenicity , suggesting that rabv s mechanisms to maintain low levels of chemokines and cytokines and/or their cognate receptors in the infected cns ( including the mechanisms described above ) , suppress inflammation and virus clearance , resulting in a lethal outcome . similar approaches have provided insights into the mechanisms by which rabv regulates inflammatory cell infiltration of the cns , highlighting a role for the blood - brain barrier ( bbb ) . in particular , infection with the attenuated cvs - f3 strain increased bbb permeability , which was accompanied by high - levels of expression of several chemokines including ccl2 , ccl5 and cxcl10 in the cerebellum as well as infiltration of cd4 t cells and cd19 b cells into this region . in contrast , infection with pathogenic shbrv maintained bbb integrity and prevented infiltration of immune effectors . these data strongly indicated that enhancement of bbb permeability is important for recruitment of immune effectors into the cns and subsequent clearance of attenuated rabv , while pathogenic strains harbor mechanisms to maintain the bbb . the key role of bbb integrity in the outcome of viral infection was also supported by the finding that in plsjl mice , which show moderate resistance to shbrv infection , reduction of bbb permeability through treatment with a steroid hormone increased the mortality rate of infected mice , while enhancement of bbb permeability by immunization with myelin basic protein resulted in decreased mortality . it was also reported that , in mouse brain infected with cvs - f3 strain , virus - specific antibody produced in situ after recruitment of b cells into the brain plays a critical role in virus clearance , highlighting the role of the humoral response in the cns . several studies have begun to delineate the mechanisms by which attenuated rabv infection results in loss of bbb integrity . a recent study demonstrated that in mouse brain infected with attenuated cvs - b2c strain , cxcl10 produced by infected neurons promotes infiltration of cxcr3 cd4 t cells , which can differentiate into ifn--producing th1 cells as well as th17 cells expressing il-17 , an important contributor to disruption of the bbb . the identification of mechanisms by which attenuated rabv infection results in recruitment of immune effectors via enhanced bbb permeability has provided insights into the strategies used by pathogenic rabv to inhibit inflammatory responses in the cns . ; this hypothesis is based on the observations that infection of mice by pathogenic shbrv only modestly induces expression of cxcl10 in the mouse brains and that infection of cultured neuroblastoma with nishigahara induces significantly lower levels of cxcl10 than infection with the attenuated ni - ce . thus , reduction of cxcl10 expression in infected neurons may be a general strategy used by pathogenic rabv strains to suppress inflammatory responses in the cns . this hypothesis is directly supported by the finding that a pathogenic cvs strain caused significantly milder symptoms in b7-h1 knockout mice than in wild - type mice . notably , it was also found that the percentages of cd3 t cells and cd8 t cells in the total t cell infiltrate in the cns of b7-h1 knockout mice were higher than those in the cns of the wild - type mice and that the proportion of apoptotic cd8 t cells was reduced in the cns of the b7-h1 knockout mice . these findings indicate that the b7-h1-mediated apoptosis of t cells infiltrating the cns fulfills a key role not only in suppression of the inflammatory response , but also in the pathogenesis of rabv . while the findings above are suggestive of a sophisticated strategy for immune evasion by rabv , there is a discrepancy that needs to be resolved : expression of the immunosuppressive b7-h1 and hla - g , both known isg products , is induced by ifn [ 14 , 27 , 28 ] , but ifn production and signaling is suppressed in neurons infected by pathogenic rabv via the function of n , m and p proteins ( see above ) . although it has been speculated that non - infected glia cells play a role in the destruction of t cells in the cns , this remains to be proven experimentally . current knowledge indicates that rabv effects a potent and multipronged strategy to disable host immunity at many levels , with a growing body of evidence from experiments using genetically modified viruses / hosts indicating critical roles in disease . in adaptive immunity / inflammation of the cns , rabv has a clear capacity to suppress host responses , with two mechanisms suggested in in vivo models , specifically blockage of immune effector infiltration via maintenance of bbb integrity , and induction of t - cell apoptosis ( fig . firstly , the roles in pathogenicity of a number of ifn - antagonist mechanisms of p protein identified in vitro , such as targeting of pml , stat3 , and microtubules remain unresolved and await research using reverse genetics approaches / animal infection models similar to those that indicated roles in disease for nes - driven p protein nuclear export . secondly , there are apparently conflicting reports with respect to the roles of ifn signaling / isg expression in infection ( where antiviral and proviral roles have been identified ) , as well as the contribution of altered bbb permeability and t - cell apoptosis in the cns to rabv suppression of t - cell immunity [ 28 , 62 ] . although such observations may relate in part to differences in experimental design / models used , it seems likely that lyssaviruses have evolved diverse mechanisms to effect immune subversion , as indicated by in vitro studies of ifn - antagonism , and that certain mechanisms may predominate in different settings due to factors , such as the viral strains / species examined , cell type infected , stage of infection , infectious dose , etc . for example , a pathogenic strain that only moderately suppresses the expression of ifn and/or cxcl10 in neurons might have evolved mechanisms to induce apoptosis of t cells . in particular , novel mutations affecting immune evasion may be combined with mutations affecting other genes / mechanisms [ 15 , 24 , 38 , 67,68,69 ] to achieve high levels of safety while retaining largely native structure and replication to generate self - adjuventing vaccines ; such advances should greatly enhance deliverability , including providing the potential means to achieve single - dose oral vaccination , thereby overcoming current limitations of multiple - dose / needle injection regimes , as well as providing potent protection in diverse host species . importantly , such vaccines should not simply be weakened , but should have the potential to induce stronger immune responses that could generate protection more efficiently . finally , another clear corollary of the above considerations is that the mechanisms underlying immune evasion may provide targets for therapeutics for rabies . several molecular interfaces have been shown to be critical to viral inhibition of immune signaling and virulence ( e.g. the ultimate outcome of such research would be molecular and structural definition of the interfaces , providing knowledge that might guide the development of inhibitors capable of preventing viral evasion of innate immunity . until the mechanisms underlying viral modulation of bbb permeability and t - cell apoptosis are resolved , potential pathways to therapeutic targeting of these processes are less clear . however , since viral targeting of innate signaling is probably linked to effects on adaptive immunity , the above mentioned therapeutics may have a dual effect on both arms of the response . it is also possible that targeting the host to enhance adaptive responses , rather than targeting viral counter - measures might be useful ; indeed , mouse studies indicated that strategies to directly alter bbb permeability can improve outcomes of infection , providing proof - of - principal to assess such possibilities towards antiviral therapies .

nanomaterials occur in nature via various geobiophysicochemical processes , such as volcanic eruptions , bacterial metabolism , and other erosion and reduction mechanisms . in recent decades , nanoparticles have been increasingly manufactured and used in daily consumer products , such as textiles , paints , pharmaceuticals , and cosmetic products , as well as in pollution treatment and remediation processes [ 1 , 2 ] . however , it is widely known that nanoparticles may cause potential adverse effects in organisms , including human and mammals [ 37 ] . consequently , the adverse effects of incompletely retrieved nanoparticles pose an increasing threat to the public [ 79 ] . therefore , it is important to develop an understanding of the effects and active mechanisms of nanoparticles . silver ( ag ) is among the most used metals in human life and is well known for its antimicrobial effect . during the recent rapid development of nanotechnology , silver nanoparticles ( ag - nps ) have been used for various types of consumer products , such as cosmetics , heath care products , and textiles . consequently , information about the toxicity and carcinogenic effects of ag is available , with it being considered nontoxic and noncarcinogenic when used in appropriate amounts . however , at relatively high concentrations , ag - nps cause cellular toxicity and other adverse impacts , such as the inhibition of mitochondrial function in rat liver cells , mouse germline stem cells , and human fibroblasts [ 1113 ] . furthermore , an increasing number of recent reports have provided evidence of the cytotoxicity of ag - nps at doses of low exposure [ 14 , 15 ] . ag - nps also have an adverse impact on aquatic organisms , such as zebrafish and medaka fish , and cause oxidative stress , cellular apoptosis , chromosomal aberrations , and other developmental toxicity effects during early life stages and adulthood . this is because changes in the functional groups on the nanoparticle surface may facilitate substances to penetrate deep into the cells of the body , causing unexpected impacts on organisms . however , it is unclear whether the effects of coating materials increase the adverse effects of nanomaterials . in this study , we investigated the effects of ag - nps in the presence of citric acid as a capping material . citric acid is an organic chemical commonly found in the natural environment and human body . therefore , the interaction of citric acid with exposed materials is a common mechanism in the natural environment . common carp ( cyprinus carpio ) is an abundant species in the freshwater environment . due to their large size , carp have a better capacity for resistance to pollutants than other laboratory fish , such as zebrafish and japanese medaka . hence , carp are not usually used in experiments to examine the fatal effects of exposure to low doses of pollutants . however , because of the dominance of this species in the natural environment , carp is considered as one of the most suitable models to assess the non - fatal effects of pollutants by evaluating fluctuations in antioxidant enzyme levels or other changes in fish histology and physiology . the objectives of this study were to investigate the adverse effects of ag - nps capped with citric acid ( ag - nps ) by measuring antioxidant enzymatic activities , including those of catalase ( cat ) , superoxide dismutase ( sod ) , and glutathione - s - transferase ( gst ) . target organs included the brain , liver , and gills of the common carp ( c. carpio ) . in addition , other biochemical parameters in the blood and histological changes in the skin , liver , and gills of the fish were examined . for this reason , we used the same biological samples as those used in the previous study to present detailed data about the toxicity of nanoparticles . all chemicals were purchased from sigma - aldrich and were of analytical grade or better . citrate - capped ag - nps were purchased from abcnanotech ( daejeon , korea ) in a suspension form . the average ph of the original solution was 6.5 , with a particle content of 20% by weight . the dispersion of the particles was confirmed using a transmission electron microscope ( tem ; jeol , japan ) and the dynamic light scattering method ( dls ; els-8000 , otsuka , japan ) . a stock solution of ag - nps was prepared at 10 mg / l . working solutions ( 25 , 50 , 100 , and 200 g / l ) were obtained by diluting the stock solution with respective amounts of di water . the concentration of elemental ag was measured using an inductive coupled plasma mass spectrometer ( icp - ms , perkin elmer , usa ) . mean concentrations were obtained by using the data to plot a standard curve with ag standard solution ( scp science , qc , canada ) and 5% hno3 solution ( electronic grade , scp science , qc , canada ) . the ranges of the standard were 0.0 , 1.0 , 5.0 , 10.0 , 20.0 , 40.0 , 60.0 , 80.0 , and 100.0 juvenile carp were obtained from the national academy of agricultural science , korea . as shown in table 1 , the body weight and length of the fish were 18.6~33.8 g and 10.9~13.5 cm , respectively . water temperature was kept at 24 1c , hardness was maintained at 40~60 mg caco3/l , ph of the water was 7.2 , and oxygen saturation was more than 85% . fish were exposed to ag - nps for a period of 48 h and 96 h , according to the oecd test guideline 203 : fish acute toxicity test . the carp were exposed to ag - np concentrations of 25 , 50 , 100 , and 200 g / l . a blank control test ( without ag - nps ) was performed at the same time and under the same exposure conditions . all the experiments were conducted in an 80-l glass tank containing 60 l of water , with 10 fish in each exposure tank . the water was not changed during the test . after exposure , the gills , liver , and brain of the fish were collected , frozen , and stored separately at 20c for further treatment . the samples were rinsed with 0.15 mm kcl solution and homogenized on ice with 50 mm phosphate buffer ( ph 7.0 ) . the suspension was sonicated in an ultrasonic bath for 30 min and then centrifuged at 10,000 g at 4c for 10 min . gst activity was measured using a gst tag assay kit ( novagen , germany ) , in which a sample was combined with 1-chloro-2,4-dinitrobenzene substrate in the reaction buffer . the absorbance of the reaction was monitored at 340 nm by using a uv spectrophotometer ( tecan infinite f200 , austria ) . cat activity was measured using the abei method , in which 50 mm h2o2 was used as a substrate . a solution of 50 mm peroxide was prepared in 50 mm potassium phosphate buffer . the decomposition of h2o2 catalyzed by catalase can be followed using uv spectroscopy on the basis of the absorbance of h2o2 at a wavelength of 240 nm . sod activity was measured by using an sod assay kit ( dojindo laboratories , kumamoto , japan ) . total protein concentration was measured using the bradford method , with bovine serum albumin as the standard protein . other biochemical parameters were examined in this study , namely , ammonia ( nh3 ) , glucose ( glu ) , total cholesterol ( tcho ) , alkaline phosphates ( alp ) , glutamic oxaloacetic transaminase / aspartate aminotransferase ( got / ast ) , alanine aminotransferase ( gpt / alt ) , -glutamyltransferase ( ggt ) , albumin ( alb ) , blood urea nitrogen ( bun ) , creatinine ( cre ) , and total bilirubin ( tbil ) . after exposure , the skin , gills , and liver of the carp were dissected and fixed in bouin 's solution for 12 h or overnight . subsequently , these samples were washed with water and then dehydrated using a graded ethanol series ( 70% , 75% , 80% , 90% , and 100% ) . the samples were embedded in paraffin at 62c and sectioned at a thickness of 5 m by using a precision microtome ( mt-990 ; rmc products , usa ) . the sections were stained using the hematoxylin and eosin ( h&e ) staining method with alcian blue and the periodic acid - schiff ( ab - pas ) reaction . an olympus bx-51 light microscope ( olympus corp . , japan ) coupled with an artcam-150 piii digital camera was used to examine any abnormalities in the samples . data were calculated and analyzed using excel ( microsoft corporation , wa , usa ) software and plotted using sigma plot ( spss inc . the differences between the samples and blank controls were evaluated using one - way analysis of variance and the student 's t - test . rna extraction and dna microarray analysis were performed according to the protocol previously described by lee et al . . the sequences of each clone i d were aligned on the basis of sequence homology by using the basic local alignment search tool ( blast ) in the national center for biotechnology information database . the translated nucleotide sequences were compared with the protein databases by using the blastx analysis software . the size distribution and morphology of ag - nps were examined using tem and dls . as shown in figure 1 , ag - nps became agglomerated in the solution , and the nominal size measured using tem was ~12 nm . however , the mean size of the agglomerated particles varied as much as several hundred nanometers . the dls results showed that the mean diameter of ag - nps at 5 mg / l and 10 mg / l was ~70 nm and ~90 nm , respectively ( table 2 ) . however , particles with a diameter of less than 20 nm were frequently observed in the solution . elemental ag was measured using icp - ms , and the results are shown in table 3 , with relative standard deviations ranging around 10% . in general , ag - nps were well dispersed in di water solution , with some agglomeration and aggregation . the antioxidant enzymes , including those of gst , cat , and sod , were measured , and the results are shown in figures 2 , 3 , and 4 . generally , enzymatic activities changed with different ag - np concentrations . after 48 h of exposure , gst activities in the liver , gills , and brain were not significantly different than that observed when the fish were exposed to 25 g / l and 50 g / l of ag - nps ( figure 2(a ) ) . when the concentration of ag - nps increased to 100 g / l and 200 g / l , gst activity was more significantly reduced in the gills than in the liver and brain . after 96 h of exposure , gst activity was elevated in the liver ; this was correlated with an increase in ag - np concentration in the media ( figure 2(b ) ) . gst activity in liver samples exposed to 100 g / l and 200 g / l was about 190% higher than that observed in the control fish . in comparison , gst activity in the liver of fish after 96 h of exposure was also higher than that in fish subjected to 48 h of exposure , especially at 100 g / l of ag - nps . after 96 h of exposure , gst activity was also higher in the liver than in the brain and gills . as shown in figure 3 , higher levels of cat activity were detected in the liver than in the brain and gills of the experimental fish . similar to gst activity , cat activity in the liver changed at different ag - np concentrations , but it remained relatively stable in the brain and gills . cat concentrations in the liver of the fish after 48 h of exposure showed no significant difference to that in the control group ( figure 3(a ) ) . however , cat activity declined at lower ag - np concentrations ( i.e. , 25 g / l and 50 g / l ) and increased at higher ag - np concentrations ( i.e. , 100 g / l and 200 g / l ) . however , after 96 h of exposure , cat activity was significantly reduced in the liver at 25 g / l and 50 g / l , recovered at 100 g / l , and then decreased again at 200 sod activity differed from that of gst and cat in that it was relatively stable in all examined tissues ( figure 4 ) . however , similar to gst and cat , higher levels of sod activity were detected in the liver than in the brain and gills . there was no significant difference in sod levels among the different exposure groups . in this study , the biochemical parameters in the blood of the exposed fish were also analyzed to understand the changes in metabolism and fish response to the stressor ( i.e. , ag - nps ) . detailed information was published in our previous study , where the concentration of biochemical parameters such as ammonia ( nh3 ) , glutamic oxaloacetic transaminase ( got ) , and total cholesterol ( tcho ) declined at the beginning of exposure and gradually recovered with increase in ag - np concentration . other parameters such as alanine aminotransferase ( gpt ) , -glutamyl transferase ( ggt ) , albumin ( alb ) , blood urea nitrogen ( bun ) , creatinine ( cre ) , and total bilirubin ( tbil ) remained stable or fluctuated around the levels of the control groups , without any significant difference . in comparison , alkaline phosphatase ( alp ) levels increased with increase in ag - nps concentration . after reaching the highest concentration when exposed to 50 g / l of ag - nps , alp concentration began to decline when the fish were exposed to 100 /l and 200 the histological structure of the skin ( figure 5 ) is composed of an epidermal layer and a dermal layer . the epidermis is the outermost layer of the skin , where the epithelial and secretory cells are distributed . when dyed with h&e stain , the vacuolated mucous cells appeared as blank circles or elliptical in shape and were marked with a blue color ( color index 313c ) as a result of the ab - pas ( ph 2.5 ) reaction . vacuolated club cells also appeared elliptical in shape when using the ab - pas ( ph 2.5 ) reaction . as illustrated in figure 5(a ) , the skin histology of the control fish showed that normal - sized mucous cells ( mcs ) were present , with appropriate amounts of club cells ( ccs ) in the epidermal layer . exposure to 50 g / l of ag - nps ( figure 5(b ) ) increased the size and number of mucous cells marked with alcian blue by using the ab - pas ( ph 2.5 ) positive reaction . on increasing the level of exposure to 100 g / l of ag - nps , the amount of club cells in the epidermal layer significantly increased . the size of the mucous cells was also larger than those of the control fish ( figure 5(c ) ) . at 200 g / l of ag - nps , hyperplasia ( ) was frequently recorded in the epidermal layer of the exposed samples ( figure 5(d ) ) . carp have one pair of gills on each side of the body , like other teleosts . the gill raker is lined up at the front of the gill arch . as shown in figure 6(a ) , numerous gill lamellae are lined up along either side of the gill filament . the gill filament consists of a cartilaginous support , which is a multilayered epithelium . in comparison , after staining with h&e , vacuolated mucous cells appeared as blank circles or elliptical in shape but were marked with a blue color ( color index 313c ) as a result of the ab - pas ( ph 2.5 ) reaction . as shown in figure 6(b ) , exposure to 50 g / l of ag - nps caused the bifurcation of the filament ( black arrow ) . figure 6(c ) shows an increased number of mucosa cells ( black arrow ) in the gills of the fish exposed to 100 g / l of ag - nps . the exposure of the fish to 200 g / l of ag - nps caused hyperplasia ( ) of the epithelium in the lamella ; this finding was not observed in the control group ( figure 6(d ) ) . this phenomenon was also recorded at other concentrations of ag - nps , but it was most clearly observed at a concentration of 200 the liver of carp consists of numerous lobules , along with various bile ducts , capillaries , and the pancreas . hepatocytes from the control group were clearly observed to have a round polygon shape with a nucleus ( figure 7(a ) ) . exposure to 100 g / l of ag - nps caused extreme atrophy of the hepatocyte nucleus in the liver of the fish ( figure 7(b ) ) . fish exposed to 200 g / l of ag - nps showed atrophy of the hepatocyte nucleus and an accumulation of eosinophilic granules ( color index 2415c , black arrowhead ) in the liver ( figure 7(c ) ) . as shown in figure 8 , ag - nps penetrated the skin of common carp after 96 h of exposure , regardless of particle concentrations . ag - nps were present in every layer of the skin , including the epithelium ( figure 8(b ) ) , epidermis ( figure 8(a ) ) , and mitochondrion ( figure 8(c ) ) . the particles stayed either in the junctions of the cells , on the cell membrane , or inside the cell . penetration by a large number of ag - nps in the skin would result in damaging the cell junction , membrane , or other cell structures , depending on the size and concentration of the particles . the penetration of large agglomerations of particles into the skin ( as shown in figure 8(b ) ) may destroy the structure and connection between the skin layers , while small amounts of single particles ( as shown in figure 8(c ) ) may stick on the membrane of cells , causing them to malfunction . in this study , the penetration of ag - nps in the gills of common carp after exposure was evaluated . figure 9 shows the presence of nanoparticles in the gill structures , including the gill lamella , the nucleus of gill cells , and blood corpuscles . if the skin is the first defensive structure of the fish body , the gill is an important organ that allows gaseous transfer and filtration of foreign materials . ag - nps were detected in every part of the gill at varying levels of penetration , such as between blood corpuscles and inside cell nuclei . the effects of nanoparticles on cell function might be more severe because of deep penetration . the presence of nanoparticles in different parts of the gills indicated that common carp is significantly affected by exposure to ag - nps . the effects may be directly expressed by damage to the tissue structure or indirectly expressed through abnormal levels of other biochemical markers . in this study , we also investigated the penetration of ag - nps into other important organs of the fish , including the brain and liver . the results are shown in figure 10 , indicating that particles entered the brain and liver of common carp exposed to 200 g / l of ag - nps for 96 h. as shown in figure 10 , the particles were able to penetrate deep into the brain cells . this result indicated that the particles were carried through the body of the fish by the circulation of blood , subsequently accumulating in different important organs . liver is an important organ of the body that facilitates the detoxification of substances , whereas the brain controls the activity of the whole body . damage to these organs may result in the malfunction of fish activities and , more seriously , may cause fish mortality . the findings of this study are comparable to those of a previous report by park et al . [ 23 , 24 ] , who observed that titanium dioxide nanoparticles caused chronic inflammatory disease and oxidative stress by intratracheal instillation in mice . alteration of the level of genomic expression towards 100 g / l of ag - nps exposure for 96 h in the liver of the common carp was analyzed using the dna microarray method . in response to ag - np exposure , there was a 2-fold upregulation and downregulation of 502 genes and 1,850 genes , respectively . differentially expressed genes ( > 2-fold , p < 0.05 ) with defined functions are summarized in table 4 . among the upregulated genes , 12 genes , including that for the myc associate protein x ( max ) , were shown to be involved in cell apoptosis , proliferation , protein synthesis , and energy production ( table 4 ) . generally , max has been known to participate in cell death and cell proliferation , with the interaction of myc . among the downregulated genes , 11 genes , including that for glutathione peroxidase 4a ( gpx ) , gst , and the retinol binding protein ( rbp ) , were shown to play a role in oxidative stress response , cellular defense , cell migration , detoxification , and fibrinolysis ( table 4 ) . both gpx and gst have protective effects against oxidative stress ( caused by the reactive oxygen species ros ) , while rbp has a major function in retinol metabolism [ 26 , 27 ] . our findings indicate that ag - nps might cause biological malfunctions or dysregulation in cellular processes , including cell death , proliferation , and resistance to oxidative stress . ag - nps also contribute towards changing the gene expression of particular sets of genes , which might be considered as plausible biomarkers of ag - nps toxicity . because of its strong antibacterial activity , ag is regularly used in preservative and hygiene products . ag - nps , categorized at a nanoscale , are expected to possess many physical and chemical properties that are different from those of their bulk particles . however , the novel properties of these particles require study , particularly because of their potential effects when exposed to organisms , including human beings . one of the novel properties of nanoparticles in general ( ag - nps in particular ) is their minute diameter , in comparison with that of their bulky counterparts [ 28 , 29 ] . because of the ultrafine particle - size , ag - nps may penetrate deep into the organs and , therefore , alter normal metabolic and bodily functions . fortunately , naturally occurring nanoparticles usually form aggregations , agglomerations , or complexes with other substances , such as natural organic materials . these complexes and/or aggregations / agglomerations reduce the size advantage of nanoparticles , thus reducing their toxicity or other adverse effects on aquatic organisms . to evaluate the impact of other materials on the toxicity of nanoparticles , synthesized particles with coated materials stabilizers of nanoparticles include ligands , surfactants , and polymers with different functional groups , such as cooh and nh2 . however , the toxicity and adverse effects of ag - nps capped with natural organic substances remain unclear . in this study , we used citric acid as a capping agent to stabilize ag - nps in solution . as shown in table 1 , this stabilizer keeps the particles well separated , with a nominal size of 10~20 nm . in addition , citric acid is a common organic substance in the natural environment and our daily life ( e.g. , fruit juice ) . hence , developing an understanding of the mechanisms and effects of citrate - ag - nps would contribute towards evaluating the fate and impacts of ag - nps in environmental matrixes . the antioxidant enzyme system is responsible for the elimination of oxidative stress during the early stage of the body 's defensive mechanism . abnormal changes in enzymes reflect the level of oxidative stress encountered by the body . in the current study , higher levels of gst , cat , and sod activities were detected in the liver compared to the brain and gills . this is because the liver is the major detoxification organ of the body . in this study , after 48 h of exposure , gst and cat activities in the liver were observed to vary , but they generally declined at lower ag - np concentrations and recovered at higher ag - np concentrations . gst is known for its catalysis function in the reduction process of glutathione ( gsh ) , in which endogenous and xenobiotic chemicals are detoxified . the reduction of gst occurs as a result of the overutilization of existing enzymes to overcome oxidative stress caused by ag - nps , which causes an increase in gsh concentration in specific organs . in this study , the carp showed a significant reduction in gst activity in the gills after 48 h of exposure to 200 g / l of ag - nps , but recovered after 96 h of exposure ( figure 2 ) . oxidative stress increased with a reduction in gst concentration ; this may cause the development of toxic effects or even carcinogenic effects . however , higher ag - np concentrations may trigger the production of enzymes to counteract the severe effects of the particles . hence , an increase in gst concentration may be used as an indicator for the depletion of gsh in detoxification processes . the main function of cat is to catalyze the decomposition of h2o2 . in this study , the activity of cat detected in the liver was significantly higher than that in the brain and gills ( figure 3 ) . cat concentration in the liver also declined when the fish were exposed to lower ag - np concentrations and then recovered with an increase in ag - np concentrations . g / l of ag - nps , cat levels in all the examined organs ( i.e. , brain , liver , and gills ) were significantly lower than those in the control group , as well as for fish exposed to 100 g / l of ag - nps ( figure 3(b ) ) . this phenomenon was not observed during the 48 h test under the same concentration . these results indicate that the impact of ag - nps on the fish was more severe under longer periods of exposure . the results of cat and gst activities also indicated that when the fish were exposed to short periods ( 48 h ) of ag - nps at a concentration of < 100 thus , the depletion of this enzyme may indicate that the antioxidant defense system is overwhelmed . in the present study , sod activity in the brain and gills was not significantly different between the control and exposed fish . however , sod activity was significantly lower in the liver of fish exposed to 200 g / l of ag - nps compared to the liver of fish in the control group ( figure 4(a ) ) . it has been suggested that the antioxidant defense system of the liver is severely affected at this concentration , as illustrated by the reduction of other enzymes ( i.e. , cat and gst ) in the liver of fish exposed to lower ag - nps concentrations . however , after 96 h of exposure , sod activity returned to the control level ( figure 4(b ) ) . hence , we hypothesize that gst and cat were produced in sufficient quantities to counteract the oxidative stress caused by ag - nps . however , the correlation and combined effects of gst , cat , and sod , as well as other antioxidant enzymes , require further study . as shown in the results of the biochemical analysis , the concentrations of nh3 and got declined and recovered , similar to that observed for other antioxidant enzymes ( e.g. , cat and gst ) . it is known that nh3 in the blood is associated with hepatic detoxification ( i.e. , the urea cycle ) . therefore , fluctuations in blood nh3 concentrations may be related to the antioxidant defense system . g / l of ag - nps ; hence , changes in the enzyme system may alter the capability of urine transformation . however , other biochemical parameters were not significantly altered . along with slight fluctuations in antioxidant enzymes , this result indicates that < 200 g / l of ag - nps exposure did not have a severe effect on the fish . the histological analysis of the tissues also showed that ag - nps have potential adverse effects on exposed fish . as shown in figure 5 , the histopathology of the skin samples showed an increase in the number and size of mucous and club cells , as well as vacuolation of the epithelium . mucous and club cells located in the epidermal layer of the skin are responsible for the excretion of waste , respiration , ionic and osmotic regulation , disease resistance , communication , and other protection functions . an increase in the number and size of these cells may be a necessary response of the body to counteract the effects of exogenous chemicals , such as nanoparticles . however , if nanoparticle concentrations exceed the resistance level of the skin and excretory systems , they may have a lethal effect on cells . for example , when the fish were exposed to 200 g / l of ag - nps ( as shown in figure 5(d ) ) , vacuolation of the epidermal epithelium was observed . malfunctioning or injured skin may also cause nanoparticles to have a fatal effect on the fish . therefore , in this study , stresses caused by the exposure of common carp to ag - nps may have potentially toxic effects . in this study , however , chronic exposure to ag - nps may cause other types of damage that kill fish . one impact of ag - nps is the inhibition of the oxygen / carbon dioxide exchange process because of injury to the respiratory organ ( gills ) . as shown in figure 6 , the exposure of fish to ag - nps initially caused the bifurcation of the filament ( figure 6(b ) ) , an increase in the number and size of mucous cells ( figure 6(c ) ) , and hyperplasia of the lamellar epithelium ( figure 6(d ) ) . similar to the effects on the skin , these slight changes to the gills did have a severe effect on the fish , but it could result in potentially fatal effects if the carp were exposed to higher concentrations of ag - nps over an extended period . the adverse effects observed in the liver of the exposed carp ( figure 7 ) , such as atrophy of the hepatocyte nucleus and eosinophilic granule formation , may therefore be used as indicators . we used genomic analysis to evaluate changes in gene expression towards ag - np exposure in the liver of common carp , and we subsequently selected several biomarkers of ag - np toxicity . in our microarray data , however , the upregulation of max blocks apoptosis in endothelial cells and is involved in the maintenance of healthy morphology in the retinal ganglion cells of rats , indicating the presence of protective cellular functions . in addition , this observation might indicate that programmed cell death and cell morphology are mediated and orientated by max , leading to cellular defense against ag - np toxicity . in our microarray data , repression of rbp in the epidermis might cause a reduction in the amount of retinol in keratinocytes , indicating cornification of the epidermis . another study documented that the underexpression of rbp is associated with inhibiting differentiation and the formation of large tumors in hepatocellular carcinoma . these observations might indicate that ag - nps cause abnormal retinoid metabolism , resulting in tumorigenesis . in our microarray data , gst has been reported to play a critical role in the detoxification of various types of toxicants ( such as carcinogens ) and has a protective effect on ros - mediated dna damage [ 4145 ] . a recent assessment indicated that gst activity might be useful towards evaluating oxidative stress [ 46 , 47 ] . hence , ag - nps might induce the accumulation of intracellular oxidative stress , giving rise to abnormal biological functions of responsive components , including gst . gene expression level of gst obtained from microarray analysis was found to be downregulated while the gst activity was induced in presence of ag - nps , particularly after 96 h exposure to 100 g / l in common carp liver tissue . possible explanation is that the inhibitory gene expression at mrna level manifests at initial stage due to the ag - nps toxic effect whereas the induction of gst activity would play an important role as defensive mechanism in response to the ag - nps - induced oxidative stress at later stage . at present , while the functions of many common carp genes remain under investigation , the inferred functions of unknown genes might be used as putative bioindicators of ag - np toxicity . the results of the current study indicated that max , gst , and rbp might be viable candidates as ag - np biomarkers . however , detailed study of the toxicity - mediated molecular mechanisms of ag - nps and further functional analysis of common carp genes are necessary to elucidate cellular responses to ag - nps and the detoxification processes . citrate - capped ag nanoparticles dispersed well in freshwater , with an estimated average particle size of 721 nm ( tem ) and 7291 nm ( dls analysis ) . despite the formation of aggregations / agglomerations in the solution , nanosized particles of citrate - ag - nps were dominant . among the examined tissues , liver was the most susceptible to changes in particle concentration . the antioxidant enzyme system was mostly active in the liver . according to the analysis of the antioxidant enzyme system and other biochemical parameters , liver was the most severely affected organ when the carp were exposed to ag - nps . the activities of antioxidant enzymes , such as gst and cat , fluctuated with different ag - np concentrations , whereas sod activity remained stable . the histopathology showed the following : ( 1 ) in the skin : an increase in the number and size of club cells and mucous cells and vacuolation of the epithelium ; ( 2 ) in the gill : bifurcation of the filament , an increase in the number of mucous cells , and hyperplasia of the lamellar epithelium ; ( 3 ) in the liver : atrophy of the hepatocyte nucleus and accumulation of eosinophilic granules . the adverse effects recorded in the current study were not severe enough to cause carp mortality , but they might potentially lead to lethal effects if the fish were exposed to higher concentrations of ag - nps over a longer period .

juvenile common carp ( cyprinus carpio ) were used as a model to investigate acute toxicity and oxidative stress caused by silver nanoparticles ( ag - nps ) . the fish were exposed to different concentrations of ag - nps for 48 h and 96 h. after exposure , antioxidant enzyme levels were measured , including glutathione - s - transferase ( gst ) , superoxidase dismutase , and catalase ( cat ) . other biochemical parameters and histological abnormalities in different tissues ( i.e. , the liver , gills , and brain ) were also examined . the results showed that ag - nps agglomerated in freshwater used during the exposure experiments , with particle size remaining < 100 nm . ag - nps had no lethal effect on fish after 4 days of exposure . biochemical analysis showed that enzymatic activities in the brain of the fish exposed to 200 g / l of ag - nps were significantly reduced . varied antioxidant enzyme activity was recorded in the liver and gills . varied antioxidant enzyme activity was recorded for cat in the liver and gst in the gills of the fish . however , the recovery rate of fish exposed to 200 g / l of ag - nps was slower than when lower particle concentrations were used . other biochemical indices showed no significant difference , except for nh3 and blood urea nitrogen concentrations in fish exposed to 50 g / l of ag - nps . this study provides new evidence about the effects of nanoparticles on aquatic organisms .

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

during the recent rapid development of nanotechnology , silver nanoparticles ( ag - nps ) have been used for various types of consumer products , such as cosmetics , heath care products , and textiles . consequently , information about the toxicity and carcinogenic effects of ag is available , with it being considered nontoxic and noncarcinogenic when used in appropriate amounts . however , at relatively high concentrations , ag - nps cause cellular toxicity and other adverse impacts , such as the inhibition of mitochondrial function in rat liver cells , mouse germline stem cells , and human fibroblasts [ 1113 ] . furthermore , an increasing number of recent reports have provided evidence of the cytotoxicity of ag - nps at doses of low exposure [ 14 , 15 ] . ag - nps also have an adverse impact on aquatic organisms , such as zebrafish and medaka fish , and cause oxidative stress , cellular apoptosis , chromosomal aberrations , and other developmental toxicity effects during early life stages and adulthood . in this study , we investigated the effects of ag - nps in the presence of citric acid as a capping material . common carp ( cyprinus carpio ) is an abundant species in the freshwater environment . however , because of the dominance of this species in the natural environment , carp is considered as one of the most suitable models to assess the non - fatal effects of pollutants by evaluating fluctuations in antioxidant enzyme levels or other changes in fish histology and physiology . the objectives of this study were to investigate the adverse effects of ag - nps capped with citric acid ( ag - nps ) by measuring antioxidant enzymatic activities , including those of catalase ( cat ) , superoxide dismutase ( sod ) , and glutathione - s - transferase ( gst ) . target organs included the brain , liver , and gills of the common carp ( c. carpio ) . in addition , other biochemical parameters in the blood and histological changes in the skin , liver , and gills of the fish were examined . a stock solution of ag - nps was prepared at 10 mg / l . working solutions ( 25 , 50 , 100 , and 200 g / l ) were obtained by diluting the stock solution with respective amounts of di water . as shown in table 1 , the body weight and length of the fish were 18.6~33.8 g and 10.9~13.5 cm , respectively . fish were exposed to ag - nps for a period of 48 h and 96 h , according to the oecd test guideline 203 : fish acute toxicity test . the carp were exposed to ag - np concentrations of 25 , 50 , 100 , and 200 g / l . after exposure , the gills , liver , and brain of the fish were collected , frozen , and stored separately at 20c for further treatment . other biochemical parameters were examined in this study , namely , ammonia ( nh3 ) , glucose ( glu ) , total cholesterol ( tcho ) , alkaline phosphates ( alp ) , glutamic oxaloacetic transaminase / aspartate aminotransferase ( got / ast ) , alanine aminotransferase ( gpt / alt ) , -glutamyltransferase ( ggt ) , albumin ( alb ) , blood urea nitrogen ( bun ) , creatinine ( cre ) , and total bilirubin ( tbil ) . after exposure , the skin , gills , and liver of the carp were dissected and fixed in bouin 's solution for 12 h or overnight . the size distribution and morphology of ag - nps were examined using tem and dls . as shown in figure 1 , ag - nps became agglomerated in the solution , and the nominal size measured using tem was ~12 nm . the dls results showed that the mean diameter of ag - nps at 5 mg / l and 10 mg / l was ~70 nm and ~90 nm , respectively ( table 2 ) . in general , ag - nps were well dispersed in di water solution , with some agglomeration and aggregation . the antioxidant enzymes , including those of gst , cat , and sod , were measured , and the results are shown in figures 2 , 3 , and 4 . after 48 h of exposure , gst activities in the liver , gills , and brain were not significantly different than that observed when the fish were exposed to 25 g / l and 50 g / l of ag - nps ( figure 2(a ) ) . when the concentration of ag - nps increased to 100 g / l and 200 g / l , gst activity was more significantly reduced in the gills than in the liver and brain . after 96 h of exposure , gst activity was elevated in the liver ; this was correlated with an increase in ag - np concentration in the media ( figure 2(b ) ) . gst activity in liver samples exposed to 100 g / l and 200 g / l was about 190% higher than that observed in the control fish . in comparison , gst activity in the liver of fish after 96 h of exposure was also higher than that in fish subjected to 48 h of exposure , especially at 100 g / l of ag - nps . after 96 h of exposure , gst activity was also higher in the liver than in the brain and gills . as shown in figure 3 , higher levels of cat activity were detected in the liver than in the brain and gills of the experimental fish . similar to gst activity , cat activity in the liver changed at different ag - np concentrations , but it remained relatively stable in the brain and gills . cat concentrations in the liver of the fish after 48 h of exposure showed no significant difference to that in the control group ( figure 3(a ) ) . however , cat activity declined at lower ag - np concentrations ( i.e. , 25 g / l and 50 g / l ) and increased at higher ag - np concentrations ( i.e. however , after 96 h of exposure , cat activity was significantly reduced in the liver at 25 g / l and 50 g / l , recovered at 100 g / l , and then decreased again at 200 sod activity differed from that of gst and cat in that it was relatively stable in all examined tissues ( figure 4 ) . however , similar to gst and cat , higher levels of sod activity were detected in the liver than in the brain and gills . in this study , the biochemical parameters in the blood of the exposed fish were also analyzed to understand the changes in metabolism and fish response to the stressor ( i.e. detailed information was published in our previous study , where the concentration of biochemical parameters such as ammonia ( nh3 ) , glutamic oxaloacetic transaminase ( got ) , and total cholesterol ( tcho ) declined at the beginning of exposure and gradually recovered with increase in ag - np concentration . other parameters such as alanine aminotransferase ( gpt ) , -glutamyl transferase ( ggt ) , albumin ( alb ) , blood urea nitrogen ( bun ) , creatinine ( cre ) , and total bilirubin ( tbil ) remained stable or fluctuated around the levels of the control groups , without any significant difference . after reaching the highest concentration when exposed to 50 g / l of ag - nps , alp concentration began to decline when the fish were exposed to 100 /l and 200 the histological structure of the skin ( figure 5 ) is composed of an epidermal layer and a dermal layer . as illustrated in figure 5(a ) , the skin histology of the control fish showed that normal - sized mucous cells ( mcs ) were present , with appropriate amounts of club cells ( ccs ) in the epidermal layer . exposure to 50 g / l of ag - nps ( figure 5(b ) ) increased the size and number of mucous cells marked with alcian blue by using the ab - pas ( ph 2.5 ) positive reaction . on increasing the level of exposure to 100 g / l of ag - nps , the amount of club cells in the epidermal layer significantly increased . at 200 g / l of ag - nps , hyperplasia ( ) was frequently recorded in the epidermal layer of the exposed samples ( figure 5(d ) ) . as shown in figure 6(b ) , exposure to 50 g / l of ag - nps caused the bifurcation of the filament ( black arrow ) . figure 6(c ) shows an increased number of mucosa cells ( black arrow ) in the gills of the fish exposed to 100 g / l of ag - nps . the exposure of the fish to 200 g / l of ag - nps caused hyperplasia ( ) of the epithelium in the lamella ; this finding was not observed in the control group ( figure 6(d ) ) . this phenomenon was also recorded at other concentrations of ag - nps , but it was most clearly observed at a concentration of 200 the liver of carp consists of numerous lobules , along with various bile ducts , capillaries , and the pancreas . exposure to 100 g / l of ag - nps caused extreme atrophy of the hepatocyte nucleus in the liver of the fish ( figure 7(b ) ) . fish exposed to 200 g / l of ag - nps showed atrophy of the hepatocyte nucleus and an accumulation of eosinophilic granules ( color index 2415c , black arrowhead ) in the liver ( figure 7(c ) ) . as shown in figure 8 , ag - nps penetrated the skin of common carp after 96 h of exposure , regardless of particle concentrations . ag - nps were present in every layer of the skin , including the epithelium ( figure 8(b ) ) , epidermis ( figure 8(a ) ) , and mitochondrion ( figure 8(c ) ) . penetration by a large number of ag - nps in the skin would result in damaging the cell junction , membrane , or other cell structures , depending on the size and concentration of the particles . in this study , the penetration of ag - nps in the gills of common carp after exposure was evaluated . figure 9 shows the presence of nanoparticles in the gill structures , including the gill lamella , the nucleus of gill cells , and blood corpuscles . the effects of nanoparticles on cell function might be more severe because of deep penetration . the presence of nanoparticles in different parts of the gills indicated that common carp is significantly affected by exposure to ag - nps . in this study , we also investigated the penetration of ag - nps into other important organs of the fish , including the brain and liver . the results are shown in figure 10 , indicating that particles entered the brain and liver of common carp exposed to 200 g / l of ag - nps for 96 h. as shown in figure 10 , the particles were able to penetrate deep into the brain cells . alteration of the level of genomic expression towards 100 g / l of ag - nps exposure for 96 h in the liver of the common carp was analyzed using the dna microarray method . among the downregulated genes , 11 genes , including that for glutathione peroxidase 4a ( gpx ) , gst , and the retinol binding protein ( rbp ) , were shown to play a role in oxidative stress response , cellular defense , cell migration , detoxification , and fibrinolysis ( table 4 ) . both gpx and gst have protective effects against oxidative stress ( caused by the reactive oxygen species ros ) , while rbp has a major function in retinol metabolism [ 26 , 27 ] . our findings indicate that ag - nps might cause biological malfunctions or dysregulation in cellular processes , including cell death , proliferation , and resistance to oxidative stress . however , the novel properties of these particles require study , particularly because of their potential effects when exposed to organisms , including human beings . one of the novel properties of nanoparticles in general ( ag - nps in particular ) is their minute diameter , in comparison with that of their bulky counterparts [ 28 , 29 ] . however , the toxicity and adverse effects of ag - nps capped with natural organic substances remain unclear . in this study , we used citric acid as a capping agent to stabilize ag - nps in solution . hence , developing an understanding of the mechanisms and effects of citrate - ag - nps would contribute towards evaluating the fate and impacts of ag - nps in environmental matrixes . in the current study , higher levels of gst , cat , and sod activities were detected in the liver compared to the brain and gills . in this study , after 48 h of exposure , gst and cat activities in the liver were observed to vary , but they generally declined at lower ag - np concentrations and recovered at higher ag - np concentrations . the reduction of gst occurs as a result of the overutilization of existing enzymes to overcome oxidative stress caused by ag - nps , which causes an increase in gsh concentration in specific organs . in this study , the carp showed a significant reduction in gst activity in the gills after 48 h of exposure to 200 g / l of ag - nps , but recovered after 96 h of exposure ( figure 2 ) . however , higher ag - np concentrations may trigger the production of enzymes to counteract the severe effects of the particles . in this study , the activity of cat detected in the liver was significantly higher than that in the brain and gills ( figure 3 ) . cat concentration in the liver also declined when the fish were exposed to lower ag - np concentrations and then recovered with an increase in ag - np concentrations . g / l of ag - nps , cat levels in all the examined organs ( i.e. , brain , liver , and gills ) were significantly lower than those in the control group , as well as for fish exposed to 100 g / l of ag - nps ( figure 3(b ) ) . these results indicate that the impact of ag - nps on the fish was more severe under longer periods of exposure . the results of cat and gst activities also indicated that when the fish were exposed to short periods ( 48 h ) of ag - nps at a concentration of < 100 thus , the depletion of this enzyme may indicate that the antioxidant defense system is overwhelmed . however , sod activity was significantly lower in the liver of fish exposed to 200 g / l of ag - nps compared to the liver of fish in the control group ( figure 4(a ) ) . it has been suggested that the antioxidant defense system of the liver is severely affected at this concentration , as illustrated by the reduction of other enzymes ( i.e. , cat and gst ) in the liver of fish exposed to lower ag - nps concentrations . however , after 96 h of exposure , sod activity returned to the control level ( figure 4(b ) ) . hence , we hypothesize that gst and cat were produced in sufficient quantities to counteract the oxidative stress caused by ag - nps . however , the correlation and combined effects of gst , cat , and sod , as well as other antioxidant enzymes , require further study . as shown in the results of the biochemical analysis , the concentrations of nh3 and got declined and recovered , similar to that observed for other antioxidant enzymes ( e.g. g / l of ag - nps ; hence , changes in the enzyme system may alter the capability of urine transformation . along with slight fluctuations in antioxidant enzymes , this result indicates that < 200 g / l of ag - nps exposure did not have a severe effect on the fish . the histological analysis of the tissues also showed that ag - nps have potential adverse effects on exposed fish . an increase in the number and size of these cells may be a necessary response of the body to counteract the effects of exogenous chemicals , such as nanoparticles . however , if nanoparticle concentrations exceed the resistance level of the skin and excretory systems , they may have a lethal effect on cells . for example , when the fish were exposed to 200 g / l of ag - nps ( as shown in figure 5(d ) ) , vacuolation of the epidermal epithelium was observed . therefore , in this study , stresses caused by the exposure of common carp to ag - nps may have potentially toxic effects . in this study , however , chronic exposure to ag - nps may cause other types of damage that kill fish . one impact of ag - nps is the inhibition of the oxygen / carbon dioxide exchange process because of injury to the respiratory organ ( gills ) . as shown in figure 6 , the exposure of fish to ag - nps initially caused the bifurcation of the filament ( figure 6(b ) ) , an increase in the number and size of mucous cells ( figure 6(c ) ) , and hyperplasia of the lamellar epithelium ( figure 6(d ) ) . similar to the effects on the skin , these slight changes to the gills did have a severe effect on the fish , but it could result in potentially fatal effects if the carp were exposed to higher concentrations of ag - nps over an extended period . the adverse effects observed in the liver of the exposed carp ( figure 7 ) , such as atrophy of the hepatocyte nucleus and eosinophilic granule formation , may therefore be used as indicators . we used genomic analysis to evaluate changes in gene expression towards ag - np exposure in the liver of common carp , and we subsequently selected several biomarkers of ag - np toxicity . gene expression level of gst obtained from microarray analysis was found to be downregulated while the gst activity was induced in presence of ag - nps , particularly after 96 h exposure to 100 g / l in common carp liver tissue . at present , while the functions of many common carp genes remain under investigation , the inferred functions of unknown genes might be used as putative bioindicators of ag - np toxicity . the results of the current study indicated that max , gst , and rbp might be viable candidates as ag - np biomarkers . however , detailed study of the toxicity - mediated molecular mechanisms of ag - nps and further functional analysis of common carp genes are necessary to elucidate cellular responses to ag - nps and the detoxification processes . despite the formation of aggregations / agglomerations in the solution , nanosized particles of citrate - ag - nps were dominant . according to the analysis of the antioxidant enzyme system and other biochemical parameters , liver was the most severely affected organ when the carp were exposed to ag - nps . the histopathology showed the following : ( 1 ) in the skin : an increase in the number and size of club cells and mucous cells and vacuolation of the epithelium ; ( 2 ) in the gill : bifurcation of the filament , an increase in the number of mucous cells , and hyperplasia of the lamellar epithelium ; ( 3 ) in the liver : atrophy of the hepatocyte nucleus and accumulation of eosinophilic granules . the adverse effects recorded in the current study were not severe enough to cause carp mortality , but they might potentially lead to lethal effects if the fish were exposed to higher concentrations of ag - nps over a longer period .

matrix metalloproteinases ( mmps ) are zinc - dependent proteases that are involved not only in the degradation and remodeling of the extracellular matrix but also in processing bioactive molecules , including cell surface receptors , neurotrophic factors , chemokines , and cytokines ( kim and joh , 2012 ) . mmps are indispensable enzymes for various physiological processes such as embryogenesis , morphogenesis , tissue reconstruction , and regulation of inflammatory processes under a variety of conditions ( nissinen and kahari , 2014 ) . mmps belong to a family of more than 25 enzymes , and each has their own activating cascade with mmp-2 and mmp-9 at the core . among mmps , gelatinases , including mmp-2 and mmp-9 , are similar in structure and are able to enzymatically process various substrates of the extracellular matrix , such as gelatin , collagen i and collagen iv ( sbardella et al . , 2012 ) . constitutively expressed in most cell types , mmp-2 has a prohomeostatic property , whereas mmp-9 is expressed at low levels in most cell types but is highly responsive to stimulation and has proinflammatory properties ( van den steen et al . , 2002 ; these enzymes are also known to be related to pathological processes such as cancer , neurodegenerative disorders , arthritis , and cardiovascular diseases ( sbardella et al . , 2012 ) . in the central nervous system , mmps play important roles in synaptogenesis , synaptic plasticity , and long - term potentiation ( ethell and ethell , 2007 ) . in contrast , there are limited studies of the role of mmps in psychiatric disorders . the mmps are endogenously inhibited by the tissue inhibitors of mmps ( timps ) . in humans , the c - terminal domain of timp-1 and timp-2 binds to the hemopexin of the proenzymes of mmp-9 and mmp-2 , respectively ( carlo , 2014 ) . this indicates that timp-1 and timp-2 are major individual inhibitors of mmp-9 and mmp-2 ( goldberg et al . , 1992 ) . mood disorders ( mds ) have been recognized as having a strong association with chronic inflammatory states ( rosenblat et al . , 2014 ) . in depression , fatigue and somatic symptoms such as gastrointestinal symptoms , autonomic symptoms , and flu - like malaise are significantly associated with signs of an activated inflammatory pathway ( maes et al . , 2012 ) . several studies have reported an association between depressive symptoms and state - dependent elevated levels of inflammatory markers , such as interleukin-1 beta , interleuki-6 , and tumor necrosis factor - alpha ( felger and lotrich , 2013 ; rosenblat et al . , 2014 ) . mmps process cytokines , which leads to alterations of their activity ( van lint and libert , 2007 ) . at the same time , the expression of mmps , mmp-9 in particular , is regulated by cytokines ( harkness et al . , 2000 ) . the current study sought to find an association between md and inflammation - related markers . the main mmps , mmp-2 and mmp-9 , and their inhibitors , timp-2 and timp-1 , were quantified in patients with md . first , serum levels of mmp-2 , mmp-9 , timp-2 , and timp-1 from patients with md who had depressive episodes and were eligible for electroconvulsive therapy ( ect ) were compared with healthy controls and patients with schizophrenia - spectrum disorders ( scz ) , who also had psychotic episodes and were also eligible for ect . serum levels of mmp and timp were then compared before and after a course of ect in patients with md and scz . finally , an association between serum levels of mmps and timps with clinical symptoms in patients with md and scz was determined . this study was conducted at the department of psychiatry of the national hospital organization kure medical center between january 2011 and april 2013 . thirty - four patients ( 12 men and 22 women , mean age sd=54.116.1 ) were recruited among inpatients planning to receive ect based on the guidelines of the american psychiatric association ( 2001 ) . ect is often proscribed when a patient exhibits episodes of severe major depression , psychosis , and catatonia or has shown insufficient improvement with prescribed pharmacotherapy treatment ( american psychiatric association , 2001 ) . twenty - one patients were diagnosed as having md , either major depressive disorders ( n=11 ) or bipolar disorders ( n=10 ) , with a current episode of major depression . thirteen patients were diagnosed with scz , either schizophrenia ( n=10 ) or schizoaffective disorder ( n=3 ) , with symptoms currently exacerbated to psychosis , such as delusions , and catatonia . various types of schizophrenia were diagnosed , including catatonic ( n=5 ) , paranoid ( n=4 ) , and undifferentiated ( n=1 ) . patients with a past or present history of substance abuse , substance dependence , significant neurological illness , or any other significant medical illness were excluded from participation in the current study . a clinical psychiatrist recommended ect to each patient according to standards recommended by the american psychiatric association task force and based on the patient s urgency and severity of illness . forty healthy subjects ( 14 men and 26 women , mean age 54.213.9 years ) , with no history of past or current mental disorders were recruited as a control group . after procedures were fully explained , written informed consent was obtained from all subjects to participate in the study . the current study was approved by the ethics committee of national hospital organization kure medical center . ect was performed according to procedures from a previous report ( shibasaki et al . , 2015 ) . before undergoing ect , each patient was screened for general health through a physical and neurological examination , blood and urine tests , electrocardiogram , chest x - ray film , and a cerebral computed tomography scan . thiamylal sodium ( 23mg / kg , i.v . ) and suxamethonium chloride ( 0.51mg / kg , i.v . ) . the ect device used was the thymatron system iv brief pulse square wave apparatus ( somatics inc ) . only one adequate seizure was required for each session , which was defined as an electroencephalographic seizure lasting more than 25 seconds with a high - amplitude , slow wave , and postictal suppression . the initial stimulus dose was determined using the half - age method ( petrides and fink , 1996 ) . if an adequate electroencephalographic seizure occurred in one session , the stimulus energy of the next session remained the same . when a missed or inadequate seizure occurred , the patient was restimulated after a 20- to 30-second pause and the stimulus increased 1.5- to 2.0-fold . if any adverse effects ( eg , cognitive dysfunction , delirium ) occurred , the frequency of the ect schedule was reduced to once or twice per week . ect continued until the patient was asymptomatic or the attending psychiatrist determined that the patient had obtained the maximum benefit within 3 to 15 sessions . after the purpose and the ect procedure were described in detail , written informed consent was obtained from patients or caregivers of patients prior to initiating ect . most of patients with md ( 20/21 patients ) received antidepressant pharmacotherapy before ect : duloxetine ( 2060mg / d ; n=8 ) , mirtazapine ( 1545mg / d ; n=8 ) , paroxetine ( 2040mg / d ; n=4 ) , sertraline ( 25100mg / d ; n=2 ) , mianserin ( 1060mg / d ; n=5 ) , trazodone ( 2575mg / d ; n=3 ) , imipramine ( 50mg / d ; n=1 ) , or nortriptyline ( 100mg / d ; n=2 ) . nine subjects were on a stable dose of antidepressant during ect , 8 subjects had antidepressant dosage decreased during the course of ect , and 3 subjects had antidepressant dosage increased during the course of ect . eleven of 21 patients with md also took an antipsychotic medication ( either olanzapine or quetiapine ) before the course of ect to control either psychomotor agitation or psychotic symptoms . eleven of 13 patients with scz received antipsychotic pharmacotherapy before the course of ect : risperidone ( 1.512mg / d ; n=7 ) , olanzapine ( 1020mg / d ; n=4 ) , quetiapine ( 300675mg / d ; n=4 ) , zotepine ( 150400mg / d ; n=3 ) , blonanserin ( 1224mg / d ; n=2 ) , aripiprazole ( 24mg / d ; n=1 ) , haloperidol ( 30mg / d ; n=1 ) , chlorpromazine ( 450mg / d ; n=1 ) , or i.v . 3 subjects were on a stable dose of antipsychotics , 7 subjects had antipsychotics dosage decreased , and 3 subjects had antipsychotics dosage increased . clinical symptomatic scores were assessed using the 17-item ( hamilton rating score for depression [ hamd ] ) for patients with md and the brief psychiatric rating scale ( bprs ) for patients with scz . hamd 17-items were assigned to the following 5 subscales : core symptom ( items 1 , 2 , 7 , 8 , 10 , 13 ) ; sleep ( items 4 , 5 , 6 ) ; activity ( items 7 , 8) ; psychic anxiety ( items 9 , 10 ) ; and somatic anxiety ( items 11 , 12 , 13 ) in accordance with a previous report ( seretti et al . each patient s symptoms were assessed prior to the first ect session ( baseline , pre - ect ) and a day after the last ect session ( post - ect ) by the same psychiatrist . responders to ect were defined as demonstrating a 50% decrease in hrsd score in md patients or a 30% decrease in bprs score in scz patients . venous blood samples were taken in the morning ( between 7:00 and 8:00 am ) at pre - ect and post - ect . they were kept at room temperature for 1 hour , and serum was separated by centrifugation at 3000rpm for 15 minutes at 4c . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 were measured using elisa ( quantikine elisa , r&d systems , minneapolis , mn ) according to the manufacturer s instructions . the data are shown as the mean sd . the normal distribution of data was tested by the shapiro - wilk and the kolmogorov - smirnov tests , and statistical analysis was performed by nonparametric tests . a kruskal - wallis test was used to evaluate the significant difference in the parameters ( clinical and laboratory values ) among the 3 groups ( md , scz , and control ) . significant differences in the parameters between the 2 groups of patients with md and scz were evaluated using the mann - whitney u - test . a wilcoxon signed - rank test was used to evaluate the statistical differences in the parameters between pre - ect and post - ect . the receiver operating characteristic ( roc ) curves for the assessment of specificity and sensitivity of mmps and timps were calculated to discriminate between the md group and the control group . the areas under the roc curve ( auc ) were calculated for each mmps and timps as well . statistical significance after logistic regression between the 2 aucs statistical analysis was conducted by logistic regression , analyzing diagnosis value of single and combined detection of mmps . analyses were performed using spss version 22.0 for windows ( ibm japan corporation , tokyo , japan ) . this study was conducted at the department of psychiatry of the national hospital organization kure medical center between january 2011 and april 2013 . thirty - four patients ( 12 men and 22 women , mean age sd=54.116.1 ) were recruited among inpatients planning to receive ect based on the guidelines of the american psychiatric association ( 2001 ) . ect is often proscribed when a patient exhibits episodes of severe major depression , psychosis , and catatonia or has shown insufficient improvement with prescribed pharmacotherapy treatment ( american psychiatric association , 2001 ) . twenty - one patients were diagnosed as having md , either major depressive disorders ( n=11 ) or bipolar disorders ( n=10 ) , with a current episode of major depression . thirteen patients were diagnosed with scz , either schizophrenia ( n=10 ) or schizoaffective disorder ( n=3 ) , with symptoms currently exacerbated to psychosis , such as delusions , and catatonia . various types of schizophrenia were diagnosed , including catatonic ( n=5 ) , paranoid ( n=4 ) , and undifferentiated ( n=1 ) . patients with a past or present history of substance abuse , substance dependence , significant neurological illness , or any other significant medical illness were excluded from participation in the current study . a clinical psychiatrist recommended ect to each patient according to standards recommended by the american psychiatric association task force and based on the patient s urgency and severity of illness . forty healthy subjects ( 14 men and 26 women , mean age 54.213.9 years ) , with no history of past or current mental disorders were recruited as a control group . after procedures were fully explained , written informed consent was obtained from all subjects to participate in the study . the current study was approved by the ethics committee of national hospital organization kure medical center . ect was performed according to procedures from a previous report ( shibasaki et al . , 2015 ) . before undergoing ect , each patient was screened for general health through a physical and neurological examination , blood and urine tests , electrocardiogram , chest x - ray film , and a cerebral computed tomography scan . thiamylal sodium ( 23mg / kg , i.v . ) and suxamethonium chloride ( 0.51mg / kg , i.v . ) . the ect device used was the thymatron system iv brief pulse square wave apparatus ( somatics inc ) . only one adequate seizure was required for each session , which was defined as an electroencephalographic seizure lasting more than 25 seconds with a high - amplitude , slow wave , and postictal suppression . the initial stimulus dose was determined using the half - age method ( petrides and fink , 1996 ) . if an adequate electroencephalographic seizure occurred in one session , the stimulus energy of the next session remained the same . when a missed or inadequate seizure occurred , the patient was restimulated after a 20- to 30-second pause and the stimulus increased 1.5- to 2.0-fold . if any adverse effects ( eg , cognitive dysfunction , delirium ) occurred , the frequency of the ect schedule was reduced to once or twice per week . ect continued until the patient was asymptomatic or the attending psychiatrist determined that the patient had obtained the maximum benefit within 3 to 15 sessions . after the purpose and the ect procedure were described in detail , written informed consent was obtained from patients or caregivers of patients prior to initiating ect . most of patients with md ( 20/21 patients ) received antidepressant pharmacotherapy before ect : duloxetine ( 2060mg / d ; n=8 ) , mirtazapine ( 1545mg / d ; n=8 ) , paroxetine ( 2040mg / d ; n=4 ) , sertraline ( 25100mg / d ; n=2 ) , mianserin ( 1060mg / d ; n=5 ) , trazodone ( 2575mg / d ; n=3 ) , imipramine ( 50mg / d ; n=1 ) , or nortriptyline ( 100mg / d ; n=2 ) . nine subjects were on a stable dose of antidepressant during ect , 8 subjects had antidepressant dosage decreased during the course of ect , and 3 subjects had antidepressant dosage increased during the course of ect . eleven of 21 patients with md also took an antipsychotic medication ( either olanzapine or quetiapine ) before the course of ect to control either psychomotor agitation or psychotic symptoms . eleven of 13 patients with scz received antipsychotic pharmacotherapy before the course of ect : risperidone ( 1.512mg / d ; n=7 ) , olanzapine ( 1020mg / d ; n=4 ) , quetiapine ( 300675mg / d ; n=4 ) , zotepine ( 150400mg / d ; n=3 ) , blonanserin ( 1224mg / d ; n=2 ) , aripiprazole ( 24mg / d ; n=1 ) , haloperidol ( 30mg / d ; n=1 ) , chlorpromazine ( 450mg / d ; n=1 ) , or i.v . 3 subjects were on a stable dose of antipsychotics , 7 subjects had antipsychotics dosage decreased , and 3 subjects had antipsychotics dosage increased . clinical symptomatic scores were assessed using the 17-item ( hamilton rating score for depression [ hamd ] ) for patients with md and the brief psychiatric rating scale ( bprs ) for patients with scz . hamd 17-items were assigned to the following 5 subscales : core symptom ( items 1 , 2 , 7 , 8 , 10 , 13 ) ; sleep ( items 4 , 5 , 6 ) ; activity ( items 7 , 8) ; psychic anxiety ( items 9 , 10 ) ; and somatic anxiety ( items 11 , 12 , 13 ) in accordance with a previous report ( seretti et al . , 1999 ) . each patient s symptoms were assessed prior to the first ect session ( baseline , pre - ect ) and a day after the last ect session ( post - ect ) by the same psychiatrist . responders to ect were defined as demonstrating a 50% decrease in hrsd score in md patients or a 30% decrease in bprs score in scz patients . venous blood samples were taken in the morning ( between 7:00 and 8:00 am ) at pre - ect and post - ect . they were kept at room temperature for 1 hour , and serum was separated by centrifugation at 3000rpm for 15 minutes at 4c . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 were measured using elisa ( quantikine elisa , r&d systems , minneapolis , mn ) according to the manufacturer s instructions . the data are shown as the mean sd . the normal distribution of data was tested by the shapiro - wilk and the kolmogorov - smirnov tests , and statistical analysis was performed by nonparametric tests . a kruskal - wallis test was used to evaluate the significant difference in the parameters ( clinical and laboratory values ) among the 3 groups ( md , scz , and control ) . significant differences in the parameters between the 2 groups of patients with md and scz were evaluated using the mann - whitney u - test . a wilcoxon signed - rank test was used to evaluate the statistical differences in the parameters between pre - ect and post - ect . the receiver operating characteristic ( roc ) curves for the assessment of specificity and sensitivity of mmps and timps were calculated to discriminate between the md group and the control group . the areas under the roc curve ( auc ) were calculated for each mmps and timps as well . statistical significance after logistic regression between the 2 aucs was determined . statistical analysis was conducted by logistic regression , analyzing diagnosis value of single and combined detection of mmps . analyses were performed using spss version 22.0 for windows ( ibm japan corporation , tokyo , japan ) . the clinical data of the 3 groups ( md , scz , and control ) are presented in table 1 . patients with md were significantly older and their duration of illness was shorter compared with the scz group . duration of current episode , number of ects , and duration of the ect course did not differ between the md and scz groups . there were no differences between the dose equivalence of imipramine of pre - ect and post - ect in the md group and also no differences between the dose equivalence of chlorpromazine of pre - ect and post - ect in the scz group . subject clinical data abbreviations : bprs , brief psychiatric rating scale ; cpz , chlorpromazine ; ect , electroconvulsive therapy ; hamd , hamilton rating score for depression ; imi , imipramine ; md , mood disorder ; scz : schizophrenia - spectrum disorder . comparison between scores at pre - ect and those at post - ect by wilcoxon signed - rank test . serum levels of mmp-2 in the md group with depressive episodes at pre - ect were significantly lower than those of the control group ( figure 1a ; p=.023 ) , whereas there were no significant differences for the serum levels of timp-2 , mmp-9 , and timp-1 between the md group and the control group ( figure 1b - d ) . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 in the md group at pre - ect were not significant different compared with those of the scz group with psychotic episodes at pre - ect . additionally , serum levels of mmps and timps were not significantly different between md patients with either md ( unipolar ) or bipolar disorder ( bipolar ) at pre - ect ( mmp-2 , unipolar=165.925.0ng / ml vs bipolar=159.426.8ng / ml , p=.512 ; timp-2 , 63.17.6ng / ml vs 66.66.2ng / ml , p=.259 ; mmp-9 , 553.9218.8ng / ml vs 604.6310.5ng / ml , p=.668 ; timp-1 , 150.539.4ng / ml vs 148.023.2ng / ml , p=.571 ) . scatter plot of serum levels of matrix metalloproteinases ( mmps ) and tissue inhibitors of mmps ( timps ) . mmp-2 ( a ) , timp-2 ( b ) , mmp-9 ( c ) , and timp-1 ( d ) in controls ( ) , mood disorders ( md ) group before a course of ect ( pre - ect , ) and after a course of ect ( post - ect , ) , and schizophrenia - spectrum disorders ( scz ) group pre - ect ( ) and post - ect ( ) . the horizontal bars represent the mean values . in the roc curve analysis of diagnosis , the auc of mmp-2 , mmp-9 , timp-1 , and timp-2 at pre - ect between the md and control groups were 0.738 , 0.603 , 0.674 , and 0.675 , respectively . the scores for depressive symptoms evaluated by hamd in the md group and the scores of psychotic symptoms evaluated by bprs in the scz group significantly decreased following ect ( table 1 ) . both nonresponding scz patients were diagnosed with paranoid - type scz . there was a statistically significant increase in serum levels of mmp-2 in the md group over the course of ect ( p=.046 ) but no significant change observed in the scz group ( p=.650 ) ( figure 1a ) . there were statistically significant decreases in serum levels of mmp-9 in both the md and scz groups following ect ( md group , p=.003 ; scz group , p=.019 ) ( figure 1c ) . among the 2 nonresponders to ect in the scz group , one patient had an increase in serum level of mmp-9 and the other had a decrease in serum level of mmp-9 after the course of ect ( data not shown ) . serum levels of timp-2 and timp-1 did not significantly change over the course of ect in both the md and scz groups ( figure 1b , d ) . no significant correlations were observed in the md group between subject characteristics ( gender , age , age of onset , duration of illness , duration of current episode , or dose of medication ) and serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 at either pre - ect or post - ect ( data not shown ) . correlation coefficients were calculated between serum levels of mmps and timps and clinical symptomatic scores evaluated by hamd for md and by bprs for scz , combining results from pre - ect and post - ect ( table 2 ) . in the scz group , there was no significant correlation between serum levels of mmps , timps , and total bprs scores ( table 2 ) . in the md group , serum levels of timp-1 and timp-2 did not correlate with total hamd score ( table 2 ) . furthermore , no significant correlations were observed between timps and subscale hamd scores ( table 3 ) . correlation between mmp and timp serum levels and clinical symptomatic scores of md and scz groups abbreviations : bprs : brief psychiatric rating scale ; hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; scz , schizophrenia - spectrum disorder ; timp , tissue inhibitor of mmp . correlation between mmp and timp serum levels and subscale hamd scores in md groups abbreviations : hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; timp , tissue inhibitor of mmp . however , in the md group , there was a significant negative correlation between serum levels of mmp-2 and total hamd score ( p=.041 ) ( table 2 ; figure 2a ) and a significant positive correlation between serum levels of mmp-9 and total hamd score ( p=.006 ) ( table 2 ; figure 2b ) . furthermore , negative correlations between serum levels of mmp-2 and subscale hamd scores ( core symptom , activity , somatic anxiety ) were observed as well as positive correlations between serum levels of mmp-9 and the same subscale hamd scores ( core symptom , activity , psychic anxiety , somatic anxiety ) as observed with mmp-2 ( table 3 ) . a significant negative correlation between serum levels of mmp-2 and mmp-9 was observed in the md group ( p=.001 , rho=-0.479 ) , but not in the scz group ( p=.368 , rho=-0.184 ) or in the control group ( p=.592 , rho=0.087 ) ( figure 2c ) . correlation between serum levels of matrix metalloproteinases ( mmps ) and total hamilton rating scale for depression ( hamd ) score in patients with mood disorders ( md ) before and after a course of ect . ( a ) significant negative correlation between serum levels of mmp-2 and total hamd score . ( b ) pre - ect values ( ) and post - ect values ( ) ; n=21 , * p < .05 the clinical data of the 3 groups ( md , scz , and control ) are presented in table 1 . patients with md were significantly older and their duration of illness was shorter compared with the scz group . duration of current episode , number of ects , and duration of the ect course did not differ between the md and scz groups . there were no differences between the dose equivalence of imipramine of pre - ect and post - ect in the md group and also no differences between the dose equivalence of chlorpromazine of pre - ect and post - ect in the scz group . subject clinical data abbreviations : bprs , brief psychiatric rating scale ; cpz , chlorpromazine ; ect , electroconvulsive therapy ; hamd , hamilton rating score for depression ; imi , imipramine ; md , mood disorder ; scz : schizophrenia - spectrum disorder . comparison between scores at pre - ect and those at post - ect by wilcoxon signed - rank test . serum levels of mmp-2 in the md group with depressive episodes at pre - ect were significantly lower than those of the control group ( figure 1a ; p=.023 ) , whereas there were no significant differences for the serum levels of timp-2 , mmp-9 , and timp-1 between the md group and the control group ( figure 1b - d ) . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 in the md group at pre - ect were not significant different compared with those of the scz group with psychotic episodes at pre - ect . additionally , serum levels of mmps and timps were not significantly different between md patients with either md ( unipolar ) or bipolar disorder ( bipolar ) at pre - ect ( mmp-2 , unipolar=165.925.0ng / ml vs bipolar=159.426.8ng / ml , p=.512 ; timp-2 , 63.17.6ng / ml vs 66.66.2ng / ml , p=.259 ; mmp-9 , 553.9218.8ng / ml vs 604.6310.5ng / ml , p=.668 ; timp-1 , 150.539.4ng / ml vs 148.023.2ng / ml , p=.571 ) . scatter plot of serum levels of matrix metalloproteinases ( mmps ) and tissue inhibitors of mmps ( timps ) . mmp-2 ( a ) , timp-2 ( b ) , mmp-9 ( c ) , and timp-1 ( d ) in controls ( ) , mood disorders ( md ) group before a course of ect ( pre - ect , ) and after a course of ect ( post - ect , ) , and schizophrenia - spectrum disorders ( scz ) group pre - ect ( ) and post - ect ( ) . the horizontal bars represent the mean values . in the roc curve analysis of diagnosis , the auc of mmp-2 , mmp-9 , timp-1 , and timp-2 at pre - ect between the md and control groups were 0.738 , 0.603 , 0.674 , and 0.675 , respectively . the scores for depressive symptoms evaluated by hamd in the md group and the scores of psychotic symptoms evaluated by bprs in the scz group significantly decreased following ect ( table 1 ) . there was a statistically significant increase in serum levels of mmp-2 in the md group over the course of ect ( p=.046 ) but no significant change observed in the scz group ( p=.650 ) ( figure 1a ) . there were statistically significant decreases in serum levels of mmp-9 in both the md and scz groups following ect ( md group , p=.003 ; scz group , p=.019 ) ( figure 1c ) . among the 2 nonresponders to ect in the scz group , one patient had an increase in serum level of mmp-9 and the other had a decrease in serum level of mmp-9 after the course of ect ( data not shown ) . serum levels of timp-2 and timp-1 did not significantly change over the course of ect in both the md and scz groups ( figure 1b , d ) . no significant correlations were observed in the md group between subject characteristics ( gender , age , age of onset , duration of illness , duration of current episode , or dose of medication ) and serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 at either pre - ect or post - ect ( data not shown ) . correlation coefficients were calculated between serum levels of mmps and timps and clinical symptomatic scores evaluated by hamd for md and by bprs for scz , combining results from pre - ect and post - ect ( table 2 ) . in the scz group , there was no significant correlation between serum levels of mmps , timps , and total bprs scores ( table 2 ) . in the md group , serum levels of timp-1 and timp-2 furthermore , no significant correlations were observed between timps and subscale hamd scores ( table 3 ) . correlation between mmp and timp serum levels and clinical symptomatic scores of md and scz groups abbreviations : bprs : brief psychiatric rating scale ; hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; scz , schizophrenia - spectrum disorder ; timp , tissue inhibitor of mmp . correlation between mmp and timp serum levels and subscale hamd scores in md groups abbreviations : hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; timp , tissue inhibitor of mmp . however , in the md group , there was a significant negative correlation between serum levels of mmp-2 and total hamd score ( p=.041 ) ( table 2 ; figure 2a ) and a significant positive correlation between serum levels of mmp-9 and total hamd score ( p=.006 ) ( table 2 ; figure 2b ) . furthermore , negative correlations between serum levels of mmp-2 and subscale hamd scores ( core symptom , activity , somatic anxiety ) were observed as well as positive correlations between serum levels of mmp-9 and the same subscale hamd scores ( core symptom , activity , psychic anxiety , somatic anxiety ) as observed with mmp-2 ( table 3 ) . a significant negative correlation between serum levels of mmp-2 and mmp-9 was observed in the md group ( p=.001 , rho=-0.479 ) , but not in the scz group ( p=.368 , rho=-0.184 ) or in the control group ( p=.592 , rho=0.087 ) ( figure 2c ) . correlation between serum levels of matrix metalloproteinases ( mmps ) and total hamilton rating scale for depression ( hamd ) score in patients with mood disorders ( md ) before and after a course of ect . ( a ) significant negative correlation between serum levels of mmp-2 and total hamd score . ( b ) pre - ect values ( ) and post - ect values ( ) ; n=21 , * p < .05 the current study demonstrated that serum levels of mmp-2 were specifically reduced in md patients with depressive symptoms applicable to ect and that serum levels of both mmp-2 and mmp-9 were significantly altered in opposing directions in a depressive state - dependent manner following ect . in addition , alterations of mmp-2 and mmp-9 appear to be associated with certain depressive symptoms such as not only core symptom , but also somatic anxiety and activity . the current study is the first to demonstrate a significant negative association between serum levels of mmp-2 and clinical scores of depressive symptoms during the course of ect in md patients . a previous proteomic study demonstrated that the plasma levels of mmp-2 in depressive patients were significantly lower than those of control subjects , and plasma levels of mmp-2 in schizophrenic patients were the same as those of control subjects ( domenici et al . , 2010 ) . the study , however , measured mmps at only one time point , utilized a proteomics method , and did not evaluate patient symptoms . despite these limitations , the previous findings parallel the current findings . it is currently unknown why circulating levels of mmp-2 declined and are associated with levels of mmp-9 in the md patients in a depressive state . mmp-2 is constitutively expressed in almost all human tissues , but mainly by endothelial and epithelial cells and fibroblasts ( sbardella et al . , astrocytes are a major source of mmp-2 and presumably drive physiological remodeling of the blood brain barrier ( bbb ) ( del zoppo et al . mmp-2 is detectable in significant serum concentrations under physiological conditions and is linked to homeostatic functioning ( sbardella et al . , 2012 ) . the current study showed a reduction of mmp-2 in md patients before ect and an increase in mmp-2 after a course of ect . a number of studies have demonstrated significant reductions of glia , mainly astrocytes , in the postmortem brain of md patients ( ongur et al . , 1998 ; cotter et al . , 2001 ; gittins and harrison , 2011 ) . another postmortem brain study demonstrated that coverage of blood vessels by astrocyte endfeet in the prefrontal cortex was significantly reduced in patients with md ( rybakowski et al . , 2013 ) . the decline in glia within brain vasculature areas associated with mood function suggests the possibility of reductions in microenvironmental cellular components of the bbb , which is composed of astrocytes and vascular endothelial cells , in md patients . a clinical finding pointed out that serum levels of mmp-2 were decreased in patients suffering from subarachnoid hemorrhage , in which the bbb is in fact compromised ( horstmann et al . , 2006 ) . decreased mmp-2 in the circulation in md patients , then , suggests a small compromise of the bbb compared with a gross compromise observed with subarachnoid hemorrhage . the increase of serum levels of mmp-2 in md patients after the ect course could be associated with the remodeling of gliovascular units , which is supported by previous ect findings showing gliogenesis and angiogenesis ( hellsten et al . , 2005 ; wennstrom et al . , 2006 ; bouckaert et al . , 2014 further , this mmp-2-related phenomenon could lead to increased incidences of inflammatory states such as production of mmp-9 through a disruption in the bbb in the brain of md patients , because expression of mmp-9 is regulated not only by immune cells in response to inflammatory events ( van den steen et al . , 2002 ; sbardella et al . , 2012 ) but also directly by mmp-2 activity ( dzwonek et al . , 2004 ; romi et al . , thus , future studies will be needed to clearly establish evidence of a mmp-2-related dysregulated bbb and concurrent alteration of mmp-9 in md patients by examining samples from patients such as postmortem brain and cerebrospinal fluid . in the current study , the serum levels of mmp-9 in md patients was not significantly different from that of the control group before ect , but mmp-9 significantly decreased following ect . similar to the current findings , a previous study found that hamd scores and serum levels of mmp-9 were positively correlated and there was no difference between depressed patients and controls in serum levels of mmp-9 ( yoshida et al . , 2012 ) . others , however , have found that plasma levels of mmp-9 in depressed patients were significantly higher than those of the controls in a proteomic study ( domenici et al . , 2010 ) , and serum levels of mmp-9 in depressed young patients with bipolar disorder were significantly higher than those of the controls , which remained elevated even following drug treatment ( rybakowski et al . , 2013 ) . possible reasons for the discrepancy between the current findings and the findings of increased mmp-9 in depressed patients include differences in subject age , severity of symptoms , choice of symptom rating scale , and treatment protocol . it is also possible that circulating mmp-9 is considerably sensitive to psychosocial stress even in healthy individuals ( garvin et al . , 2009 ) . an inflammatory state has been proposed as the substrate of md , since circulating proinflammatory cytokines increase with the severity of mood disturbance symptoms ( felger and lotrich , 2013 ; rosenblat et al . , it is possible that in a depressive episode , the homeostatic state maintained by the immune system could be altered by changes in expression of proinflammatory cytokines and mmps , and these alterations could be normalized through ect treatment . the decreased mmp-9 levels in both md and scz groups following ect could reflect a common involvement of mmp-9 in psychiatric disorders . fragile x syndrome , psychiatric disorders associated with anxiety , compulsive - repetitive behaviors , and social communication deficits have been associated with changes in mmp-9 levels . knockout of the mmp-9 gene improved symptoms in a mouse model of fragile x syndrome and reduced dendritic spine abnormalities ( sidhu et al . , 2014 ) . a clinical trial of the antibiotic minocycline decreased mmp-9 levels and appeared to attenuate the symptoms of fragile x syndrome ( dziembowska et al . , 2013 ) . these findings , combined with the current findings , suggest that decreasing mmp-9 alleviates some types of psychiatric disorders . as biomarkers for md , roc analysis in the current study demonstrated that the auc for both mmps and timps prior to ect was found to be 0.745 at its peak , which does not suggest that either ligand would have high diagnostic value as a biomarker . because serum levels of mmp-2 was negatively correlated to certain depressive symptoms , to which mmp-9 was positively correlated , both mmp-2 and mmp-9 could serve as biomarkers for specific depressive states or symptoms with greater accuracy if possibly combined with other inflammatory ligands such as cytokines . to place the current findings into perspective , there are a few limitations that should be mentioned . while the total sample size of the current study was small , significant correlations were nonetheless observed . second , it is unclear if blood concentrations of mmp and timp reflect those in the brain . there are some reports that found no correlation between blood and csf concentrations of mmps ( horstmann et al . , 2010 ; niebroj - dobosz et al . , therefore , it is possible that the dynamics of mmps , as well as timp , in the brain are different from those in peripheral tissues . to overcome this issue , direct assessment of mmps in csf from md patients could be highly useful . finally , the effect of medications on serum mmps levels is not well understood . although there were no statistically significantly differences before and after ect on the equivalence of both imipramine and chlorpromazine , patients were allowed to use medication during the course of ect . a future investigation could include drug - nave patients to evaluate the effects of medications on mmp , at least before treatments . serum levels of mmp-2 were significantly decreased in md patients with depressive episodes , and a course of ect in these patients significantly increased mmp-2 but decreased mmp-9 . also in md patients , there were significant correlations between depressive symptoms and serum levels of mmp-2 and mmp-9 , in opposite directions , in response to ect . the findings suggest that alteration of homeostasis linked to inflammation lead to changes in levels of mmp-2 and mmp-9 , which occurs in a disease - specific manner and depend on the severity of depressive symptoms , which are in turn sensitive to ect treatment . shigeto yamawaki md , phd has received donation for research from astellas , pfizer and msd honoraria for lectures from glaxo smith kline , astellas , daiichi sankyo , japan eli lilly , takeda , otsuka , eisai , dai nippon sumitomo , shionogi , meiji seika , mochida , and pfizer .

background : inflammatory processes could underlie mood disorders . matrix metalloproteinases ( mmps ) and tissue inhibitors of mmps ( timp ) are inflammation - related molecules . the current study sought an association between mood disorders and systemic levels of mmps and timps.methods:serum was obtained from patients with mood disorders ( n=21 ) and patients with schizophrenia ( n=13 ) scheduled to undergo electroconvulsive therapy . serum was also obtained from healthy controls ( n=40 ) . clinical symptoms were assessed by the hamilton rating score for depression and the brief psychiatric rating scale . serum levels of mmps and timps were quantified by elisa.results:the serum levels of mmp-2 in mood disorder patients , but not in schizophrenia patients , prior to the first electroconvulsive therapy session ( baseline ) was significantly lower than that of healthy controls . at baseline , levels of mmp-9 and timp-2 , -1 were not different between patients with mood disorder and schizophrenia and healthy controls . after a course of electroconvulsive therapy , mmp-2 levels were significantly increased in mood disorder patients , but mmp-9 levels were significantly decreased in both mood disorder and schizophrenia patients . in mood disorder patients , there was a significant negative correlation between depressive symptoms and serum levels of mmp-2 and a positive correlation between depressive symptoms and mmp-9 . in addition , alterations of serum levels of mmp-2 and mmp-9 were significantly correlated each other and were associated with certain depressive symptoms.conclusion:a change in inflammatory homeostasis , as indicated by mmp-2 and mmp-9 , could be related to mood disorders , and these markers appear to be sensitive to electroconvulsive therapy .

Introduction Methods Subjects ECT Treatment Procedures Assessment of Clinical Symptoms MMPs and TIMPs Assay Statistical Analysis Results Clinical Data Serum Levels of MMPs and TIMPs at Pre-ECT in the MD Group Compared with the SCZ Group and Control Group Alterations of Serum Levels of MMPs and TIMPs in the MD and SCZ Groups over the Course of ECT Relationship between Serum Levels of MMPs and TIMPs at Pre-ECT and Post-ECT and Subject Characteristics of the MD Group Correlation between Serum Levels of MMPs and TIMPs and Clinical Symptomatic Scores in the MD and SCZ Groups Discussion Conclusion Interest Statement

matrix metalloproteinases ( mmps ) are zinc - dependent proteases that are involved not only in the degradation and remodeling of the extracellular matrix but also in processing bioactive molecules , including cell surface receptors , neurotrophic factors , chemokines , and cytokines ( kim and joh , 2012 ) . , 2002 ; these enzymes are also known to be related to pathological processes such as cancer , neurodegenerative disorders , arthritis , and cardiovascular diseases ( sbardella et al . the mmps are endogenously inhibited by the tissue inhibitors of mmps ( timps ) . in humans , the c - terminal domain of timp-1 and timp-2 binds to the hemopexin of the proenzymes of mmp-9 and mmp-2 , respectively ( carlo , 2014 ) . this indicates that timp-1 and timp-2 are major individual inhibitors of mmp-9 and mmp-2 ( goldberg et al . several studies have reported an association between depressive symptoms and state - dependent elevated levels of inflammatory markers , such as interleukin-1 beta , interleuki-6 , and tumor necrosis factor - alpha ( felger and lotrich , 2013 ; rosenblat et al . the current study sought to find an association between md and inflammation - related markers . the main mmps , mmp-2 and mmp-9 , and their inhibitors , timp-2 and timp-1 , were quantified in patients with md . first , serum levels of mmp-2 , mmp-9 , timp-2 , and timp-1 from patients with md who had depressive episodes and were eligible for electroconvulsive therapy ( ect ) were compared with healthy controls and patients with schizophrenia - spectrum disorders ( scz ) , who also had psychotic episodes and were also eligible for ect . serum levels of mmp and timp were then compared before and after a course of ect in patients with md and scz . finally , an association between serum levels of mmps and timps with clinical symptoms in patients with md and scz was determined . after the purpose and the ect procedure were described in detail , written informed consent was obtained from patients or caregivers of patients prior to initiating ect . clinical symptomatic scores were assessed using the 17-item ( hamilton rating score for depression [ hamd ] ) for patients with md and the brief psychiatric rating scale ( bprs ) for patients with scz . each patient s symptoms were assessed prior to the first ect session ( baseline , pre - ect ) and a day after the last ect session ( post - ect ) by the same psychiatrist . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 were measured using elisa ( quantikine elisa , r&d systems , minneapolis , mn ) according to the manufacturer s instructions . the receiver operating characteristic ( roc ) curves for the assessment of specificity and sensitivity of mmps and timps were calculated to discriminate between the md group and the control group . after the purpose and the ect procedure were described in detail , written informed consent was obtained from patients or caregivers of patients prior to initiating ect . clinical symptomatic scores were assessed using the 17-item ( hamilton rating score for depression [ hamd ] ) for patients with md and the brief psychiatric rating scale ( bprs ) for patients with scz . each patient s symptoms were assessed prior to the first ect session ( baseline , pre - ect ) and a day after the last ect session ( post - ect ) by the same psychiatrist . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 were measured using elisa ( quantikine elisa , r&d systems , minneapolis , mn ) according to the manufacturer s instructions . the receiver operating characteristic ( roc ) curves for the assessment of specificity and sensitivity of mmps and timps were calculated to discriminate between the md group and the control group . subject clinical data abbreviations : bprs , brief psychiatric rating scale ; cpz , chlorpromazine ; ect , electroconvulsive therapy ; hamd , hamilton rating score for depression ; imi , imipramine ; md , mood disorder ; scz : schizophrenia - spectrum disorder . serum levels of mmp-2 in the md group with depressive episodes at pre - ect were significantly lower than those of the control group ( figure 1a ; p=.023 ) , whereas there were no significant differences for the serum levels of timp-2 , mmp-9 , and timp-1 between the md group and the control group ( figure 1b - d ) . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 in the md group at pre - ect were not significant different compared with those of the scz group with psychotic episodes at pre - ect . additionally , serum levels of mmps and timps were not significantly different between md patients with either md ( unipolar ) or bipolar disorder ( bipolar ) at pre - ect ( mmp-2 , unipolar=165.925.0ng / ml vs bipolar=159.426.8ng / ml , p=.512 ; timp-2 , 63.17.6ng / ml vs 66.66.2ng / ml , p=.259 ; mmp-9 , 553.9218.8ng / ml vs 604.6310.5ng / ml , p=.668 ; timp-1 , 150.539.4ng / ml vs 148.023.2ng / ml , p=.571 ) . scatter plot of serum levels of matrix metalloproteinases ( mmps ) and tissue inhibitors of mmps ( timps ) . mmp-2 ( a ) , timp-2 ( b ) , mmp-9 ( c ) , and timp-1 ( d ) in controls ( ) , mood disorders ( md ) group before a course of ect ( pre - ect , ) and after a course of ect ( post - ect , ) , and schizophrenia - spectrum disorders ( scz ) group pre - ect ( ) and post - ect ( ) . in the roc curve analysis of diagnosis , the auc of mmp-2 , mmp-9 , timp-1 , and timp-2 at pre - ect between the md and control groups were 0.738 , 0.603 , 0.674 , and 0.675 , respectively . there was a statistically significant increase in serum levels of mmp-2 in the md group over the course of ect ( p=.046 ) but no significant change observed in the scz group ( p=.650 ) ( figure 1a ) . there were statistically significant decreases in serum levels of mmp-9 in both the md and scz groups following ect ( md group , p=.003 ; scz group , p=.019 ) ( figure 1c ) . no significant correlations were observed in the md group between subject characteristics ( gender , age , age of onset , duration of illness , duration of current episode , or dose of medication ) and serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 at either pre - ect or post - ect ( data not shown ) . correlation coefficients were calculated between serum levels of mmps and timps and clinical symptomatic scores evaluated by hamd for md and by bprs for scz , combining results from pre - ect and post - ect ( table 2 ) . in the scz group , there was no significant correlation between serum levels of mmps , timps , and total bprs scores ( table 2 ) . correlation between mmp and timp serum levels and clinical symptomatic scores of md and scz groups abbreviations : bprs : brief psychiatric rating scale ; hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; scz , schizophrenia - spectrum disorder ; timp , tissue inhibitor of mmp . correlation between mmp and timp serum levels and subscale hamd scores in md groups abbreviations : hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; timp , tissue inhibitor of mmp . however , in the md group , there was a significant negative correlation between serum levels of mmp-2 and total hamd score ( p=.041 ) ( table 2 ; figure 2a ) and a significant positive correlation between serum levels of mmp-9 and total hamd score ( p=.006 ) ( table 2 ; figure 2b ) . furthermore , negative correlations between serum levels of mmp-2 and subscale hamd scores ( core symptom , activity , somatic anxiety ) were observed as well as positive correlations between serum levels of mmp-9 and the same subscale hamd scores ( core symptom , activity , psychic anxiety , somatic anxiety ) as observed with mmp-2 ( table 3 ) . a significant negative correlation between serum levels of mmp-2 and mmp-9 was observed in the md group ( p=.001 , rho=-0.479 ) , but not in the scz group ( p=.368 , rho=-0.184 ) or in the control group ( p=.592 , rho=0.087 ) ( figure 2c ) . correlation between serum levels of matrix metalloproteinases ( mmps ) and total hamilton rating scale for depression ( hamd ) score in patients with mood disorders ( md ) before and after a course of ect . ( a ) significant negative correlation between serum levels of mmp-2 and total hamd score . subject clinical data abbreviations : bprs , brief psychiatric rating scale ; cpz , chlorpromazine ; ect , electroconvulsive therapy ; hamd , hamilton rating score for depression ; imi , imipramine ; md , mood disorder ; scz : schizophrenia - spectrum disorder . serum levels of mmp-2 in the md group with depressive episodes at pre - ect were significantly lower than those of the control group ( figure 1a ; p=.023 ) , whereas there were no significant differences for the serum levels of timp-2 , mmp-9 , and timp-1 between the md group and the control group ( figure 1b - d ) . serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 in the md group at pre - ect were not significant different compared with those of the scz group with psychotic episodes at pre - ect . additionally , serum levels of mmps and timps were not significantly different between md patients with either md ( unipolar ) or bipolar disorder ( bipolar ) at pre - ect ( mmp-2 , unipolar=165.925.0ng / ml vs bipolar=159.426.8ng / ml , p=.512 ; timp-2 , 63.17.6ng / ml vs 66.66.2ng / ml , p=.259 ; mmp-9 , 553.9218.8ng / ml vs 604.6310.5ng / ml , p=.668 ; timp-1 , 150.539.4ng / ml vs 148.023.2ng / ml , p=.571 ) . scatter plot of serum levels of matrix metalloproteinases ( mmps ) and tissue inhibitors of mmps ( timps ) . mmp-2 ( a ) , timp-2 ( b ) , mmp-9 ( c ) , and timp-1 ( d ) in controls ( ) , mood disorders ( md ) group before a course of ect ( pre - ect , ) and after a course of ect ( post - ect , ) , and schizophrenia - spectrum disorders ( scz ) group pre - ect ( ) and post - ect ( ) . there was a statistically significant increase in serum levels of mmp-2 in the md group over the course of ect ( p=.046 ) but no significant change observed in the scz group ( p=.650 ) ( figure 1a ) . there were statistically significant decreases in serum levels of mmp-9 in both the md and scz groups following ect ( md group , p=.003 ; scz group , p=.019 ) ( figure 1c ) . among the 2 nonresponders to ect in the scz group , one patient had an increase in serum level of mmp-9 and the other had a decrease in serum level of mmp-9 after the course of ect ( data not shown ) . no significant correlations were observed in the md group between subject characteristics ( gender , age , age of onset , duration of illness , duration of current episode , or dose of medication ) and serum levels of mmp-2 , mmp-9 , timp-1 , and timp-2 at either pre - ect or post - ect ( data not shown ) . correlation coefficients were calculated between serum levels of mmps and timps and clinical symptomatic scores evaluated by hamd for md and by bprs for scz , combining results from pre - ect and post - ect ( table 2 ) . in the scz group , there was no significant correlation between serum levels of mmps , timps , and total bprs scores ( table 2 ) . correlation between mmp and timp serum levels and clinical symptomatic scores of md and scz groups abbreviations : bprs : brief psychiatric rating scale ; hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; scz , schizophrenia - spectrum disorder ; timp , tissue inhibitor of mmp . correlation between mmp and timp serum levels and subscale hamd scores in md groups abbreviations : hamd , hamilton rating score for depression ; md , mood disorder ; mmp , matrix metalloproteinase ; timp , tissue inhibitor of mmp . however , in the md group , there was a significant negative correlation between serum levels of mmp-2 and total hamd score ( p=.041 ) ( table 2 ; figure 2a ) and a significant positive correlation between serum levels of mmp-9 and total hamd score ( p=.006 ) ( table 2 ; figure 2b ) . furthermore , negative correlations between serum levels of mmp-2 and subscale hamd scores ( core symptom , activity , somatic anxiety ) were observed as well as positive correlations between serum levels of mmp-9 and the same subscale hamd scores ( core symptom , activity , psychic anxiety , somatic anxiety ) as observed with mmp-2 ( table 3 ) . a significant negative correlation between serum levels of mmp-2 and mmp-9 was observed in the md group ( p=.001 , rho=-0.479 ) , but not in the scz group ( p=.368 , rho=-0.184 ) or in the control group ( p=.592 , rho=0.087 ) ( figure 2c ) . correlation between serum levels of matrix metalloproteinases ( mmps ) and total hamilton rating scale for depression ( hamd ) score in patients with mood disorders ( md ) before and after a course of ect . ( a ) significant negative correlation between serum levels of mmp-2 and total hamd score . ( b ) pre - ect values ( ) and post - ect values ( ) ; n=21 , * p < .05 the current study demonstrated that serum levels of mmp-2 were specifically reduced in md patients with depressive symptoms applicable to ect and that serum levels of both mmp-2 and mmp-9 were significantly altered in opposing directions in a depressive state - dependent manner following ect . in addition , alterations of mmp-2 and mmp-9 appear to be associated with certain depressive symptoms such as not only core symptom , but also somatic anxiety and activity . the current study is the first to demonstrate a significant negative association between serum levels of mmp-2 and clinical scores of depressive symptoms during the course of ect in md patients . a previous proteomic study demonstrated that the plasma levels of mmp-2 in depressive patients were significantly lower than those of control subjects , and plasma levels of mmp-2 in schizophrenic patients were the same as those of control subjects ( domenici et al . it is currently unknown why circulating levels of mmp-2 declined and are associated with levels of mmp-9 in the md patients in a depressive state . the current study showed a reduction of mmp-2 in md patients before ect and an increase in mmp-2 after a course of ect . the increase of serum levels of mmp-2 in md patients after the ect course could be associated with the remodeling of gliovascular units , which is supported by previous ect findings showing gliogenesis and angiogenesis ( hellsten et al . in the current study , the serum levels of mmp-9 in md patients was not significantly different from that of the control group before ect , but mmp-9 significantly decreased following ect . similar to the current findings , a previous study found that hamd scores and serum levels of mmp-9 were positively correlated and there was no difference between depressed patients and controls in serum levels of mmp-9 ( yoshida et al . others , however , have found that plasma levels of mmp-9 in depressed patients were significantly higher than those of the controls in a proteomic study ( domenici et al . , 2010 ) , and serum levels of mmp-9 in depressed young patients with bipolar disorder were significantly higher than those of the controls , which remained elevated even following drug treatment ( rybakowski et al . possible reasons for the discrepancy between the current findings and the findings of increased mmp-9 in depressed patients include differences in subject age , severity of symptoms , choice of symptom rating scale , and treatment protocol . , it is possible that in a depressive episode , the homeostatic state maintained by the immune system could be altered by changes in expression of proinflammatory cytokines and mmps , and these alterations could be normalized through ect treatment . as biomarkers for md , roc analysis in the current study demonstrated that the auc for both mmps and timps prior to ect was found to be 0.745 at its peak , which does not suggest that either ligand would have high diagnostic value as a biomarker . because serum levels of mmp-2 was negatively correlated to certain depressive symptoms , to which mmp-9 was positively correlated , both mmp-2 and mmp-9 could serve as biomarkers for specific depressive states or symptoms with greater accuracy if possibly combined with other inflammatory ligands such as cytokines . serum levels of mmp-2 were significantly decreased in md patients with depressive episodes , and a course of ect in these patients significantly increased mmp-2 but decreased mmp-9 . also in md patients , there were significant correlations between depressive symptoms and serum levels of mmp-2 and mmp-9 , in opposite directions , in response to ect . the findings suggest that alteration of homeostasis linked to inflammation lead to changes in levels of mmp-2 and mmp-9 , which occurs in a disease - specific manner and depend on the severity of depressive symptoms , which are in turn sensitive to ect treatment .

mesial temporal lobe epilepsy ( mtle ) is the most common form of partial epilepsy in humans and is generally refractory to treatment . it is characterized by seizures that originate in limbic structures , namely , the hippocampus , the parahippocampal gyrus and the amygdala . in approximately 65% of people suffering from this form of epilepsy , the underlying pathology is ammon 's horn sclerosis characterized by neuronal loss , gliosis and atrophy of the hippocampus . while mtle classically begins in teenagers and sometimes even adulthood , the initial insult is thought to be neurodevelopmental and to happen in early life , namely , after prolonged febrile seizures ( fss ) . two prevailing hypotheses exist to explain the possible relationship between prolonged fs , hippocampal sclerosis , and mtle . the second , supported by a wide body of recent evidence , suggests that prolonged fs may in fact arise from an already predisposed brain due to anatomical and/or genetic alterations , but it is the prolonged fs that leads to hippocampal sclerosis and mtle later in life . to study the pathophysiology of mtle , experimental animal modeling stands on the important assumption that understanding fundamental mechanisms of action will help us in the elaboration of more effective treatments and therapeutic strategies for human diseases . the translational impact of experimental evidence from the study of fs and mtle has been limited by the complexity of these clinical conditions , more specifically their uncertain causal relationship . however , recent clinical data appear to support the fact that prolonged fs , more specifically febrile status epilepticus , directly leads to hippocampal injury and mtle . here , we will review several animal models that have been developed to study the putative biological substrate and risk factors behind the development of mtle in humans . we will focus on two important developmental risk factors , namely , prolonged febrile seizures and cortical malformations as we propose a two - hit model of mtle . to conclude we will discuss the impact of these findings on future clinical management . febrile seizures ( fss ) are a common neurological disorder that usually involves 2 to 5% of children between the age of 6 months and 6 years old with a peak incidence in toddlers of 12 to 18 months [ 57 ] . simple fss are generalized and brief seizures ( lasting < 15 min ) that do not recur within 24 hours . atypical fss are prolonged ( > 15 min ) , recurrent within 24 hours , or lateralized seizures or express more than one of these characteristics . in contrast to simple fss that generally have no long - term consequences , prolonged fss , more specifically febrile status epilepticus ( lasting > 30 min ) , have been associated with mtle [ 3 , 5 , 810 ] . based on retrospective clinical studies , it has been shown that up to 30 to 60% of patients with mtle have a past history of prolonged fss [ 2 , 7 , 1113 ] . in one important yet controversial series , children with atypical febrile seizures showed an eightfold increased risk of developing epilepsy compared to those with simple fss and controls . thus , one needs to understand what causes some individuals to experience prolonged fss in order to try to prevent them . to study this , several experimental paradigms have been used to mimic the increase in core body temperature occurring during episodes of fever . multiple studies have artificially evoked hyperthermia - induced seizures ( hss ) to determine how fss are generated . the most stable and most accepted model is hyperthermia - induced by hot dry air [ 11 , 1517 ] . hss have been provoked in rats by many other methods such as exposure to an infrared lamp , infrared rays , microwaves [ 20 , 21 ] , a heated pad , or warm water . however , the use of these apparatus was restricted because of high morbidity , mortality , and clinical variability . in contrast , models of hss induced by exposure to heated dry air develop highly stereotypical generalized seizures that are reproducible and easy to characterize , with minimal or no mortality [ 2 , 3 , 7 , 9 , 11 , 12 , 15 , 17 , 24 ] . like in humans , this model leads to the development of age - specific seizures that , when brief , do not lead to the development of spontaneous seizures later in life . however , when seizures are prolonged , up to 33% of naive rats develop electroclinical seizures in adulthood [ 11 , 24 ] . the original studies have reported changes in hippocampal excitability , gene expression , and network effects but without the typical changes observed in mtle such as neuronal loss , mossy fiber sprouting , or neurogenesis [ 2527 ] . however , in these models , the duration of the seizure is determined by the duration of the exposure to high temperature rather than by individual vulnerability . many experimental models have used hyperthermia to induce convulsions as a model to study fss [ 15 , 18 , 28 , 29 ] . this is because most of the developing animals experience seizures when they reach a high core temperature and because hyperthermia and fever share common mechanisms to elicit seizures such as the release of cytokines including interleukin-1 ( il-1 ) [ 11 , 27 , 30 ] . this particular cytokine seems key to generate fss in young rats based on the evidence that rats lacking the interleukin-1 receptor type i ( il-1r1 ) gene exhibit a much higher temperature threshold necessary to develop fss [ 11 , 27 ] . in the hippocampus , il-1 receptors are expressed in high density and their stimulation triggers a cascade of downstream effects through mitogen - activated protein ( map ) kinase and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) signalling . this could alter gene expression and transform normal neuronal circuitry into a proconvulsive epileptic network [ 5 , 8 , 32 ] . however , even if the il-1 pathway seems to be a crucial and shared mechanism of both hyperthermia and fever , its activation may not fully or appropriately imitate fss as fever reflects a regulated increase of body temperature resulting from a broader immune challenge . the common precipitating event in both simple and prolonged fss is an infection with a bacterial or viral agent . this induces a febrile response , which involves the elaboration of several inflammatory cytokines that include not only interleukin-1 but also il-6 and tumor necrosis factor ( tnf ) . these cytokines , released by activated leukocytes , lead to the production of prostaglandins such as cycloxygenase-2/3 and prostaglandin e2 in the preoptic nuclei of the hypothalamus . this then results in an upregulation in thermostatic set point for body temperature and then fever [ 33 , 34 ] . however , apart from the fibrogenic properties of inflammatory cytokines , there is increasing evidence that they play a direct role in the generation of fss . clinical studies have shown that peripheral leukocytes obtained from children with fss show an exaggerated il-1 release to a challenge with lipopolysaccharide ( lps ) [ 3537 ] or viral rna . injection of bacterial lipopolysaccharide ( lps ) in vivo in animals is another interesting experimental approach to study the role of proinflammatory cytokines in fever challenge at the systemic level . however , the rise in temperature is somewhat limited and is not sufficient to induce seizures in naive animals . were the first to demonstrate a causal relationship between il-1 and fss in an experimental model of fss using lps [ 28 , 30 ] . in this study immature rats were first injected with lps , which produced a mild fever without a seizure but by giving a subconvulsive dose of kainite , seizures are induced in 50% of pups pretreated with lps . il-1 is thought to lead to the generation of fss through its effects on inhibition , by reducing gabaa receptor currents , or through promoting glutamatergic mediated excitatory effects , by increasing calcium conductance through n - methyl - d - aspartate ( nmda ) receptors . some clinical studies have pointed that the human herpes virus 6 ( hhv-6 ) could be a putative link between fss and mtle . other studies found hhv-6 dna in brain tissue removed during surgery for mtle [ 41 , 42 ] . however , only a minority of primary hhv-6 infections may be associated with fss [ 5 , 43 , 44 ] . another virus of the herpes family , the herpes simplex virus type 1 ( hsv-1 ) , causes the limbic seizures by reducing dynorphin expression in the dentate gyrus of hippocampus in rats , leading to seizures [ 45 , 46 ] . inherited dynorphin promoter polymorphisms are associated with temporal lobe epilepsy and febrile seizures in human . in animals , these findings show a vulnerability of hippocampal dynorphin during herpes infection , and this may highlight a neurochemical basis for limbic seizures following viral infections . overall the evidence summarized here indicates that prolonged fss contribute to epileptogenesis rather than being simply a marker of an epileptic tendency . in addition , the duration of the fss seems to be an important factor of the development of subsequent epilepsy in the nonpredisposed brain . however , it has recently been shown that prolonged fss are often unrecognized in the emergency room . therefore , early identification of children at risk for prolonged fss and epileptogenesis could be a better strategy to prevent mtle . in order to understand how underlying cortical malformations may be implicated in epileptogenesis and developmental delay , various animal models were developed . a good animal model for malformation of cortical development should display ( 1 ) hyperexcitable brain regions and ( 2 ) macroscopic as well as microscopic structural abnormalities that are similar to the human pathology . many of these models have been yielding interesting results . methylazoxymethanol acetate or mam is a teratogenic alkylating neurotoxin which specifically blocks mitosis of neuroepithelial cells actively dividing during development , without affecting the postmitotic cells . when administered to pregnant rats ( intraperitoneal injection at embryonic day 15 ( e15 ) ) , mam causes multifocal cerebral malformations in the rat pups including microcephaly , cortical thinning , loss of lamination , and cortical and hippocampal heterotopia [ 49 , 50 ] . the heterotopic neurons displayed hyperexcitable properties , and these animals showed a diminished seizure threshold to various proconvulsant agents such as kainic acid [ 48 , 51 ] or hippocampal electrical kindling . interestingly , no long - term spontaneous recurrent seizures ( srss ) were generally reported in this model , although harrington et al . described some electrographic seizures in 2 out of 11 mam - treated animals . some of the molecular and cell mechanisms of mam - induced hyperexcitability include altered cell firing due to smaller calcium - activated potassium ( k ) currents affecting membrane potential after - hyperpolarization [ 53 , 54 ] , lack of fast a - type kv4.2 k currents on heterotopic neurons , modification of n - methyl - d - aspartate receptor subtype 2a / b ( nr2a / b ) expression in heterotopic neurons , and diminution in inhibitory synaptic activity in heterotopic neurons suggesting profound changes of heterotopic neurons . the mam model has the advantage of having a specific effect on neuroepithelial cells , not affecting astrocytic cells and not affecting cells from other organs which have a different ontogenic precursor . however , in order for the mam administration to be reliable , the first day of gestation must always accurately be identified . in any case , this model yields a more diffuse cortical dysplasia than what is observed clinically and does not show spontaneous recurrent seizures alone . nonetheless , mam - treated pups are more susceptible to the epileptogenic effects of prolonged fss with all animals developing epilepsy . the in utero irradiation model is obtained by exposing pregnant rats at e17 to radiation doses as low as 100 centigray ( cgy ) to as high as 225 cgy of external gamma radiation from a linear accelerator source [ 61 , 62 ] . the irradiated cortex shows diffuse cortical dysplasia , similar to the mam model , along with microcephaly characterized by a 50% diminution in cortical thickness , agenesis , hypoplasia , and the presence of heterotopic neurons , sign of a severe migrational abnormality . treated animals have been shown to display interictal epileptiform activity visible in the cortex as well as in the hippocampus ; however , spontaneous recurrent seizures occurred only in a subset of irradiated animals , depending on the radiation dose . the manifestations of these clinical seizures was quite typical of limbic seizures including staring , facial twitches , wet dog shakes , and limb clonus [ 61 , 64 , 66 ] . looking at the network and cellular levels , slices obtained from radiation - treated animals are more excitable as seen by spontaneous and evoked field potentials in slices of neocortex . furthermore , electrophysiological recordings have shown that the excitatory activity in slices coming from irradiated animals is greater relative to untreated controls and that the inhibitory activity is diminished , which may be explained by a diminution in activity of somatostatin and parvalbumin containing inhibitory interneurons in the irradiated group . therefore , an imbalance between excitation and inhibition is involved in the neocortical hyperactivity leading to the presence of epileptiform events in this model . the in utero irradiation model has the advantage of being noninvasive to the offspring , which induces less stress ; however , it yields a diffuse type of cortical dysplasia distinct from the typical clinical situation . in any case , studies looking at the vulnerability of irradiated pups to fss have , to our knowledge , not yet been done . the freeze - lesion - induced cortical malformation in rats was developed by dvorak and feit and closely resembles the polymicrogyrus observed in humans , in that it yields the formation of a four - layer neocortex rather than the typical six . to achieve this model , one - day - old rat pups are anaesthetized with isoflurane , their scalp cut at the midline and opened , and a frozen 2 mm large probe is placed on the soft cranium overlying the sensorimotor cortex for a period of ten seconds [ 9 , 17 ] . it should be noted however that the probe width , the lesion duration , and the number of lesions may vary from one study to another . in all cases , contact with the frozen probe causes an immediate focal necrotic lesion , followed by neuronal migration to repair the damaged region , which explains why lesions should be done at a very young age when cells are still in a migratory state . indeed , glial fibrillary acidic protein ( gfap ) as well as bromodeoxyuridine ( brdu ) expressing cells were found in high levels within the dysplastic cortex suggesting the presence of still proliferating astrocytic cells . the polymicrogyrus later obtained following the freeze lesion in rat is very similar to what would be observed in a focal human neuronal migration disorder [ 71 , 72 ] . fiber reorganization occurs within the cortical and subcortical layers of lesion rats as thalamocortical and corticothalamic projections are shown to be affected , possibly implicated in the process of epileptogenesis . disorganized projections were also seen by brill and huguenard who noted more inputs coming from infra- and supragranular cortical layers synapsing onto layer v pyramidal cells than in controls . on top of the macroscopic modifications taking place in the dysplastic cortex , other changes at the molecular level occur and seem to unbalance the excitation / inhibition equilibrium favoring excitation . looking at the expression of excitatory glutamate receptors , an autoradiography study showed that nmda , ampa , and ka receptor levels were elevated within the dysplastic cortex [ 75 , 76 ] , while they were unchanged when measures were taken in the surrounding normal cortex , suggesting the presence of a spatial gradient of ionotropic glutamate receptors with a greater concentration within the polymicrogyrus . amongst the nmda receptors , the nr2b subunit seems to be of great importance to the epileptogenicity of the lesion as the nr2b currents are functionally enhanced , and specific nr2b antagonists limit the spread of the epileptiform activity [ 71 , 78 ] , although it was shown that an ampar antagonist may block more widespread epileptiform activity measured extracellularly . on the other hand , looking at inhibitory activity , the same autoradiography study showed lowered gabaa and gabab binding within the dysplastic cortex and a downregulation of gabaa inhibition has been shown electrophysiologically in the freeze model . however , no interneuron cell loss was reported near or far from the lesion [ 80 , 81 ] . this widespread modification in various gabaa subunits can be the cause of the decrease in inhibitory activity . it is , however , also plausible that the gabaa inhibition downregulation may not be directly involved in the hyperexcitability observed , as the somatostatin - positive interneuron loss occurred after the onset of epileptiform activity in their model . however , despite the hyperexcitability observed in brain slices from lesion animals , there are in this model no recurrent seizures occurring spontaneously in vivo which is an important prerequisite to a good experimental model of human mtle . when the cortical polymicrogyrus model precedes another insult , this represents a two - hit model and mimics the human condition described in our clinical series . a few models , having in common a cortical polymicrogyrus as a first hit followed by another insult [ 17 , 60 , 8486 ] , may lead to tle development at a later age . in the case of the freeze lesion and hss model , the fss constitute the second hit occurring postnatally while the first hit is thought to occur at an early stage of brain development . therefore , in this model , the freeze lesion is performed at p1 , while the hss are induced at p10 ( figure 1 ) . at the time of hss induction , the temperature necessary to induce a generalized convulsion during hyperthermia was diminished in rats with a cortical lesion compared to rats without lesion , and the latency to attain the generalized convulsion was also shorter . more importantly , only the lesioned pups developed status epilepticus following a brief exposure to hyperthermia . furthermore , a brain and ipsilateral hippocampal atrophy was already measurable ten days following hss at p20 . this suggests that the lesion alone seems to predispose the brain and the hippocampus to prolonged fss and their consequences . at p80 , the hippocampal atrophy is more severe than at p20 ; however , it may be prevented by limiting seizure duration with diazepam at p10 . furthermore , using in vitro electrophysiological recordings , ca1 pyramidal cell hyperexcitability has been shown , yielding greater evoked excitatory postsynaptic potentials ( epsps ) and more frequent spontaneous excitatory postsynaptic currents ( sepscs ) specifically in the double - hit group onto pyramidal cells and onto ca1 interneurons . as the excitatory activity , the inhibitory activity is also altered with greater amplitude gabaa and gabab inhibitory postsynaptic potentials ( ipsps ) and evoked inhibitory postsynaptic currents ( eipscs ) on ca1 pyramidal cells in the double - hit group . this would suggest an excitatory / inhibitory imbalance favoring excitation already at p20 , prior to the occurrence of spontaneous recurrent seizures at p80 . in adulthood , we have found that the double hit results in the occurrence of spontaneous seizures occurring in 86100% of male rats with the seizures arising ipsilateral to the lesion [ 2 , 9 , 12 ] , numbers similar to the mam model and more significant than the 33% observed in naive rats exposed to prolonged fss . ipsilateral hippocampal atrophy persists in the double - hit group and is associated in adults with neuronal loss and memory deficits in performance of a hippocampus - dependent task . our data indicate that ionotropic glutamate receptor expression , especially the nmda subtype , is upregulated in the double - hit animals : nr2b subtype being overexpressed at approximately p20 and nr2a at p80 . this is in accordance with findings from other two - hit models where high levels of nr2b expression are also found : such as in the mam + pilocarpine model or in the prenatal freeze lesion + electrical kindling . the fact that lesion - only animals show higher nr2b levels and greater nmda currents at p20 suggests that the lesion itself may underlie the hyperexcitability at p20 and may be a predisposing factor to prolonged hss susceptibility at p10 , which is supported by a study in the freeze - lesion - only model . furthermore , it is possible that the il-1 expression during hyperthermia , as earlier stated , further exacerbates the effect of the lesion on nmdar . in parallel with the excitatory changes , whether the changes in inhibitory circuits reduce or increase the risk of recurrent seizures remains to be determined . in summary , the lesion alone and the hyperthermia alone appear to each leave the developing brain more vulnerable , but the presence of two hits strongly promotes epileptogenesis . in humans , development of mtle is more and more thought to be a multistage process taking place in early life and including a history of childhood prolonged febrile seizures . a retrospective study from our group demonstrated that 66% of children affected with mtle and a history of fss had dual pathology with the coexistence of hippocampal sclerosis and of a cortical malformation on pathology . more recently , the febstat study group was able to distinguish two subpopulations of fss with those experiencing prolonged fss being younger and with developmental delay . in an earlier publication , they had demonstrated that these same children were more likely ( or = 4.3 ) to have imaging abnormalities on mri including cortical malformations . although the models described here involve disorders of neuronal migration , other predisposing factors such as genetic susceptibility can represent the first hit . indeed , it has been shown that , in familial mtle , mesial temporal sclerosis develops in those who have experienced prolonged fss in early life . more so , prospective studies suggest that fs duration is a key factor in leading to hippocampal injury and that developmental abnormalities are indeed also present in children with febrile status and mtle . therefore , we believe that any child who presents with a febrile status epilepticus could benefit from a thorough imaging evaluation , including high - resolution mri . however , experimental evidence suggests a potential role for nr2b antagonists as not only a good seizure medication but also a potential antiepileptogenic treatment in the developing brain . the role of other potential first hits such as early - life stress in the development of mtle remains to be properly studied . only few animal models of mtle models implying early - life stress paradigms exist in the present literature [ 9498 ] . both corticotropin releasing factor ( crf ) and the glucocorticoid cortisol ( or corticosterone in rodents ) appear to exert potent proconvulsive or hyperexcitable effects on limbic structures in the developing brain [ 96 , 99107 ] . although there is no clear evidence that isolated early - life stressors can induce epileptogenesis , the anatomical and physiological changes produced by these hormones could predispose the developing brain to a second hit . in conclusion , a better understanding of the pathophysiology of mtle in the developing brain will help us develop age - specific treatments not only to control the seizures but also to prevent their occurrence altogether , an important step toward our ultimate goal of no seizure , no side effect .

febrile seizures occurring in the neonatal period , especially when prolonged , are thought to be involved in the later development of mesial temporal lobe epilepsy ( mtle ) in children . the presence of an often undetected , underlying cortical malformation has also been reported to be implicated in the epileptogenesis process following febrile seizures . this paper highlights some of the various animal models of febrile seizures and of cortical malformation and portrays a two - hit model that efficiently mimics these two insults and leads to spontaneous recurrent seizures in adult rats . potential mechanisms are further proposed to explain how these two insults may each , or together , contribute to network hyperexcitability and epileptogenesis . finally the clinical relevance of the two - hit model is briefly discussed in light of a therapeutic and preventive approach to mtle .

1. Introduction 2. Animal Studies 3. Clinical Relevance of the Two-Hit Models

mesial temporal lobe epilepsy ( mtle ) is the most common form of partial epilepsy in humans and is generally refractory to treatment . in approximately 65% of people suffering from this form of epilepsy , the underlying pathology is ammon 's horn sclerosis characterized by neuronal loss , gliosis and atrophy of the hippocampus . while mtle classically begins in teenagers and sometimes even adulthood , the initial insult is thought to be neurodevelopmental and to happen in early life , namely , after prolonged febrile seizures ( fss ) . two prevailing hypotheses exist to explain the possible relationship between prolonged fs , hippocampal sclerosis , and mtle . the second , supported by a wide body of recent evidence , suggests that prolonged fs may in fact arise from an already predisposed brain due to anatomical and/or genetic alterations , but it is the prolonged fs that leads to hippocampal sclerosis and mtle later in life . to study the pathophysiology of mtle , experimental animal modeling stands on the important assumption that understanding fundamental mechanisms of action will help us in the elaboration of more effective treatments and therapeutic strategies for human diseases . however , recent clinical data appear to support the fact that prolonged fs , more specifically febrile status epilepticus , directly leads to hippocampal injury and mtle . here , we will review several animal models that have been developed to study the putative biological substrate and risk factors behind the development of mtle in humans . we will focus on two important developmental risk factors , namely , prolonged febrile seizures and cortical malformations as we propose a two - hit model of mtle . to conclude we will discuss the impact of these findings on future clinical management . febrile seizures ( fss ) are a common neurological disorder that usually involves 2 to 5% of children between the age of 6 months and 6 years old with a peak incidence in toddlers of 12 to 18 months [ 57 ] . simple fss are generalized and brief seizures ( lasting < 15 min ) that do not recur within 24 hours . atypical fss are prolonged ( > 15 min ) , recurrent within 24 hours , or lateralized seizures or express more than one of these characteristics . in one important yet controversial series , children with atypical febrile seizures showed an eightfold increased risk of developing epilepsy compared to those with simple fss and controls . multiple studies have artificially evoked hyperthermia - induced seizures ( hss ) to determine how fss are generated . the most stable and most accepted model is hyperthermia - induced by hot dry air [ 11 , 1517 ] . hss have been provoked in rats by many other methods such as exposure to an infrared lamp , infrared rays , microwaves [ 20 , 21 ] , a heated pad , or warm water . in contrast , models of hss induced by exposure to heated dry air develop highly stereotypical generalized seizures that are reproducible and easy to characterize , with minimal or no mortality [ 2 , 3 , 7 , 9 , 11 , 12 , 15 , 17 , 24 ] . like in humans , this model leads to the development of age - specific seizures that , when brief , do not lead to the development of spontaneous seizures later in life . however , when seizures are prolonged , up to 33% of naive rats develop electroclinical seizures in adulthood [ 11 , 24 ] . the original studies have reported changes in hippocampal excitability , gene expression , and network effects but without the typical changes observed in mtle such as neuronal loss , mossy fiber sprouting , or neurogenesis [ 2527 ] . however , in these models , the duration of the seizure is determined by the duration of the exposure to high temperature rather than by individual vulnerability . this is because most of the developing animals experience seizures when they reach a high core temperature and because hyperthermia and fever share common mechanisms to elicit seizures such as the release of cytokines including interleukin-1 ( il-1 ) [ 11 , 27 , 30 ] . in the hippocampus , il-1 receptors are expressed in high density and their stimulation triggers a cascade of downstream effects through mitogen - activated protein ( map ) kinase and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) signalling . this could alter gene expression and transform normal neuronal circuitry into a proconvulsive epileptic network [ 5 , 8 , 32 ] . however , even if the il-1 pathway seems to be a crucial and shared mechanism of both hyperthermia and fever , its activation may not fully or appropriately imitate fss as fever reflects a regulated increase of body temperature resulting from a broader immune challenge . the common precipitating event in both simple and prolonged fss is an infection with a bacterial or viral agent . these cytokines , released by activated leukocytes , lead to the production of prostaglandins such as cycloxygenase-2/3 and prostaglandin e2 in the preoptic nuclei of the hypothalamus . this then results in an upregulation in thermostatic set point for body temperature and then fever [ 33 , 34 ] . however , apart from the fibrogenic properties of inflammatory cytokines , there is increasing evidence that they play a direct role in the generation of fss . clinical studies have shown that peripheral leukocytes obtained from children with fss show an exaggerated il-1 release to a challenge with lipopolysaccharide ( lps ) [ 3537 ] or viral rna . injection of bacterial lipopolysaccharide ( lps ) in vivo in animals is another interesting experimental approach to study the role of proinflammatory cytokines in fever challenge at the systemic level . however , the rise in temperature is somewhat limited and is not sufficient to induce seizures in naive animals . were the first to demonstrate a causal relationship between il-1 and fss in an experimental model of fss using lps [ 28 , 30 ] . il-1 is thought to lead to the generation of fss through its effects on inhibition , by reducing gabaa receptor currents , or through promoting glutamatergic mediated excitatory effects , by increasing calcium conductance through n - methyl - d - aspartate ( nmda ) receptors . however , only a minority of primary hhv-6 infections may be associated with fss [ 5 , 43 , 44 ] . another virus of the herpes family , the herpes simplex virus type 1 ( hsv-1 ) , causes the limbic seizures by reducing dynorphin expression in the dentate gyrus of hippocampus in rats , leading to seizures [ 45 , 46 ] . inherited dynorphin promoter polymorphisms are associated with temporal lobe epilepsy and febrile seizures in human . overall the evidence summarized here indicates that prolonged fss contribute to epileptogenesis rather than being simply a marker of an epileptic tendency . in addition , the duration of the fss seems to be an important factor of the development of subsequent epilepsy in the nonpredisposed brain . however , it has recently been shown that prolonged fss are often unrecognized in the emergency room . therefore , early identification of children at risk for prolonged fss and epileptogenesis could be a better strategy to prevent mtle . in order to understand how underlying cortical malformations may be implicated in epileptogenesis and developmental delay , various animal models were developed . a good animal model for malformation of cortical development should display ( 1 ) hyperexcitable brain regions and ( 2 ) macroscopic as well as microscopic structural abnormalities that are similar to the human pathology . many of these models have been yielding interesting results . when administered to pregnant rats ( intraperitoneal injection at embryonic day 15 ( e15 ) ) , mam causes multifocal cerebral malformations in the rat pups including microcephaly , cortical thinning , loss of lamination , and cortical and hippocampal heterotopia [ 49 , 50 ] . the heterotopic neurons displayed hyperexcitable properties , and these animals showed a diminished seizure threshold to various proconvulsant agents such as kainic acid [ 48 , 51 ] or hippocampal electrical kindling . interestingly , no long - term spontaneous recurrent seizures ( srss ) were generally reported in this model , although harrington et al . described some electrographic seizures in 2 out of 11 mam - treated animals . some of the molecular and cell mechanisms of mam - induced hyperexcitability include altered cell firing due to smaller calcium - activated potassium ( k ) currents affecting membrane potential after - hyperpolarization [ 53 , 54 ] , lack of fast a - type kv4.2 k currents on heterotopic neurons , modification of n - methyl - d - aspartate receptor subtype 2a / b ( nr2a / b ) expression in heterotopic neurons , and diminution in inhibitory synaptic activity in heterotopic neurons suggesting profound changes of heterotopic neurons . the mam model has the advantage of having a specific effect on neuroepithelial cells , not affecting astrocytic cells and not affecting cells from other organs which have a different ontogenic precursor . however , in order for the mam administration to be reliable , the first day of gestation must always accurately be identified . in any case , this model yields a more diffuse cortical dysplasia than what is observed clinically and does not show spontaneous recurrent seizures alone . the in utero irradiation model is obtained by exposing pregnant rats at e17 to radiation doses as low as 100 centigray ( cgy ) to as high as 225 cgy of external gamma radiation from a linear accelerator source [ 61 , 62 ] . the irradiated cortex shows diffuse cortical dysplasia , similar to the mam model , along with microcephaly characterized by a 50% diminution in cortical thickness , agenesis , hypoplasia , and the presence of heterotopic neurons , sign of a severe migrational abnormality . treated animals have been shown to display interictal epileptiform activity visible in the cortex as well as in the hippocampus ; however , spontaneous recurrent seizures occurred only in a subset of irradiated animals , depending on the radiation dose . furthermore , electrophysiological recordings have shown that the excitatory activity in slices coming from irradiated animals is greater relative to untreated controls and that the inhibitory activity is diminished , which may be explained by a diminution in activity of somatostatin and parvalbumin containing inhibitory interneurons in the irradiated group . therefore , an imbalance between excitation and inhibition is involved in the neocortical hyperactivity leading to the presence of epileptiform events in this model . the in utero irradiation model has the advantage of being noninvasive to the offspring , which induces less stress ; however , it yields a diffuse type of cortical dysplasia distinct from the typical clinical situation . in any case , studies looking at the vulnerability of irradiated pups to fss have , to our knowledge , not yet been done . the freeze - lesion - induced cortical malformation in rats was developed by dvorak and feit and closely resembles the polymicrogyrus observed in humans , in that it yields the formation of a four - layer neocortex rather than the typical six . to achieve this model , one - day - old rat pups are anaesthetized with isoflurane , their scalp cut at the midline and opened , and a frozen 2 mm large probe is placed on the soft cranium overlying the sensorimotor cortex for a period of ten seconds [ 9 , 17 ] . it should be noted however that the probe width , the lesion duration , and the number of lesions may vary from one study to another . indeed , glial fibrillary acidic protein ( gfap ) as well as bromodeoxyuridine ( brdu ) expressing cells were found in high levels within the dysplastic cortex suggesting the presence of still proliferating astrocytic cells . fiber reorganization occurs within the cortical and subcortical layers of lesion rats as thalamocortical and corticothalamic projections are shown to be affected , possibly implicated in the process of epileptogenesis . on top of the macroscopic modifications taking place in the dysplastic cortex , other changes at the molecular level occur and seem to unbalance the excitation / inhibition equilibrium favoring excitation . looking at the expression of excitatory glutamate receptors , an autoradiography study showed that nmda , ampa , and ka receptor levels were elevated within the dysplastic cortex [ 75 , 76 ] , while they were unchanged when measures were taken in the surrounding normal cortex , suggesting the presence of a spatial gradient of ionotropic glutamate receptors with a greater concentration within the polymicrogyrus . amongst the nmda receptors , the nr2b subunit seems to be of great importance to the epileptogenicity of the lesion as the nr2b currents are functionally enhanced , and specific nr2b antagonists limit the spread of the epileptiform activity [ 71 , 78 ] , although it was shown that an ampar antagonist may block more widespread epileptiform activity measured extracellularly . on the other hand , looking at inhibitory activity , the same autoradiography study showed lowered gabaa and gabab binding within the dysplastic cortex and a downregulation of gabaa inhibition has been shown electrophysiologically in the freeze model . this widespread modification in various gabaa subunits can be the cause of the decrease in inhibitory activity . it is , however , also plausible that the gabaa inhibition downregulation may not be directly involved in the hyperexcitability observed , as the somatostatin - positive interneuron loss occurred after the onset of epileptiform activity in their model . however , despite the hyperexcitability observed in brain slices from lesion animals , there are in this model no recurrent seizures occurring spontaneously in vivo which is an important prerequisite to a good experimental model of human mtle . when the cortical polymicrogyrus model precedes another insult , this represents a two - hit model and mimics the human condition described in our clinical series . in the case of the freeze lesion and hss model , the fss constitute the second hit occurring postnatally while the first hit is thought to occur at an early stage of brain development . therefore , in this model , the freeze lesion is performed at p1 , while the hss are induced at p10 ( figure 1 ) . at the time of hss induction , the temperature necessary to induce a generalized convulsion during hyperthermia was diminished in rats with a cortical lesion compared to rats without lesion , and the latency to attain the generalized convulsion was also shorter . this suggests that the lesion alone seems to predispose the brain and the hippocampus to prolonged fss and their consequences . furthermore , using in vitro electrophysiological recordings , ca1 pyramidal cell hyperexcitability has been shown , yielding greater evoked excitatory postsynaptic potentials ( epsps ) and more frequent spontaneous excitatory postsynaptic currents ( sepscs ) specifically in the double - hit group onto pyramidal cells and onto ca1 interneurons . as the excitatory activity , the inhibitory activity is also altered with greater amplitude gabaa and gabab inhibitory postsynaptic potentials ( ipsps ) and evoked inhibitory postsynaptic currents ( eipscs ) on ca1 pyramidal cells in the double - hit group . this would suggest an excitatory / inhibitory imbalance favoring excitation already at p20 , prior to the occurrence of spontaneous recurrent seizures at p80 . in adulthood , we have found that the double hit results in the occurrence of spontaneous seizures occurring in 86100% of male rats with the seizures arising ipsilateral to the lesion [ 2 , 9 , 12 ] , numbers similar to the mam model and more significant than the 33% observed in naive rats exposed to prolonged fss . ipsilateral hippocampal atrophy persists in the double - hit group and is associated in adults with neuronal loss and memory deficits in performance of a hippocampus - dependent task . our data indicate that ionotropic glutamate receptor expression , especially the nmda subtype , is upregulated in the double - hit animals : nr2b subtype being overexpressed at approximately p20 and nr2a at p80 . this is in accordance with findings from other two - hit models where high levels of nr2b expression are also found : such as in the mam + pilocarpine model or in the prenatal freeze lesion + electrical kindling . the fact that lesion - only animals show higher nr2b levels and greater nmda currents at p20 suggests that the lesion itself may underlie the hyperexcitability at p20 and may be a predisposing factor to prolonged hss susceptibility at p10 , which is supported by a study in the freeze - lesion - only model . furthermore , it is possible that the il-1 expression during hyperthermia , as earlier stated , further exacerbates the effect of the lesion on nmdar . in parallel with the excitatory changes , whether the changes in inhibitory circuits reduce or increase the risk of recurrent seizures remains to be determined . in summary , the lesion alone and the hyperthermia alone appear to each leave the developing brain more vulnerable , but the presence of two hits strongly promotes epileptogenesis . in humans , development of mtle is more and more thought to be a multistage process taking place in early life and including a history of childhood prolonged febrile seizures . a retrospective study from our group demonstrated that 66% of children affected with mtle and a history of fss had dual pathology with the coexistence of hippocampal sclerosis and of a cortical malformation on pathology . more recently , the febstat study group was able to distinguish two subpopulations of fss with those experiencing prolonged fss being younger and with developmental delay . although the models described here involve disorders of neuronal migration , other predisposing factors such as genetic susceptibility can represent the first hit . indeed , it has been shown that , in familial mtle , mesial temporal sclerosis develops in those who have experienced prolonged fss in early life . more so , prospective studies suggest that fs duration is a key factor in leading to hippocampal injury and that developmental abnormalities are indeed also present in children with febrile status and mtle . however , experimental evidence suggests a potential role for nr2b antagonists as not only a good seizure medication but also a potential antiepileptogenic treatment in the developing brain . the role of other potential first hits such as early - life stress in the development of mtle remains to be properly studied . only few animal models of mtle models implying early - life stress paradigms exist in the present literature [ 9498 ] . both corticotropin releasing factor ( crf ) and the glucocorticoid cortisol ( or corticosterone in rodents ) appear to exert potent proconvulsive or hyperexcitable effects on limbic structures in the developing brain [ 96 , 99107 ] . although there is no clear evidence that isolated early - life stressors can induce epileptogenesis , the anatomical and physiological changes produced by these hormones could predispose the developing brain to a second hit . in conclusion , a better understanding of the pathophysiology of mtle in the developing brain will help us develop age - specific treatments not only to control the seizures but also to prevent their occurrence altogether , an important step toward our ultimate goal of no seizure , no side effect .

cognitive training is designed to restore , increase , or optimize capacities in persons suffering from cognitive impairment [ 1 , 2 ] . multimodal training , which combines cognitive training with psychosocial or other activities , has also gained popularity because it is often viewed as more ecological and potentially more clinically relevant . alternatively , a great deal of studies have used specific training protocols to assess in more detail the cognitive processes that show residual plasticity as the brain matures . many of these have reported positive effects of cognitive training in healthy older adults [ 3 , 4 ] and in persons with mild cognitive impairment ( mci ) [ 2 , 5 , 6 , 7 , 8 ] , a transitional stage between normal aging and dementia . more recently , an increasing number of studies have relied on biological measures , mostly brain imaging , to assess the effect of cognitive training in aging . an analysis of the implications and implicit assumptions underlying this approach is therefore timely , particularly with the increasing importance and appeal for the identification of biomarkers . a biomarker is a measurement used as an indicator of normal biological or disease processes . biomarkers should meet the criteria of being a biologically plausible measure of the disease or condition and sharing a coherent relation with the disease progression and severity . for example , brain imaging allows for the measurement of structural and functional changes that are characteristic of alzheimer s disease ( ad ) and that correlate with disease progression and severity . brain imaging measures are also modified by the normal aging process and are related to cognitive changes that occur with age . surrogate markers are used in therapeutic trials as clinically meaningful measures of the effect of a therapy [ 9 , 10 ] . surrogate biomarkers are used when optimal clinical outcomes ( for example , progression to dementia , or death ) might be undesired , require too long a follow - up , or necessitate too large a sample . by using biomarkers , one can design a study where therapeutic effects are measured in a more feasible and timely manner . in the context of cognitive training , biomarkers also can provide invaluable information on the mechanisms through which an intervention enhances cognition . in addition , biomarkers can be used to quantify cognitive and neural plasticity by revealing cognitive and neural compensatory mechanisms , or by indicating patterns of changes that suggest increased efficiency of information processing in aging . not all biological measures are appropriate biomarkers and similarly , not all biomarkers are appropriate surrogates of training efficacy . biomarkers need to be sensitive to the disease and to correlate with progression and severity , and their biological relation with the disease must be at least partially understood . surrogate markers are typically biomarkers of the disease , but they also need to be sensitive to change and reliable over time , and their mechanisms should be relevant to the therapeutic effect that they are expected to indicate . in this review , we will examine structural and functional brain imaging as potential surrogate markers for cognitive training effects . we will discuss studies that have used brain imaging as a marker of cognitive training effects in healthy aging and in mci , the early stage of ad . we also will address some challenges , limitations , and implications of using brain imaging as biomarkers of cognitive training effects in aging populations . structural brain imaging provides quantitative information on brain structure , including whole - brain volume , regional grey matter volumes , cortical thickness , and indicators of white matter integrity and microstructure . brain imaging techniques have provided valuable information regarding the effects of aging on brain structure . such studies have found that brain volume decreases with age and that those structural changes accelerate , with an annual decline of 0.35 % in older adults , compared to 0.12 % in young adults ( see dennis et al . and raz for reviews ) . normal aging does not alter all cognitive functions at the same time in the course of aging , and likewise , some brain regions are more sensitive to age than others . the caudate , cerebellum , hippocampi , and association cortices show the largest volume loss . in addition , aging brings changes in white matter integrity , with greater changes occurring after the seventh decade , and those are localized preferentially in the anterior regions ( frontal and prefrontal ) . training - related structural changes often have been reported in younger adults and middle - aged participants , but very few studies have been published on the structural modifications produced by cognitive training in older adults . in one of these studies , boyke et al . showed with voxel - based morphometry ( vbm ) that grey matter volume increased in older adults after 3 months of learning classic three - ball cascade juggling . changes were observed in the middle temporal area ( v5 ) of the visual cortex , in the left hippocampus , and in the nucleus accumbens bilaterally . interestingly , the training - related changes found in the hippocampus and nucleus accumbens were not observed in young adults . this study suggests that training can induce considerable structural plasticity in older adults , although older adults showed slightly smaller changes than younger adults . it is of note , however , that the authors did not find any correlation between changes in grey matter and performance or exercise intensity . in a more recent study , engvig and colleagues [ 14 ] examined the effects of memory training on cortical thickness in middle - aged and elderly healthy volunteers . after 8 weeks of memory training using the method of loci , participants in the training group showed improved source memory and regional increases in cortical thickness of the lateral orbitofrontal cortex bilaterally and right fusiform cortex . changes in cortical thickness in the right fusiform and lateral orbitofrontal cortex correlated positively with improvement in source memory , indicating that those structural changes are relevant markers of such cognitive changes . the training also had an impact on the alterations found in the white matter microstructure [ 15 ] . using diffusion tensor imaging ( dti ) , the authors found reduced fractional anisotropy ( fa ) in the frontal areas of untrained older adults , whereas no change was found in those who received memory training . training - associated effects on fa seemed to be accompanied by a relative decrease in radial diffusivity , which might indicate a role for myelination in white matter plasticity . in line with other dti studies showing that changes in myelination lead to fa changes in dti , findings by engvig et al . [ 14 , 15 ] suggest that training protects against age - related reductions in myelination . in this study , memory improvements were correlated with changes in anterior fa , providing support for dti as a valid surrogate marker for the effects of cognitive training . also found increased fa ( and decreased mean diffusivity ) in older adults after a multidimensional program with repeated practice on working memory , episodic memory , and perceptual speed tasks . these changes were found in the anterior portion of the corpus callosum but were not correlated with cognitive improvement . surprisingly , no study has reported changes in structural volume or connectivity after executive control or attentional training , despite the fact that these types of process - based training have been widely used with older adults . however , an interesting set of studies suggests that structural brain imaging could be used as a biomarker of the impact of attention training . erickson et al . explored whether the effects of training with the space fortress video game could be predicted by the pretraining volume of either of the two key brain regions implicated in learning and memory : the striatum and the hippocampus . they observed that only dorsal striatal volumes , but not ventral striatum , predicted early acquisition rates . taken together , these findings suggest that structural volume in certain regions of the brain could help predict effects of cognitive training , and that cognitive training can induce short - term structural changes in older adults . measures of regional grey matter volume with vbm , cortical thickness and white matter integrity with dti and fa have been shown to be sensitive to cognitive training in healthy older adults . though we found no study that had used structural brain imaging as a biomarker of training efficacy in mci , structural brain changes have been documented in the early course of ad . in particular , regional cortical thickness was found to be reduced in mci and predicted progression from mci to ad [ 18 , 19 ] . this measure might thus represent a very powerful surrogate marker to assess cognitive training effects in mci populations as well . functional imaging has proven to be a critical tool for understanding the changes in neural mechanisms occurring in aging and ad . it also has many of the features necessary to qualify as a valid surrogate marker in healthy aging and in early ad . when brain imaging is used in older adults to examine changes in cerebral blood flow associated with memory tasks , it reveals a combination of increased activation patterns ( eg , prefrontal activation during working memory tasks ) and decreased activation patterns ( eg , in the left prefrontal and median temporal lobes ) or compensatory activation patterns . compensatory patterns of activation refers to the process by which alternative or atypical brain regions compensate for reduced processing efficiency that occurs with age . compensatory recruitment can involve the hemispheric homologues ( see harold model [ hemispheric asymmetry reduction in older adults ] proposed by cabeza ) or anterior recruitment ( see pasa [ posterior anterior shift in aging ] in davis et al . ) . functional brain imaging also shows impairments early in the development of ad [ 2224 ] . reduced activation is found in the medial temporal areas but also in some regions of the prefrontal cortex of ad patients . studies of mci , the early stage of ad , have reported the presence of hyperactivation early in the process , followed by hypoactivation as patients progress from mci to ad [ 2528 ] . several studies [ 25 , 2931 ] have reported that brain activation differs markedly as a function of the severity of mci , with greater brain activation found in patients with early phase mci than at a later stage in the disease . it was also found that hippocampal activation was inversely associated with disease severity [ 29 , 31 ] . obrien et al . showed a marked decrease in activation over a 2-year period during the mci phase . thus , whereas ad is mostly characterized by reduced brain activation , a number of studies on mci have reported increased activation ; that is , greater task - related brain recruitment in persons with mci than in healthy older adults . there is also accumulating evidence that activation is inversely related to the severity of symptoms and disease . overall , milder symptoms and earlier phases of the disease are characterized by greater task - associated brain recruitment . functional brain imaging can thus qualify as a biomarker of aging and ad because it is sensitive to chronological age . it also reveals a characteristic pattern of impairment in those diagnosed with ad that changes with disease severity and progression . a few studies have used functional brain imaging as a biological surrogate of training efficacy in healthy aging populations and persons with mci . in healthy aging , most studies reporting training - related changes in brain activation patterns have used memory training . among the first studies to do so , noted improved memory performance in both older and younger adults after training with a visual - based mnemonic ( the method of loci ) . they observed increased brain activity ( using positron emission tomography [ pet ] ) during memory encoding in occipitoparietal and frontal brain regions after training in younger adults . in older adults , increased occipitoparietal activity was observed only in participants that improved after training , and no change was observed in frontal regions . valenzuela et al . used localized proton magnetic resonance spectroscopy ( mrs ) to measure changes in the biochemistry of the right hippocampus , midline parietal occipital region , and left frontal lobes after memory training with the method of loci . they observed improved performance after 5 weeks of training accompanied by increased creatine and choline signals in the hippocampus . this study shows that focused memory exercises can induce measurable and persisting biochemical changes in the hippocampus , and that use of the memory program may have led to increased resting oxidative phosphorylation in a region that plays a critical role in memory processing . training - induced changes in brain activity were also reported after attention control training using computer - based programs . erickson et al . conducted a randomized longitudinal dual - task training study of functional mri ( fmri ) activation measures in older adults . they observed training - induced changes in activation in two cortical areas commonly associated with age - related atrophy : the dorsal and ventral prefrontal cortex . interestingly , some brain regions showed equal changes among older and younger adults , whereas others showed differential training effects . for instance , in older adults , increased activity was found in the left ventrolateral prefrontal cortex ( vlpfc ) region ( near broca s area ) , suggesting an increased reliance on verbal or inner speech strategies during dual - task performance . increased verbalization also has been reported as an efficient strategy to enhance performances in training with a switching task . the authors also observed decreased activity in the right vlpfc in both younger and older adults , suggesting a reduced dependence on response selection strategies or a more efficient response - stimulus association . that both age groups showed similar reductions in activity in this region and that changes in brain activation correlated with changes in behavior suggest that patterns of changes in brain activation were important correlates of training - related improvements in behavior and age is not a factor . [ 37 ] examined the behavioral performance and neural activity following 5 weeks of intensive working memory adaptive individualized training . brain activity was measured before and after training , using fmri , while participants performed a working memory task in two difficulty conditions . neocortical brain activity decreased post - training only in the training group , which indicates intervention - related increases in neural efficiency . a few studies have used functional brain imaging as a biological surrogate of training efficacy in mci , and those studies indicate that fmri is sensitive to changes following cognitive training in this group of individuals . [ 38 ] found that memory - related activation increased after strategy memory training in persons with mci . researchers also found that training increased activation in specialized brain regions involved in memory and in compensatory brain regions not typically activated by the verbal memory task ( eg , the right inferior parietal lobe ) . relative to healthy older adults , a number of brain regions that were dysfunctional before training were no longer different following training . importantly , activation of the right inferior parietal lobe was found to correlate with the efficacy of memory training in persons with mci . this indicates that the biological change has a valid relation to the expected clinical outcome . training in this study did not increase activation of the hippocampus , which is surprising considering the importance of this structure in ad . this might be due to the fact that the training program relied heavily on the teaching of visual - based strategies and thus promoted the recruitment of prefrontal and posterior brain regions . [ 39 ] used a memory training method that relied on the identification of salient visual cues to learn new face name associations . in a pilot study , they reported increased activation in the default - mode network , which comprised the medial frontal , parietal , and occipital brain areas , as well as increased connectivity between the temporal lobe , occipital regions , and precuneus following training in mci populations . in a subsequent study , the researchers used a form of memory training relying on mental imagery to learn object location associations and compared it to an exposure - control condition [ 40 ] . before training , they found reduced hippocampal activation in mci participants relative to controls . [ 39 , 40 ] found changes almost exclusively in areas that showed impaired activation before the training . rosen and collaborators randomly assigned persons with mci to either the posit science program ( posit science corporation , san francisco , ca ) , designed to improve speed and temporal auditory processing , or a control condition where patients participated in diverse computerized activities . there was a tendency for a significant correlation between brain changes and behavioral changes , but this was not significant , perhaps due to the very small sample size . to our knowledge , only two studies have examined brain - related changes following executive training in mci participants . carlson et al . randomly assigned 17 individuals with low education , low income , and low mini mental state examination to participate in experience corps , a program promoting social engagement , or to a control condition . brain activation associated with performing an executive function test ( flanker - task ) had increased in the left dorsolateral prefrontal cortex and anterior cingulate gyrus in trained participants . clare et al . reported a single case study where goal - oriented training of one mci patient led to decreased activation in sensory regions and increased activation in memory - related regions during an associative face pet can measure neural activity by recording the uptake of [ 18f]fluorodeoxyglucose ( fdg ) . reduced fdg - pet at rest may reveal early cerebrometabolic changes in ad and could represent a valid biomarker of neuronal injury in early ad and mci [ 44 , 45 ] . a 6-month multicomponent cognitive training program was found to reduce decline in brain glucose metabolism in mci and early ad participants [ 46 ] . the strongest attenuated decline was found in the left anterior temporal lobe and left anterior cingulate in persons with mci . however , there was no correlation between cognitive improvement and changes in glucose metabolism . in turn , small and collaborators found that a multimodal intervention ( cognitive stimulation , physical activity , stress reduction , healthy diet ) decreased fdg - pet uptake in the left dorsolateral prefrontal cortex of non - demented older adults this can be interpreted as supporting more efficient processing following training in older adults , whereas the data with mci might suggest an impact on the pathological process underlying ad . the present review of recent studies provides support for the use of structural and functional brain imaging as sensitive surrogate biomarkers for the effects of cognitive training ( see table 1 for summary findings ) . however , further studies are required to generalize these findings to larger groups and more diverse training protocols . at the structural level , reliable training effects have been reported for regional brain volume , cortical thickness , and white matter microstructure . at the functional level , task - related brain activation ( using fmri and pet ) and fdg - pet at rest were found to be sensitive to training effects . in general , cognitive training led to increased brain volume and increased density and coherence of white matter tracts . functionally , training increased brain metabolism at rest and task - related brain activation in persons with mci ; however healthy older adults showed patterns of increased and decreased activation.table 1summary of findingsbiomarker usedeffect observedgrey matter volume ( vbm)increased volumecortical thicknessincreased thicknesswhite matter integrity ( dti)increased fabiochemistry ( mrs)increased creatine and choline signalglucose metabolism ( fdg - pet)reduced activation in healthy agingincreased activation in mcitask - related activationincreased & decreased activation in healthy agingincreased activation in mcivbm voxel - based morphometry ; dti diffusion tensor imaging ; fa fractional anisotropy ; mrs magnetic resonance spectroscopy ; fdg - pet fluorodeoxyglucose positron emission tomography ; mci mild cognitive impairment vbm voxel - based morphometry ; dti diffusion tensor imaging ; fa fractional anisotropy ; mrs magnetic resonance spectroscopy ; fdg - pet fluorodeoxyglucose positron emission tomography ; mci mild cognitive impairment apart from being sensitive to change , there are a number of other important characteristics to guide the use of biomarkers as surrogate markers of training efficacy . training efficacy is assessed by repeated measurement , and thus , measures ought to be consistent at the individual and/or group level . some of the aforementioned studies have implemented a replication component in their design to assess changes in an independent group of nontreated participants . [ 14 ] , though not a reliability study as such , provides support for the use of cortical thickness as a replicable measure in healthy older adults . studies in young adults have shown that many of the dti measures have good test formal reliability studies have shown that fmri is reliable for use with older adults and persons with mci [ 49 , 50 ] . clement and belleville found very few changes in the areas and levels of activation when comparing two measures separated by a 2-month interval , a timeframe typically found in cognitive training studies . furthermore , it was found that persons with mci , older adults , and young adults showed comparable overlap ratios , a reproducibility index measuring the amount of voxels activated in one versus both sessions . importantly , however , the magnitude of the overlap ratio only indicates moderate between - session agreement . this suggests that treatment effects can be more reliably measured by comparing condition contrasts ( or region of interest [ roi ] ) across sessions rather than by comparing voxel activation ( see also putcha et al . ) . similarly , intraclass correlations show important intrasubject variability , indicating that while reliable at the group level , care should be taken when interpreting fmri treatment data at the level of individuals . one other important characteristic when selecting a surrogate marker is that the selected biological measure reflects a clinically relevant change . one way to determine this is by examining the correlation between changes in the biological marker and changes in the relevant clinical or cognitive outcome . in some of these studies , correlations were found between changes in the biomarker and changes in cognitive or clinical status ( for example , [ 15 , 38 , 35 ] ) . however , this was not systematically found ( for example , [ 13 , 16 , 41 , 46 ] ) or even documented . lack of a relation might be attributable to many factors , which need to be better understood , including insufficient power , inappropriate selection of clinical variables , or the presence of a nonselective effect . relevance also can be determined by selecting biomarkers that reflect neuropathological processes underlying the disease , and in that case , it is important to fully understand the relationship between the surrogate marker and the pathophysiology of the condition of interest . most studies have used markers that are considered valid measures of the processes that underlie normal aging or ad . however , some critical biomarkers of early ad have surprisingly not been tested as surrogate markers of training . it is unclear whether short - term cognitive training is likely to have an effect on beta - amyloid deposition . however , some recent studies have reported that having engaged in cognitively stimulating activities in early and middle life is associated with reduced beta - amyloid accumulation in older adults . it has been suggested that the increased synaptic activity provided by cognitive stimulation might protect against amyloid deposition . in conclusion , this review supports the notion that brain - imaging techniques could be reliably used to assess training - related changes in brain structure and function in healthy older adults and patients with mci or ad . however , future studies are needed to identify all the potential biomarkers and surrogate biomarkers of brain plasticity induced by cognitive training interventions . we believe that following a structured methodology that meets the prerequisites provided in the present review would effectively guide future research and help to increase the body of knowledge on brain changes associated with cognitive training in older adults .

an increasing number of studies have relied on brain imaging to assess the effects of cognitive training in healthy aging populations and in persons with early alzheimer s disease or mild cognitive impairment ( mci ) . at the structural level , cognitive training in healthy aging individuals has been associated with increased brain volume , cortical thickness , and density and coherence of white matter tracts . at the functional level , task - related brain activation ( using fmri and pet ) and fluorodeoxyglucose positron emission tomography ( fdg - pet ) were found to be sensitive to the effects of training . in persons with mci , cognitive training increased brain metabolism and task - related brain activation , whereas healthy older adults showed patterns of increased and decreased activation . further studies are required to generalize these findings to larger groups and to investigate more diverse training protocols . research will also need to address important methodological issues regarding the use of biomarkers in cognitive aging , including reliability , clinical validity , and relevance to the pathophysiological process .

Introduction Structural Imaging as a Biomarker of Cognitive Training Functional Imaging as a Biomarker of Cognitive Training Implications for Future Research Conclusions

cognitive training is designed to restore , increase , or optimize capacities in persons suffering from cognitive impairment [ 1 , 2 ] . multimodal training , which combines cognitive training with psychosocial or other activities , has also gained popularity because it is often viewed as more ecological and potentially more clinically relevant . alternatively , a great deal of studies have used specific training protocols to assess in more detail the cognitive processes that show residual plasticity as the brain matures . many of these have reported positive effects of cognitive training in healthy older adults [ 3 , 4 ] and in persons with mild cognitive impairment ( mci ) [ 2 , 5 , 6 , 7 , 8 ] , a transitional stage between normal aging and dementia . more recently , an increasing number of studies have relied on biological measures , mostly brain imaging , to assess the effect of cognitive training in aging . an analysis of the implications and implicit assumptions underlying this approach is therefore timely , particularly with the increasing importance and appeal for the identification of biomarkers . biomarkers should meet the criteria of being a biologically plausible measure of the disease or condition and sharing a coherent relation with the disease progression and severity . for example , brain imaging allows for the measurement of structural and functional changes that are characteristic of alzheimer s disease ( ad ) and that correlate with disease progression and severity . brain imaging measures are also modified by the normal aging process and are related to cognitive changes that occur with age . in the context of cognitive training , biomarkers also can provide invaluable information on the mechanisms through which an intervention enhances cognition . not all biological measures are appropriate biomarkers and similarly , not all biomarkers are appropriate surrogates of training efficacy . biomarkers need to be sensitive to the disease and to correlate with progression and severity , and their biological relation with the disease must be at least partially understood . surrogate markers are typically biomarkers of the disease , but they also need to be sensitive to change and reliable over time , and their mechanisms should be relevant to the therapeutic effect that they are expected to indicate . in this review , we will examine structural and functional brain imaging as potential surrogate markers for cognitive training effects . we will discuss studies that have used brain imaging as a marker of cognitive training effects in healthy aging and in mci , the early stage of ad . we also will address some challenges , limitations , and implications of using brain imaging as biomarkers of cognitive training effects in aging populations . structural brain imaging provides quantitative information on brain structure , including whole - brain volume , regional grey matter volumes , cortical thickness , and indicators of white matter integrity and microstructure . brain imaging techniques have provided valuable information regarding the effects of aging on brain structure . such studies have found that brain volume decreases with age and that those structural changes accelerate , with an annual decline of 0.35 % in older adults , compared to 0.12 % in young adults ( see dennis et al . normal aging does not alter all cognitive functions at the same time in the course of aging , and likewise , some brain regions are more sensitive to age than others . the caudate , cerebellum , hippocampi , and association cortices show the largest volume loss . in addition , aging brings changes in white matter integrity , with greater changes occurring after the seventh decade , and those are localized preferentially in the anterior regions ( frontal and prefrontal ) . training - related structural changes often have been reported in younger adults and middle - aged participants , but very few studies have been published on the structural modifications produced by cognitive training in older adults . changes were observed in the middle temporal area ( v5 ) of the visual cortex , in the left hippocampus , and in the nucleus accumbens bilaterally . interestingly , the training - related changes found in the hippocampus and nucleus accumbens were not observed in young adults . this study suggests that training can induce considerable structural plasticity in older adults , although older adults showed slightly smaller changes than younger adults . in a more recent study , engvig and colleagues [ 14 ] examined the effects of memory training on cortical thickness in middle - aged and elderly healthy volunteers . after 8 weeks of memory training using the method of loci , participants in the training group showed improved source memory and regional increases in cortical thickness of the lateral orbitofrontal cortex bilaterally and right fusiform cortex . changes in cortical thickness in the right fusiform and lateral orbitofrontal cortex correlated positively with improvement in source memory , indicating that those structural changes are relevant markers of such cognitive changes . the training also had an impact on the alterations found in the white matter microstructure [ 15 ] . using diffusion tensor imaging ( dti ) , the authors found reduced fractional anisotropy ( fa ) in the frontal areas of untrained older adults , whereas no change was found in those who received memory training . training - associated effects on fa seemed to be accompanied by a relative decrease in radial diffusivity , which might indicate a role for myelination in white matter plasticity . [ 14 , 15 ] suggest that training protects against age - related reductions in myelination . in this study , memory improvements were correlated with changes in anterior fa , providing support for dti as a valid surrogate marker for the effects of cognitive training . also found increased fa ( and decreased mean diffusivity ) in older adults after a multidimensional program with repeated practice on working memory , episodic memory , and perceptual speed tasks . these changes were found in the anterior portion of the corpus callosum but were not correlated with cognitive improvement . surprisingly , no study has reported changes in structural volume or connectivity after executive control or attentional training , despite the fact that these types of process - based training have been widely used with older adults . however , an interesting set of studies suggests that structural brain imaging could be used as a biomarker of the impact of attention training . explored whether the effects of training with the space fortress video game could be predicted by the pretraining volume of either of the two key brain regions implicated in learning and memory : the striatum and the hippocampus . taken together , these findings suggest that structural volume in certain regions of the brain could help predict effects of cognitive training , and that cognitive training can induce short - term structural changes in older adults . measures of regional grey matter volume with vbm , cortical thickness and white matter integrity with dti and fa have been shown to be sensitive to cognitive training in healthy older adults . though we found no study that had used structural brain imaging as a biomarker of training efficacy in mci , structural brain changes have been documented in the early course of ad . in particular , regional cortical thickness was found to be reduced in mci and predicted progression from mci to ad [ 18 , 19 ] . this measure might thus represent a very powerful surrogate marker to assess cognitive training effects in mci populations as well . functional imaging has proven to be a critical tool for understanding the changes in neural mechanisms occurring in aging and ad . it also has many of the features necessary to qualify as a valid surrogate marker in healthy aging and in early ad . when brain imaging is used in older adults to examine changes in cerebral blood flow associated with memory tasks , it reveals a combination of increased activation patterns ( eg , prefrontal activation during working memory tasks ) and decreased activation patterns ( eg , in the left prefrontal and median temporal lobes ) or compensatory activation patterns . compensatory patterns of activation refers to the process by which alternative or atypical brain regions compensate for reduced processing efficiency that occurs with age . compensatory recruitment can involve the hemispheric homologues ( see harold model [ hemispheric asymmetry reduction in older adults ] proposed by cabeza ) or anterior recruitment ( see pasa [ posterior anterior shift in aging ] in davis et al . ) functional brain imaging also shows impairments early in the development of ad [ 2224 ] . studies of mci , the early stage of ad , have reported the presence of hyperactivation early in the process , followed by hypoactivation as patients progress from mci to ad [ 2528 ] . several studies [ 25 , 2931 ] have reported that brain activation differs markedly as a function of the severity of mci , with greater brain activation found in patients with early phase mci than at a later stage in the disease . thus , whereas ad is mostly characterized by reduced brain activation , a number of studies on mci have reported increased activation ; that is , greater task - related brain recruitment in persons with mci than in healthy older adults . there is also accumulating evidence that activation is inversely related to the severity of symptoms and disease . overall , milder symptoms and earlier phases of the disease are characterized by greater task - associated brain recruitment . functional brain imaging can thus qualify as a biomarker of aging and ad because it is sensitive to chronological age . a few studies have used functional brain imaging as a biological surrogate of training efficacy in healthy aging populations and persons with mci . in healthy aging , most studies reporting training - related changes in brain activation patterns have used memory training . they observed increased brain activity ( using positron emission tomography [ pet ] ) during memory encoding in occipitoparietal and frontal brain regions after training in younger adults . in older adults , increased occipitoparietal activity was observed only in participants that improved after training , and no change was observed in frontal regions . used localized proton magnetic resonance spectroscopy ( mrs ) to measure changes in the biochemistry of the right hippocampus , midline parietal occipital region , and left frontal lobes after memory training with the method of loci . they observed improved performance after 5 weeks of training accompanied by increased creatine and choline signals in the hippocampus . this study shows that focused memory exercises can induce measurable and persisting biochemical changes in the hippocampus , and that use of the memory program may have led to increased resting oxidative phosphorylation in a region that plays a critical role in memory processing . conducted a randomized longitudinal dual - task training study of functional mri ( fmri ) activation measures in older adults . they observed training - induced changes in activation in two cortical areas commonly associated with age - related atrophy : the dorsal and ventral prefrontal cortex . interestingly , some brain regions showed equal changes among older and younger adults , whereas others showed differential training effects . for instance , in older adults , increased activity was found in the left ventrolateral prefrontal cortex ( vlpfc ) region ( near broca s area ) , suggesting an increased reliance on verbal or inner speech strategies during dual - task performance . increased verbalization also has been reported as an efficient strategy to enhance performances in training with a switching task . the authors also observed decreased activity in the right vlpfc in both younger and older adults , suggesting a reduced dependence on response selection strategies or a more efficient response - stimulus association . that both age groups showed similar reductions in activity in this region and that changes in brain activation correlated with changes in behavior suggest that patterns of changes in brain activation were important correlates of training - related improvements in behavior and age is not a factor . brain activity was measured before and after training , using fmri , while participants performed a working memory task in two difficulty conditions . neocortical brain activity decreased post - training only in the training group , which indicates intervention - related increases in neural efficiency . a few studies have used functional brain imaging as a biological surrogate of training efficacy in mci , and those studies indicate that fmri is sensitive to changes following cognitive training in this group of individuals . [ 38 ] found that memory - related activation increased after strategy memory training in persons with mci . researchers also found that training increased activation in specialized brain regions involved in memory and in compensatory brain regions not typically activated by the verbal memory task ( eg , the right inferior parietal lobe ) . relative to healthy older adults , a number of brain regions that were dysfunctional before training were no longer different following training . importantly , activation of the right inferior parietal lobe was found to correlate with the efficacy of memory training in persons with mci . this indicates that the biological change has a valid relation to the expected clinical outcome . training in this study did not increase activation of the hippocampus , which is surprising considering the importance of this structure in ad . this might be due to the fact that the training program relied heavily on the teaching of visual - based strategies and thus promoted the recruitment of prefrontal and posterior brain regions . [ 39 ] used a memory training method that relied on the identification of salient visual cues to learn new face name associations . in a pilot study , they reported increased activation in the default - mode network , which comprised the medial frontal , parietal , and occipital brain areas , as well as increased connectivity between the temporal lobe , occipital regions , and precuneus following training in mci populations . rosen and collaborators randomly assigned persons with mci to either the posit science program ( posit science corporation , san francisco , ca ) , designed to improve speed and temporal auditory processing , or a control condition where patients participated in diverse computerized activities . there was a tendency for a significant correlation between brain changes and behavioral changes , but this was not significant , perhaps due to the very small sample size . to our knowledge , only two studies have examined brain - related changes following executive training in mci participants . randomly assigned 17 individuals with low education , low income , and low mini mental state examination to participate in experience corps , a program promoting social engagement , or to a control condition . brain activation associated with performing an executive function test ( flanker - task ) had increased in the left dorsolateral prefrontal cortex and anterior cingulate gyrus in trained participants . reported a single case study where goal - oriented training of one mci patient led to decreased activation in sensory regions and increased activation in memory - related regions during an associative face pet can measure neural activity by recording the uptake of [ 18f]fluorodeoxyglucose ( fdg ) . reduced fdg - pet at rest may reveal early cerebrometabolic changes in ad and could represent a valid biomarker of neuronal injury in early ad and mci [ 44 , 45 ] . a 6-month multicomponent cognitive training program was found to reduce decline in brain glucose metabolism in mci and early ad participants [ 46 ] . the strongest attenuated decline was found in the left anterior temporal lobe and left anterior cingulate in persons with mci . in turn , small and collaborators found that a multimodal intervention ( cognitive stimulation , physical activity , stress reduction , healthy diet ) decreased fdg - pet uptake in the left dorsolateral prefrontal cortex of non - demented older adults this can be interpreted as supporting more efficient processing following training in older adults , whereas the data with mci might suggest an impact on the pathological process underlying ad . the present review of recent studies provides support for the use of structural and functional brain imaging as sensitive surrogate biomarkers for the effects of cognitive training ( see table 1 for summary findings ) . however , further studies are required to generalize these findings to larger groups and more diverse training protocols . at the structural level , reliable training effects have been reported for regional brain volume , cortical thickness , and white matter microstructure . at the functional level , task - related brain activation ( using fmri and pet ) and fdg - pet at rest were found to be sensitive to training effects . in general , cognitive training led to increased brain volume and increased density and coherence of white matter tracts . functionally , training increased brain metabolism at rest and task - related brain activation in persons with mci ; however healthy older adults showed patterns of increased and decreased activation.table 1summary of findingsbiomarker usedeffect observedgrey matter volume ( vbm)increased volumecortical thicknessincreased thicknesswhite matter integrity ( dti)increased fabiochemistry ( mrs)increased creatine and choline signalglucose metabolism ( fdg - pet)reduced activation in healthy agingincreased activation in mcitask - related activationincreased & decreased activation in healthy agingincreased activation in mcivbm voxel - based morphometry ; dti diffusion tensor imaging ; fa fractional anisotropy ; mrs magnetic resonance spectroscopy ; fdg - pet fluorodeoxyglucose positron emission tomography ; mci mild cognitive impairment vbm voxel - based morphometry ; dti diffusion tensor imaging ; fa fractional anisotropy ; mrs magnetic resonance spectroscopy ; fdg - pet fluorodeoxyglucose positron emission tomography ; mci mild cognitive impairment apart from being sensitive to change , there are a number of other important characteristics to guide the use of biomarkers as surrogate markers of training efficacy . training efficacy is assessed by repeated measurement , and thus , measures ought to be consistent at the individual and/or group level . some of the aforementioned studies have implemented a replication component in their design to assess changes in an independent group of nontreated participants . [ 14 ] , though not a reliability study as such , provides support for the use of cortical thickness as a replicable measure in healthy older adults . studies in young adults have shown that many of the dti measures have good test formal reliability studies have shown that fmri is reliable for use with older adults and persons with mci [ 49 , 50 ] . clement and belleville found very few changes in the areas and levels of activation when comparing two measures separated by a 2-month interval , a timeframe typically found in cognitive training studies . furthermore , it was found that persons with mci , older adults , and young adults showed comparable overlap ratios , a reproducibility index measuring the amount of voxels activated in one versus both sessions . this suggests that treatment effects can be more reliably measured by comparing condition contrasts ( or region of interest [ roi ] ) across sessions rather than by comparing voxel activation ( see also putcha et al . ) similarly , intraclass correlations show important intrasubject variability , indicating that while reliable at the group level , care should be taken when interpreting fmri treatment data at the level of individuals . in some of these studies , correlations were found between changes in the biomarker and changes in cognitive or clinical status ( for example , [ 15 , 38 , 35 ] ) . lack of a relation might be attributable to many factors , which need to be better understood , including insufficient power , inappropriate selection of clinical variables , or the presence of a nonselective effect . relevance also can be determined by selecting biomarkers that reflect neuropathological processes underlying the disease , and in that case , it is important to fully understand the relationship between the surrogate marker and the pathophysiology of the condition of interest . most studies have used markers that are considered valid measures of the processes that underlie normal aging or ad . however , some critical biomarkers of early ad have surprisingly not been tested as surrogate markers of training . it is unclear whether short - term cognitive training is likely to have an effect on beta - amyloid deposition . however , some recent studies have reported that having engaged in cognitively stimulating activities in early and middle life is associated with reduced beta - amyloid accumulation in older adults . it has been suggested that the increased synaptic activity provided by cognitive stimulation might protect against amyloid deposition . in conclusion , this review supports the notion that brain - imaging techniques could be reliably used to assess training - related changes in brain structure and function in healthy older adults and patients with mci or ad . however , future studies are needed to identify all the potential biomarkers and surrogate biomarkers of brain plasticity induced by cognitive training interventions . we believe that following a structured methodology that meets the prerequisites provided in the present review would effectively guide future research and help to increase the body of knowledge on brain changes associated with cognitive training in older adults .

microbial metabolism is regarded as the most important mechanism of pesticide degradation in soil [ 12 , 13 ] , and it constitutes the basis for all bioremediation and bioaugmentation strategies . therefore , conditions that favor microbial growth and activity in soil , such as temperature , moisture , nutrient status , ph , and aeration , will also generally promote metabolic degradation of pesticides . as suggested by gadd amongst others , the composition and size of soil microbial populations , as well as the status of metabolic activity , are the determining factors as to whether or not biodegradation is feasible as a remediation option . biodegradation refers to the metabolic ability of microorganisms to transform organic contaminants into less harmful compounds . according to mcfarland et al . , bioremediation techniques aim to accelerate the naturally occurring biodegradation process by optimizing the conditions under which it occurs . in many contaminated areas , even though suitable microbial populations may be available for biodegradation of a given contaminant , environmental conditions may limit or even inhibit this process . in such cases , biostimulation of the degrading potential of native microbial populations and/or the addition of selected degrading microorganisms to contaminated soil ( bioaugmentation ) have been effective at enhancing pesticide metabolism [ 2 , 17 ] . biostimulation typically involves the addition of limited nutrients ( e.g. , carbon and nitrogen sources , o2 ) , acid or bases for ph optimization , or water or specific substrates to stimulate specific enzymes . it is an effective bioremediation strategy [ 15 , 16 ] , although it may have poor reproducibility and be dependent on the characteristics of microbial populations . indeed , the major advantage offered by bioaugmentation is the ability to choose the introduced species based on the goals of the process and the conditions of the matrix . in this case , the success of bioremediation is mainly dependent on the competition / proliferation capability of the introduced species and the bioavailability of the xenobiotic compounds . bioavailability here refers to the acquisition and subsequent transformation / degradation of the compound and is closely related to its chemical properties , as well as to a wide range of soil physical and chemical parameters . in addition to mineralization , which implies the use of xenobiotics as a carbon source , microorganisms can also co - metabolize pesticides , e.g. , transforming them into metabolites while growing and obtaining energy from other substrates found in soil [ 3 , 9 ] . many of the reactions involved in co - metabolism of pesticides , including oxidation - reduction , de - halogenation , ring - cleavage , and hydrolysis , occur simultaneously . transformation can lead to complete detoxification , breakdown of products , which may be further attacked by other microbial groups , or in some cases , to more toxic metabolites . triazines , among other halogenated aromatic compounds , are often co - metabolized into more toxic metabolites [ 9 , 10 ] . for example , microbial enzyme - mediated methylation reactions usually increase herbicide lipophilicity and thus the potential for bioaccumulation in the food chain . the use of bioremediation to remove pollutants is typically less expensive than equivalent physical - chemical methods . this technology offers the potential to treat contaminated soil and groundwater on site without the need for excavation [ 3 , 18 ] , requires relatively little energy input , and preserves the soil structure . perhaps the most attractive feature of bioremediation is its reduced impact on natural ecosystems , which should be well received by the public . for fungal systems , bioremediation requires the soil to be aerobic with the provision of enough oxygen to enable effective colonization to occur . for any xenobiotic compound , the threshold concentration above which remediation becomes necessary is referred to as the so - called " remediation trigger level . " however , for many pesticides as well as xenobiotic compounds , the threshold concentration has not yet been established . generally , the target concentration is assumed to be in the 1 ppm ( mg / l ) range , but in practice it can vary from site to site and region to region [ 3 , 10 ] . very often , urban application of pesticides is carried out at an excessively high concentration , resulting in pesticide waste characterized by prolonged persistence . when applied at normal agricultural rates , which can be between 1~4.5 kg / ha , pesticide degradation in soil suggested that even at these concentrations , top - soil residues have been found to last for several years , ranging from 0.5 to 2.5 ppm . unfortunately , even when present in soil at the ppb level ( g / l ) , many recalcitrant compounds often migrate through leaching and reach groundwater . at present , bioremediation conducted on a commercial scale predominantly utilizes prokaryotes , with comparatively few recent attempts using white rot fungi . bacteria are very sensitive to fluctuating environmental conditions in soil since their growth requires films of water to form in soil pores . however , filamentous fungi offer major advantages over bacteria regarding the diversity of compounds they are able to oxidize . in addition , they are robust organisms that are generally more tolerant to high concentrations of polluting chemicals than bacteria . filamentous fungi are also more tolerant of environmental stress and can produce copious amounts of extracellular enzymes during hyphal colonization of soil , resulting in enhanced rates of bioremediation [ 22 , 23 ] . therefore , white rot fungi potentially represent a powerful tool for soil bioremediation , with some species already patented . interestingly , only a few companies have incorporated ligninolytic fungi for soil remediation into their program , e.g. , " earthfax development corp . " in usa or " gebruder huber bodenrecycling " in germany . however , many research studies have been carried out to examine the efficacy of bioremediation fungi in degrading single xenobiotic compounds . the application of fungi for the cleanup of contaminated soil first came to attention in the mid-1980s when the white rot fungus phanerochaete chrysosporium was shown to metabolize a range of organic environmental contaminants [ 25 , 26 ] . later , this ability was demonstrated for other white rot fungi , including trametes versicolor and pleurotus ostreatus . white rot fungi are the most widely studied and understood ligninolytic fungi to date [ 2 , 3 , 27 - 29 ] . in nature , these fungi colonize and degrade lignocellulosic materials ( normally tree wood ) and are responsible for causing white rot of wood . lignin is a three - dimensional , naturally occurring polymer present in woody plants , and it constitutes one of the most structurally complex and therefore resistant materials to microbial degradation [ 9 , 30 ] . the ability of white rot fungi to mineralize lignin is generally attributed to the secretion of extracellular ligninolytic enzymes , mostly laccase ( lac ) , lignin peroxidase ( lip ) , and manganese peroxidase ( mnp ) [ 9 , 27 , 29 ] . it is accepted that both of these peroxidases catalyze the oxidation of ( endogenously - produced ) low - molecular weight mediators using h2o2 as an oxidant . these powerful mediators then oxidize lignin , leaving it partially modified and open to further attack by other enzymes such as lac [ 9 , 10 ] . a main feature of lac is its highly non - specific nature with regard to the breakdown of substrates . further , xenobiotics share at least one of many sub - structures ( e.g. , combination of functional groups ) present in the lignin molecule . this explains the ability of white rot fungi to tolerate and degrade such a wide range of environmental organic pollutants , even when at high concentrations [ 3 , 9 ] . it has been shown that both lac and peroxidases co - metabolize these compounds with lignin through similar oxidative mechanisms [ 2 , 9 , 30 ] , although with no net energy gain . in fact , oxidation of lignin is performed in order for these fungi to have access to wood polysaccharides , which is their main energy source and which other microorganisms can not access . this implies that the presence of lignocellulosic substrates is a requirement for the degradation of xenobiotic compounds . although lac activity is involved in lignin degradation , it is also associated with microbial growth or specific interactions between microorganisms , particularly with regard to t. versicolor . determining the activity of lac in soil inoculated with white rot species provides a measure of the colonizing ability of the fungus and can be used to monitor the bioremediation of numerous soil contaminants , among them triazine pesticides [ 28 , 30 , 31 ] . other applications of lac activity were reported by schmidt et al . , who studied the impact of fungal inoculum properties on t. versicolor growth and activity in soil . measured lac activity to demonstrate the correlation between its production and the degradation of polycyclic aromatic hydrocarbons ( pahs ) by several strains of white rot fungi in both liquid culture and soil . paszczynski and crawford , gadd , and aek et al . reviewed a series of applications involving white rot fungi , among them t. versicolor , for environmental remediation of pesticides , disinfectants ( e.g. , pentachlorophenol ) synthetic dyes , benzene derivatives ( e.g. , petrol and diesel ) , pahs , explosives ( trinitrotoluene , tnt derivatives ) , and industrial solvents ( e.g. , polychlorinated biphenyls ) . they also offered descriptions on the use of these fungi for the biotransformation of coal , treatment of effluents from paper and olive - processing plants , and the degradation of synthetic polymers ( e.g. , plastics ) and other materials ( e.g. , nylon ) . all of these applications were found to be related to the production of lacs , mn - peroxidase or ( less frequently ) ligninperoxidases , both alone or in combination , which has been corroborated by other studies [ 28 , 30 , 31 , 34 ] . however , most studies performed thus far have focused on the screening of white rot fungi for the bioremediation of xenobiotic contaminants in liquid culture media , bioreactors , sterile soil or soil extract broth . in non - sterile soil , where pesticide degradation may be influenced by other factors other than the fungus , our knowledge is more limited . recently , fragoeiro and magan reported the successful application of three white rot species , including t. versicolor , for the bioremediation of mixtures of pesticides in non - sterile sandy loam soil under low water potential conditions . this demonstrated that differential breakdown of mixtures of pesticides occurred possibly due to environmental factors . lac activity in the biodegradation of xenobiotic compounds with lignin - like structures has already attracted considerable interest , and its biodegradative effects on different contaminants have been exhaustively studied . specifically , lac enzyme is a copper - containing phenoloxidase involved in the degradation of lignin , and it oxidizes phenol and phenolic lignin sub - structures . the catabolic role of fungal lac in lignin biodegradation is not well understood [ 38 , 40 ] , but there have been some successful instances of this enzyme performing decontamination . for example , dye decoloration by trametes hispida , degradation of azo - dyes by pyricularia oryzae , and textile effluent degradation by t. versicolor have all been attributed to lac activity . esposito et al . also reported that lac from cerrena unicolor results in complete transformation of 2,4 dcp in soil colloids . demir examined the biological degradation of benzene and toluene by t. versicolor as well as biomass determinations . complete removal of benzene and toluene was observed after 4 hr when the initial toluene concentration was 50 mg / l , whereas it took 36 hr for complete degradation at an initial concentration of 300 mg / l . regarding benzene , biodegradation was completed after 4 hr at an initial concentration of 50 mg / l , whereas it took 42 hr to completely remove benzene at 300 mg addition of veratryl alcohol , a lac inducer , to the basic feed medium enhanced the performance of the enzyme system and shortened the overall time period of biodegradation completed in a shorter time period . after 36 hr , about 46 and 65% of the compound added at initial concentrations of 100 mg / l to shaken and static fungal cultures were removed , respectively . although the removal percentage was highest ( 76.7% ) at 10mg / l , the transformation rate was maximal ( 0.82mg / hr ) at 100mg / l of phenanthrene in the fungal culture . however , when purified lac of t. versicolor was reacted with phenanthrene , the compound was not transformed . another interesting example of contaminant degradation and enzyme activity was seen in the study described by barr and aust . they found cyanide to be quite toxic to spores of p. chrysosporium ( 50% inhibition of glucose metabolism at 2.6 mg / l ) due to the absence of lips , which can rapidly metabolize cyanide , as ligninolytic 6-day - old cultures were able to tolerate considerably higher cyanide concentrations ( 50% inhibition of glucose metabolism at 182 their results showed that purified lips and mnps were capable of mineralization in a multistep pathway . esposito et al . showed that different actinomycetes were able to degrade diuron in soil using mnps . despite many reports that the degradation of xenobiotics by white rot fungi is mediated by enzymes involved in lignin degradation , some authors have presented contradictory evidence . for example , jackson et al . reported the degradation of tnt by non - ligninolytic strains of p. chrysosporium . . showed > 86% degradation of atrazine and terbuthylazine by white rot fungi in liquid culture and found no relationship between degradation rate and ligninolytic activity . other studies featuring p. chrysosporium in liquid culture have reported biotransformation of the insecticide lindane independently of the production of ligninolytic enzymes . these researchers ruled out the involvement of peroxidases in lindane biotransformation and mineralization , and they assessed the activity of cytochrome p450 monooxygenase , an enzymatic system used by many organisms for detoxification . they found that the p450 inactivator 1-aminobenzotriazole drastically reduced pesticide metabolism , whereas phobarbital , a p450 inducer , did not increase lindane breakdown . whether the degradation of pesticides is carried out by lignin - degrading enzymes or other enzymatic systems , or both , the use of fungi in bioremediation is clearly very promising , and further studies should be conducted to understand which enzymes are involved . such information could be very useful in establishing the best conditions for enzyme production followed by fungal bioremediation in situ . it is also necessary to assess the production of these enzymes in soil , since it is where bioremediation occurs under field conditions and since there are considerably more studies on enzyme production in liquid cultures . additionally , there is little information on the degradation of mixtures of xenobiotics , which is more common in nature than single ones . recent work on the p. chrysosporium genome has shown that cytochrome p450 monooxygenases constitute the largest and most important group of p450 genes in any fungal species and that they are differentially expressed depending on xenobiotic type and nutrition [ 47 , 48 ] . this has resulted in the development of powerful tools useful in understanding the roles of key enzymes produced by p. chrysosporium in the bioremediation of different xenobiotic compounds under various environmental conditions . however , these tools have seldom been applied to the examination of how environmental stress factors such as water potential and temperature effects on the expression of p450 gene clusters involved in enzyme production both in vitro and in situ . the in vitro environmentally relevant screening of white rot species using both single and mixtures of pesticides can be used to identify potential candidates when followed by in situ microcosm studies using different inoculant formulations . this can be instructive and useful in understanding the approaches and strategies needed for the development of effective bioremediation systems using white rot fungi . tolerance and growth of white rot fungi in the presence of individual and mixtures of xenobiotic compounds may vary depending upon the type and concentration of the xenobiotic as well as the nutritional and environmental conditions . it is important to use realistic nutritional media such as soil extract - based systems , in which the water potential , ph , and temperature can be modified , since these conditions can have a significant impact on the relative tolerance of a potential fungal bioremediation agent . table 1 shows an example of the concentrations of single and mixtures of pesticides ( effective concentration ; ec50 values ) required to control a range of white rot fungal isolates in response to changes in water potential . the results highlight the importance of examining such factors when determining the relative tolerance / sensitivity to single and mixtures of pesticides as well as when choosing appropriate isolates for subsequent use in situ . in this case , -0.7 and -2.8mpa water potential were used to represent conditions under which water was available and plants would grow , with the latter being twice the wilting point of plants . this result can be compared with previous in vitro modifications of soil extract media in which the water potential was modified matrically using peg8000 as a solute . we previously showed that many trametes and related species are more sensitive to matric than solute stress [ 46 , 49 , 50 ] . table 1 shows an example of the relative growth levels of isolates of t. versicolor and p. ostreatus in response to individual and mixtures of pesticides . the results show that under matric stress conditions , these fungi are able to grow at the wilting point of plants but not at twice this level . this approach is important in determining whether or not the candidate isolates may be effective in situ . the applicability of fungi to the bioremediation of soil contaminated with pesticides depends on the capacity of fungi to grow in the presence of such compounds as well as their ability to produce degradative enzymes . additionally , complementary information on the capacity and ability of fungi to produce the key extracellular enzymes required for degradation of individual and mixtures of xenobiotic compounds is required . a significant amount of research on white rot fungi has been conducted in liquid and/or synthetic media , but less is known about its bioremediation capabilities in soil , especially under different environmental conditions . reported that field conditions do not always enable white rot fungi such as p. chrysosporium to achieve optimum activity , and therefore it is not a good competitor in a soil environment [ 37 , 40 ] . , who reported that bacteria from polluted and agricultural soil antagonize the growth of p. chrysosporium on solid media . nevertheless , some studies have described the successful application of p. chrysosporium as a bioremediation agent in soil . for example , mcfarland et al . described complete alachlor transformation by this fungus within 56 days of treatment . reddy and mathew also showed that this species was able to degrade ddt , lindane , and atrazine . recently , we demonstrated that under different osmotic stress regimes , white rot fungi are able to differentially degrade mixtures of pesticides in soil extract broth ( table 2 ) . we also demonstrated an increase in the range of hydrolytic enzyme production , including ligninases and cellulases , even under water stress conditions ( table 3 ) . although it is accepted that extracellular ligninolytic enzymes are at least in part responsible for the critical initial reactions of pollutant transformation , the production and activity of these enzymes in contaminated soil under different field conditions have not been examined in detail , although they are critical for successful degradation [ 36 , 52 ] . there are many studies on how to optimize the biodegradation potential of white rot fungi in contaminated soil [ 16 , 35 , 53 , 54 ] . if it is accepted that extracellular ligninolytic enzymes are at least in part responsible for the critical initial reactions of pollutant transformation , then the production and activity of these enzymes in contaminated soil under field conditions are both prerequisites for the successful application of white rot fungi in soil bioremediation . additionally , bioremedial fungi should be able to secrete the necessary enzymes into the soil matrix in order to enhance the degradation of pesticide molecules that would otherwise be unable to be incorporated across cell walls . most protocols for delivering inoculum of wood rot fungi for soil bioremediation have been adopted from mushroom growers , who have perfected the art of producing fungal spawn on lignocellulosic waste . species used in mushroom production have been formulated on inexpensive substrates , including corncob , sawdust , wood chips , peat or wheat straw . when used in bioremediation , these substrates are impregnated with mycelium and mixed with contaminated soil [ 7 , 33 ] . there is little information available on the survival of white rot fungi in soil , especially those fungi not consumed by humans . several groups are investigating methods of improving the survival of wood rot fungi in polluted soils [ 9 , 35 , 53 ] . certainly , better fungal growth could help introduced fungi overcome competition from indigenous microorganisms as well as enhance bioremediation . this is important as native soil microorganisms may occupy the lignocellulosic substrate , which restrains the growth and activity of white rot fungi , inhibits fungal lignino - cellulose decomposition , and reduces enzyme release . the introduction ratio of white rot fungi has an important impact on its economics of practical application . for example , in studies by fragoeiro and magan , a ratio of 5 g of inoculant to 95 g of soil was used . the effect of using this approach on the differential breakdown of mixtures of xenobiotic compounds under different environmental regimes is shown in table 4 . for example , novotny et al . reported the degradation of dye in soil using a 50 : 50 soil : straw - based inoculant of irpex lacteus ; canet et al . used straw - based inoculum with an incorporation rate of 40% incorporation ; ryan and bumpus used a 25% straw - based inoculum ; meysami and baheri used 10% straw - based inoculum ; and morgan et al . used 4 we believe that some of these formulations are very unrealistic for the bioremediation of xenobiotics in contaminated soils from a practical and economic point of view . furthermore , few , if any , have examined the effect of water potential on mixtures of pesticides . used the same species as the present study along with pleurotus ostreatus and found that the latter was better than both p. chrysosporium and t. versicolor . however , they used sterile soil only , which is devoid of all natural microbial communities otherwise present . boyle reported increases in growth and carbon dioxide production in natural soil supplemented with carbon . he observed that mineralization of [ c ] pentachlorophenol ( degradation to co2 ) was much faster in soil that had been amended with alfalfa and benomyl and inoculated with t. versicolor . another study showed that the addition of straw increases the hyphal length of white rot fungi in soil . . investigated ligninolytic enzyme production by the white rot fungi p. chrysosporium and t. versicolor after pre - cultivation on various insoluble lignocellulosic materials , including grape seeds , barley bran , and wood shavings . cultures of p. chrysosporium pregrown on grape seeds and barley bran showed maximum lip and mnp activities ( 1,000 and 1,232 units / l , respectively ) . on the other hand , t. versicolor pre - cultivated with the same lignocellulosic residues showed maximum lac activity ( approx . 250 units / l ) . in vitro decoloration of the polymeric dye poly r-478 using the extracellular liquid obtained in the above - mentioned cultures was carried out in order to determine the respective capabilities of lac , lip , and mnp . it is noteworthy that the degrading capability of lip upon pre - cultivation of p. chrysosporium with barley bran gives a percentage of decoloration of about 80% after 100 sec . utilization of these solid substrates in soil may also be advantageous as a means of evenly distributing fungal inoculum in large volumes of soil , and according to singleton , growth amendments could also exert beneficial effects by adsorbing pollutants and hence decreasing the bioavailability of toxic pollutants . composting matrices and composts are rich sources of xenobiotic - degrading microorganisms , including bacteria , actinomycetes , and ligninolytic fungi , all of which can degrade pollutants to innocuous compounds such as carbon dioxide and water . these microorganisms can also biotransform pollutants into less toxic substances and/or immobilize pollutants within an organic matrix , thereby reducing pollutant bioavailability . spent mushroom compost ( smc ) is a byproduct of mushroom production and is produced in large amounts . about 600 g of spent compost is produced , or 5 kg of smc generated for every 1 kg of edible mushrooms according to law et al . . most either discretely burn or discard it , and thus its exploitation as a potential bioremediation adjuvant has received significant attention . mushroom cultivation involves the pure culture of spawn , composting , pasteurization of the substrate , and careful regulation of growing conditions . the substrates are lignocellulosic residues , such as straw , horse manure , chicken manure , and activators . the purpose of composting the substrate is to exclude microorganisms that may interfere with mushroom growth . following mushroom harvest , smc is likely to contain not only a large and diverse group of microorganisms but also a wide range of extracellular enzymes that are active against wheat straw . singh et al . reported the extraction of cellulase , hemicellulose , -glucosidase , lignin peroxidises , and lac from smc . it also contains very high organic content ( 20% ) , including cellulose , hemicellulose , and lignin , from the unused lignocellulosic substrate . previous research has provided some interesting findings using this type of compost as a bioremediation adjuvant . law et al . reported that smc of pleurotus pulmonarius could remove 89.0 + /- 0.4% of 100 mg of pcp / l within 2 days at room temperature predominantly by biodegradation . kuo and regan used sterilized smc as an adsorption medium for the removal of a mixture of pesticides ( carbaryl , carbofuran , and aldicarb ) with a concentration range of 0~30 mg / l and found that smc was able to successfully adsorb carbamate pesticides from aqueous solutions , possibly due to increased organic matter content . with mushroom production being the largest solid - state fermentation industry in the world , and with so much waste being produced , it is extremely important to find a use for smc . thus , smc as a soil amendment for the improvement of pesticide bioremediation is an interesting area . furthermore , there is no information on the effect of smc addition on soil enzymes , soil respiration , and soil populations , or on how these metabolic parameters are affected by the presence of pesticides and water availability . we previously examined the addition of a mixture of 5 g of smc and 95 g of unsterile sandy loam soil treated with 10 mg / kg of soil to a combination of pesticides ( simazine , trifluralin , and dieldrin ) using a moisture adsorption curve to achieve water potentials of -0.7 and 2.8mpa . these treatments were stored for 42 and 84 days at 15. the amount of co2 produced was measured by gc analysis and by determining total ligninolytic activity , whereas high - performance liquid chromatography with uv detection was used to analyze the amount of each pesticide remaining in each of the treatments . the soil amended with smc had by far the highest microbial activity regardless of water potential and pesticide treatment . 3 shows that in all cases , the smc - amended treatments ( - and + pesticides ) had the highest ligninolytic activities after 42 and 84 days . table 5 shows the effect of smc on the differential breakdown of the three pesticides under different water potential and time conditions . the results demonstrate that there was significant enhanced breakdown of all three pesticides after 42 and 84 days and under both water potential regimes due to smc inoculation . this suggests that smc contains a mixture of ligninolytic enzymes , including lacs , actinomycetes , and in some cases , basidiomycete hyphae advantages include maintenance of an aerobic soil structure , effective rates of moisture retention , and avoidance of anaerobic conditions . application of fungal technology for the cleanup of contaminants has shown promise ever since 1985 , when the white rot species phanerochaete chrysosporium was found capable of metabolizing a number of important environmental pollutants and strains were subsequently commercialized for the treatment of contaminated soil - based xenobiotic contaminants . there have been many studies that have shown white rot fungi to be useful for the in vitro and in situ degradation of predominantly single contaminants , although in mixtures of xenobiotic compounds have been addressed occasionally . in reality , contaminated soil usually involves a mixture of xenobiotic compounds . it is thus surprising that only a few studies have addressed this important aspect , e.g. , the differential degradation of xenobiotic compounds in natural soil ecosystems . most research is carried out under largely optimal conditions for the growth / colonization of fungal inoculants . however , as we have shown , white rot fungi can grow effectively under water stress conditions where no plant growth occurs [ 23 , 67 ] . this may become very important in the future when we examine the different interacting factors that affect bioremediation by fungi , and indeed all microorganisms , including the impacts of climate change . thus , elevated co2 concentrations and slightly increased temperatures may have subtle but significant effects on the functioning of terrestrial ecosystems as well as any introduced microorganisms from a bioremedial perspective . the last area that will hasten the development of remediation approaches is the determination of the genomes of specific white rot fungi . for example , research into the p. chrysosporium genome has resulted in the elucidation of specific cytochrome p450 monooxygenases , which may be differentially expressed in the presence of xenobiotic compounds [ 14 , 47 ] . knowledge of these gene clusters and their relative expression levels can now be quantified and integrated with data on associated ecological and physiological factors , which will result in a more complete understanding of the mechanisms of action and functioning of fungal bioremediation systems . this could result in a more integrated " systems " approach for the effective exploitation of bioremedial fungi for treating xenobiotics in terrestrial ecosystems .

this review provides background information on the importance of bioremediation approaches . it describes the roles of fungi , specifically white rot fungi , and their extracellular enzymes , laccases , ligninases , and peroxidises , in the degradation of xenobiotic compounds such as single and mixtures of pesticides . we discuss the importance of abiotic factors such as water potential , temperature , and ph stress when considering an environmental screening approach , and examples are provided of the differential effect of white rot fungi on the degradation of single and mixtures of pesticides using fungi such as trametes versicolor and phanerochaete chrysosporium . we also explore the formulation and delivery of fungal bioremedial inoculants to terrestrial ecosystems as well as the use of spent mushroom compost as an approach . future areas for research and potential exploitation of new techniques are also considered .

Bioremediation Approaches White Rot Fungi: Evidence of Enzyme-mediated Degradation of Xenobiotic Compounds Screening of White Rot Fungi for Tolerance to Single and Mixtures of Xenobiotic Compounds Inoculant Production for Soil Incorporation of Bioremedial Fungi Use of Spent Mushroom Composts Conclusions and Future Strategies

microbial metabolism is regarded as the most important mechanism of pesticide degradation in soil [ 12 , 13 ] , and it constitutes the basis for all bioremediation and bioaugmentation strategies . therefore , conditions that favor microbial growth and activity in soil , such as temperature , moisture , nutrient status , ph , and aeration , will also generally promote metabolic degradation of pesticides . as suggested by gadd amongst others , the composition and size of soil microbial populations , as well as the status of metabolic activity , are the determining factors as to whether or not biodegradation is feasible as a remediation option . it is an effective bioremediation strategy [ 15 , 16 ] , although it may have poor reproducibility and be dependent on the characteristics of microbial populations . indeed , the major advantage offered by bioaugmentation is the ability to choose the introduced species based on the goals of the process and the conditions of the matrix . in this case , the success of bioremediation is mainly dependent on the competition / proliferation capability of the introduced species and the bioavailability of the xenobiotic compounds . bioavailability here refers to the acquisition and subsequent transformation / degradation of the compound and is closely related to its chemical properties , as well as to a wide range of soil physical and chemical parameters . in addition to mineralization , which implies the use of xenobiotics as a carbon source , microorganisms can also co - metabolize pesticides , e.g. many of the reactions involved in co - metabolism of pesticides , including oxidation - reduction , de - halogenation , ring - cleavage , and hydrolysis , occur simultaneously . for example , microbial enzyme - mediated methylation reactions usually increase herbicide lipophilicity and thus the potential for bioaccumulation in the food chain . the use of bioremediation to remove pollutants is typically less expensive than equivalent physical - chemical methods . perhaps the most attractive feature of bioremediation is its reduced impact on natural ecosystems , which should be well received by the public . for any xenobiotic compound , the threshold concentration above which remediation becomes necessary is referred to as the so - called " remediation trigger level . " however , for many pesticides as well as xenobiotic compounds , the threshold concentration has not yet been established . generally , the target concentration is assumed to be in the 1 ppm ( mg / l ) range , but in practice it can vary from site to site and region to region [ 3 , 10 ] . very often , urban application of pesticides is carried out at an excessively high concentration , resulting in pesticide waste characterized by prolonged persistence . at present , bioremediation conducted on a commercial scale predominantly utilizes prokaryotes , with comparatively few recent attempts using white rot fungi . filamentous fungi are also more tolerant of environmental stress and can produce copious amounts of extracellular enzymes during hyphal colonization of soil , resulting in enhanced rates of bioremediation [ 22 , 23 ] . therefore , white rot fungi potentially represent a powerful tool for soil bioremediation , with some species already patented . however , many research studies have been carried out to examine the efficacy of bioremediation fungi in degrading single xenobiotic compounds . the application of fungi for the cleanup of contaminated soil first came to attention in the mid-1980s when the white rot fungus phanerochaete chrysosporium was shown to metabolize a range of organic environmental contaminants [ 25 , 26 ] . later , this ability was demonstrated for other white rot fungi , including trametes versicolor and pleurotus ostreatus . white rot fungi are the most widely studied and understood ligninolytic fungi to date [ 2 , 3 , 27 - 29 ] . lignin is a three - dimensional , naturally occurring polymer present in woody plants , and it constitutes one of the most structurally complex and therefore resistant materials to microbial degradation [ 9 , 30 ] . the ability of white rot fungi to mineralize lignin is generally attributed to the secretion of extracellular ligninolytic enzymes , mostly laccase ( lac ) , lignin peroxidase ( lip ) , and manganese peroxidase ( mnp ) [ 9 , 27 , 29 ] . it is accepted that both of these peroxidases catalyze the oxidation of ( endogenously - produced ) low - molecular weight mediators using h2o2 as an oxidant . , combination of functional groups ) present in the lignin molecule . this explains the ability of white rot fungi to tolerate and degrade such a wide range of environmental organic pollutants , even when at high concentrations [ 3 , 9 ] . this implies that the presence of lignocellulosic substrates is a requirement for the degradation of xenobiotic compounds . determining the activity of lac in soil inoculated with white rot species provides a measure of the colonizing ability of the fungus and can be used to monitor the bioremediation of numerous soil contaminants , among them triazine pesticides [ 28 , 30 , 31 ] . measured lac activity to demonstrate the correlation between its production and the degradation of polycyclic aromatic hydrocarbons ( pahs ) by several strains of white rot fungi in both liquid culture and soil . reviewed a series of applications involving white rot fungi , among them t. versicolor , for environmental remediation of pesticides , disinfectants ( e.g. , petrol and diesel ) , pahs , explosives ( trinitrotoluene , tnt derivatives ) , and industrial solvents ( e.g. they also offered descriptions on the use of these fungi for the biotransformation of coal , treatment of effluents from paper and olive - processing plants , and the degradation of synthetic polymers ( e.g. however , most studies performed thus far have focused on the screening of white rot fungi for the bioremediation of xenobiotic contaminants in liquid culture media , bioreactors , sterile soil or soil extract broth . recently , fragoeiro and magan reported the successful application of three white rot species , including t. versicolor , for the bioremediation of mixtures of pesticides in non - sterile sandy loam soil under low water potential conditions . this demonstrated that differential breakdown of mixtures of pesticides occurred possibly due to environmental factors . lac activity in the biodegradation of xenobiotic compounds with lignin - like structures has already attracted considerable interest , and its biodegradative effects on different contaminants have been exhaustively studied . specifically , lac enzyme is a copper - containing phenoloxidase involved in the degradation of lignin , and it oxidizes phenol and phenolic lignin sub - structures . for example , dye decoloration by trametes hispida , degradation of azo - dyes by pyricularia oryzae , and textile effluent degradation by t. versicolor have all been attributed to lac activity . demir examined the biological degradation of benzene and toluene by t. versicolor as well as biomass determinations . regarding benzene , biodegradation was completed after 4 hr at an initial concentration of 50 mg / l , whereas it took 42 hr to completely remove benzene at 300 mg addition of veratryl alcohol , a lac inducer , to the basic feed medium enhanced the performance of the enzyme system and shortened the overall time period of biodegradation completed in a shorter time period . after 36 hr , about 46 and 65% of the compound added at initial concentrations of 100 mg / l to shaken and static fungal cultures were removed , respectively . despite many reports that the degradation of xenobiotics by white rot fungi is mediated by enzymes involved in lignin degradation , some authors have presented contradictory evidence . reported the degradation of tnt by non - ligninolytic strains of p. chrysosporium . showed > 86% degradation of atrazine and terbuthylazine by white rot fungi in liquid culture and found no relationship between degradation rate and ligninolytic activity . other studies featuring p. chrysosporium in liquid culture have reported biotransformation of the insecticide lindane independently of the production of ligninolytic enzymes . these researchers ruled out the involvement of peroxidases in lindane biotransformation and mineralization , and they assessed the activity of cytochrome p450 monooxygenase , an enzymatic system used by many organisms for detoxification . whether the degradation of pesticides is carried out by lignin - degrading enzymes or other enzymatic systems , or both , the use of fungi in bioremediation is clearly very promising , and further studies should be conducted to understand which enzymes are involved . additionally , there is little information on the degradation of mixtures of xenobiotics , which is more common in nature than single ones . recent work on the p. chrysosporium genome has shown that cytochrome p450 monooxygenases constitute the largest and most important group of p450 genes in any fungal species and that they are differentially expressed depending on xenobiotic type and nutrition [ 47 , 48 ] . this has resulted in the development of powerful tools useful in understanding the roles of key enzymes produced by p. chrysosporium in the bioremediation of different xenobiotic compounds under various environmental conditions . however , these tools have seldom been applied to the examination of how environmental stress factors such as water potential and temperature effects on the expression of p450 gene clusters involved in enzyme production both in vitro and in situ . the in vitro environmentally relevant screening of white rot species using both single and mixtures of pesticides can be used to identify potential candidates when followed by in situ microcosm studies using different inoculant formulations . this can be instructive and useful in understanding the approaches and strategies needed for the development of effective bioremediation systems using white rot fungi . tolerance and growth of white rot fungi in the presence of individual and mixtures of xenobiotic compounds may vary depending upon the type and concentration of the xenobiotic as well as the nutritional and environmental conditions . it is important to use realistic nutritional media such as soil extract - based systems , in which the water potential , ph , and temperature can be modified , since these conditions can have a significant impact on the relative tolerance of a potential fungal bioremediation agent . table 1 shows an example of the concentrations of single and mixtures of pesticides ( effective concentration ; ec50 values ) required to control a range of white rot fungal isolates in response to changes in water potential . the results highlight the importance of examining such factors when determining the relative tolerance / sensitivity to single and mixtures of pesticides as well as when choosing appropriate isolates for subsequent use in situ . in this case , -0.7 and -2.8mpa water potential were used to represent conditions under which water was available and plants would grow , with the latter being twice the wilting point of plants . table 1 shows an example of the relative growth levels of isolates of t. versicolor and p. ostreatus in response to individual and mixtures of pesticides . the applicability of fungi to the bioremediation of soil contaminated with pesticides depends on the capacity of fungi to grow in the presence of such compounds as well as their ability to produce degradative enzymes . additionally , complementary information on the capacity and ability of fungi to produce the key extracellular enzymes required for degradation of individual and mixtures of xenobiotic compounds is required . a significant amount of research on white rot fungi has been conducted in liquid and/or synthetic media , but less is known about its bioremediation capabilities in soil , especially under different environmental conditions . reported that field conditions do not always enable white rot fungi such as p. chrysosporium to achieve optimum activity , and therefore it is not a good competitor in a soil environment [ 37 , 40 ] . recently , we demonstrated that under different osmotic stress regimes , white rot fungi are able to differentially degrade mixtures of pesticides in soil extract broth ( table 2 ) . we also demonstrated an increase in the range of hydrolytic enzyme production , including ligninases and cellulases , even under water stress conditions ( table 3 ) . there are many studies on how to optimize the biodegradation potential of white rot fungi in contaminated soil [ 16 , 35 , 53 , 54 ] . if it is accepted that extracellular ligninolytic enzymes are at least in part responsible for the critical initial reactions of pollutant transformation , then the production and activity of these enzymes in contaminated soil under field conditions are both prerequisites for the successful application of white rot fungi in soil bioremediation . additionally , bioremedial fungi should be able to secrete the necessary enzymes into the soil matrix in order to enhance the degradation of pesticide molecules that would otherwise be unable to be incorporated across cell walls . there is little information available on the survival of white rot fungi in soil , especially those fungi not consumed by humans . certainly , better fungal growth could help introduced fungi overcome competition from indigenous microorganisms as well as enhance bioremediation . this is important as native soil microorganisms may occupy the lignocellulosic substrate , which restrains the growth and activity of white rot fungi , inhibits fungal lignino - cellulose decomposition , and reduces enzyme release . the introduction ratio of white rot fungi has an important impact on its economics of practical application . the effect of using this approach on the differential breakdown of mixtures of xenobiotic compounds under different environmental regimes is shown in table 4 . reported the degradation of dye in soil using a 50 : 50 soil : straw - based inoculant of irpex lacteus ; canet et al . furthermore , few , if any , have examined the effect of water potential on mixtures of pesticides . another study showed that the addition of straw increases the hyphal length of white rot fungi in soil . investigated ligninolytic enzyme production by the white rot fungi p. chrysosporium and t. versicolor after pre - cultivation on various insoluble lignocellulosic materials , including grape seeds , barley bran , and wood shavings . in vitro decoloration of the polymeric dye poly r-478 using the extracellular liquid obtained in the above - mentioned cultures was carried out in order to determine the respective capabilities of lac , lip , and mnp . utilization of these solid substrates in soil may also be advantageous as a means of evenly distributing fungal inoculum in large volumes of soil , and according to singleton , growth amendments could also exert beneficial effects by adsorbing pollutants and hence decreasing the bioavailability of toxic pollutants . composting matrices and composts are rich sources of xenobiotic - degrading microorganisms , including bacteria , actinomycetes , and ligninolytic fungi , all of which can degrade pollutants to innocuous compounds such as carbon dioxide and water . spent mushroom compost ( smc ) is a byproduct of mushroom production and is produced in large amounts . most either discretely burn or discard it , and thus its exploitation as a potential bioremediation adjuvant has received significant attention . mushroom cultivation involves the pure culture of spawn , composting , pasteurization of the substrate , and careful regulation of growing conditions . the substrates are lignocellulosic residues , such as straw , horse manure , chicken manure , and activators . reported the extraction of cellulase , hemicellulose , -glucosidase , lignin peroxidises , and lac from smc . kuo and regan used sterilized smc as an adsorption medium for the removal of a mixture of pesticides ( carbaryl , carbofuran , and aldicarb ) with a concentration range of 0~30 mg / l and found that smc was able to successfully adsorb carbamate pesticides from aqueous solutions , possibly due to increased organic matter content . with mushroom production being the largest solid - state fermentation industry in the world , and with so much waste being produced , it is extremely important to find a use for smc . furthermore , there is no information on the effect of smc addition on soil enzymes , soil respiration , and soil populations , or on how these metabolic parameters are affected by the presence of pesticides and water availability . we previously examined the addition of a mixture of 5 g of smc and 95 g of unsterile sandy loam soil treated with 10 mg / kg of soil to a combination of pesticides ( simazine , trifluralin , and dieldrin ) using a moisture adsorption curve to achieve water potentials of -0.7 and 2.8mpa . these treatments were stored for 42 and 84 days at 15. the amount of co2 produced was measured by gc analysis and by determining total ligninolytic activity , whereas high - performance liquid chromatography with uv detection was used to analyze the amount of each pesticide remaining in each of the treatments . the soil amended with smc had by far the highest microbial activity regardless of water potential and pesticide treatment . table 5 shows the effect of smc on the differential breakdown of the three pesticides under different water potential and time conditions . the results demonstrate that there was significant enhanced breakdown of all three pesticides after 42 and 84 days and under both water potential regimes due to smc inoculation . this suggests that smc contains a mixture of ligninolytic enzymes , including lacs , actinomycetes , and in some cases , basidiomycete hyphae advantages include maintenance of an aerobic soil structure , effective rates of moisture retention , and avoidance of anaerobic conditions . application of fungal technology for the cleanup of contaminants has shown promise ever since 1985 , when the white rot species phanerochaete chrysosporium was found capable of metabolizing a number of important environmental pollutants and strains were subsequently commercialized for the treatment of contaminated soil - based xenobiotic contaminants . there have been many studies that have shown white rot fungi to be useful for the in vitro and in situ degradation of predominantly single contaminants , although in mixtures of xenobiotic compounds have been addressed occasionally . in reality , contaminated soil usually involves a mixture of xenobiotic compounds . , the differential degradation of xenobiotic compounds in natural soil ecosystems . however , as we have shown , white rot fungi can grow effectively under water stress conditions where no plant growth occurs [ 23 , 67 ] . this may become very important in the future when we examine the different interacting factors that affect bioremediation by fungi , and indeed all microorganisms , including the impacts of climate change . thus , elevated co2 concentrations and slightly increased temperatures may have subtle but significant effects on the functioning of terrestrial ecosystems as well as any introduced microorganisms from a bioremedial perspective . the last area that will hasten the development of remediation approaches is the determination of the genomes of specific white rot fungi . for example , research into the p. chrysosporium genome has resulted in the elucidation of specific cytochrome p450 monooxygenases , which may be differentially expressed in the presence of xenobiotic compounds [ 14 , 47 ] . knowledge of these gene clusters and their relative expression levels can now be quantified and integrated with data on associated ecological and physiological factors , which will result in a more complete understanding of the mechanisms of action and functioning of fungal bioremediation systems . this could result in a more integrated " systems " approach for the effective exploitation of bioremedial fungi for treating xenobiotics in terrestrial ecosystems .

the fhs is a prospective cohort originally designed to assess the epidemiology of cv disease ( cvd).27,28 data have been collected from 3 generations of participants since its inception in 1948 . the original cohort involved 5209 participants , 5124 were enrolled in the second generation starting in 1971 , and 4095 in the third generation starting in 2002.27,28 our analysis included participants from the second and third generations of the fhs for whom serum tc , tg , ldl - c ( calculated using the friedewald equation : ldl - c = tc - hdl(tg/5 ) ) and hdl - c were available for both generations.29 to limit confounding of results by relatedness , we only considered the oldest offspring for each nuclear family , creating parent - offspring trios . the final study population consisted of 416 trios , with 228 females and 188 males in the offspring generation . the sardinia study is a longitudinal study designed to assess the epidemiology and genetics of aging - associated conditions.30 the study enrolled 6921 volunteers from a cluster of 4 towns on the east coast of sardinia and represents a collection of large pedigrees , containing data from up to 5 generations . from each pedigree , we only considered parents and their oldest offspring from the 2 most recent generations . because this cohort included several families with only a single parent enrolled , analyses were performed on 277 mothers , 151 fathers , 168 daughters , and 136 sons . for both cohorts , families in which any member had a tg level > 400 mg / dl were excluded . only individuals with at least 2 of the 4 lipid traits available were included in the analysis . the study was approved by the institutional review boards at vanderbilt university , boston university , dartmouth college , the national institutes of health , and the local ethical committee of lanusei , sardinia , in italy . all of the included fhs and sardinia participants provided written informed consent , including consent to use of their dna data in genetic analyses . for both cohorts , false paternity was assessed by genetics data management groups and the parental information was adjusted accordingly before release of data and therefore not considered in the current study . participants of the framingham cohort are routinely followed up , permitting access to clinical phenotype data at multiple time points . we used first patient visit data for the offspring population , because at the time of the study only 1 visit was available for this generation . for the parental population of the fhs , analyses were performed on values from visit 3 only , given that it had the largest number of individuals with available lipid data . the other phenotypes relevant to our study were age , body mass index ( bmi ) , smoking status , use of lipid - lowering medications ( ever treated vs. never treated ) , as well as the menopausal status in females . these phenotypes were used as covariates for both the offspring and parental populations . for the offspring population , we used the first adult patient visit , providing a direct adult to adult comparison . in sardinia , we analyzed the same phenotypes and covariates from visit 1 in the parental and offspring population , given that both provided the largest number of patients with available lipid data . we examined the poe on the variation of fasting lipids in the offspring populations . to identify transmission effects , we performed a series of nested , sex - stratified linear regression analyses , modeling lipid traits in offspring . the models were generated by sequentially changing the variables included ; namely , all offspring covariates and corresponding parental lipid traits ( figure1 panel a ) . we report the adjusted r values throughout , which represent the proportion of variation explained by each model with all variables included in a given model . flowchart of nested models used to determine parent of origin effects on offspring lipid traits . parent of origin effects and relevant covariates ( a ) and parent of origin effects combined with snps previously associated with lipid traits ( b ) . the models assessed , also reported in figure1 panel a , were the following : the summary of all analyses modeling offspring lipid traits , including the assessment of the effect of parental covariates , is provided in the supplemental materials ( table s1 ) . to evaluate the performance of each model , we compared the adjusted r values for each lipid trait model using a likelihood ratio test . pair - wise model comparisons were carried out for nested pairs , namely , model 3 versus model 1 ( effect of offspring covariates ) and model 3 versus model 2 ( effect of parental lipid trait ) . to estimate the effects of shared environment on lipid traits , we modeled each maternal lipid trait with the corresponding paternal lipid trait under the assumption that shared environment would be revealed by large r in this regression model ( model 4 ) . the following model was used for all parents in the trio families and then separately stratified by the sex of their offspring , to mirror the analysis above : additional models for the effects of maternal and paternal covariates on the adjusted r produced by model 4 are reported in table s2 . to assess the role of early environment on lipid profiles specifically , we determined the variance explained in same sex versus different sex sibling pairs . to examine whether the effects of parental lipid traits are explained by genetic variants in offspring , we analyzed the effects of the 95 previously validated single - nucleotide variants ( snps ) from teslovich et al.11 on the corresponding lipid levels . to assess the dependence of offspring lipid traits on snps previously associated with each lipid trait , we performed nested , sex - stratified linear regression models and compared them to the variance explained only by the corresponding parental lipid traits ( figure1 panel b ) . in the framingham cohort , genotyping was performed using the 500k affymetrix genechip , and many of the teslovich snps were not included . we therefore used proxy variants based on high linkage disequilibrium ( ld ) in european populations ( ceu and tsi ) from phase 3 of the international hapmap project.3133 for each nongenotyped snp , we chose a variant on the same chromosome in strong ld ( r>0.75 ) , having the highest minor allele frequency ( list of snps used can be found in table s3 ) . only 63 of the 95 snps were available either through direct genotyping or as proxies . in contrast , in the sardinia cohort , genotyping information was available from 4 different illumina arrays , one of which , cardio - metabochip , included the majority of the teslovich snps ( pistis et al.34 ) . overall , in the sardinia cohort , 92 of the 95 snps reported in teslovich et al . were available and analyzed ( table s3 ) . in addition , to make all analyses directly comparable between the 2 cohorts , we also evaluated the same subset of 63 snps , original and proxies , as in the framingham cohort . to further assess the effects of using proxies , as opposed to the original teslovich snps , in sardinia we also considered models using only the original 63 teslovich snps for which we had either direct genotype data or proxies available in the framingham cohort ( table s3 ) . we only used the subset of snps previously associated with each lipid trait phenotype ( table s3 ) . because genotypes were not available for all participants , the number of observations in model 1 included in this comparison differs from the one above ( tables7 and 8 vs. tables3 and 4 , respectively ) . analyses adding offspring covariates and using the other snp sets for the sardinia cohort were also performed . effect sizes and potential redundancy among the influence of known genes and parental lipid trait measures were evaluated by comparing the results of likelihood ratio tests of model 6 versus model 1 ( effect of offspring snps ) and model 6 versus model 5 ( effect of parental lipid trait ) . all analyses were conducted using stata ( 11.1 ; statacorp lp , college station , tx ) and r software ( r foundation for statistical computing , vienna , austria ) . the fhs is a prospective cohort originally designed to assess the epidemiology of cv disease ( cvd).27,28 data have been collected from 3 generations of participants since its inception in 1948 . the original cohort involved 5209 participants , 5124 were enrolled in the second generation starting in 1971 , and 4095 in the third generation starting in 2002.27,28 our analysis included participants from the second and third generations of the fhs for whom serum tc , tg , ldl - c ( calculated using the friedewald equation : ldl - c = tc - hdl(tg/5 ) ) and hdl - c were available for both generations.29 to limit confounding of results by relatedness , we only considered the oldest offspring for each nuclear family , creating parent - offspring trios . the final study population consisted of 416 trios , with 228 females and 188 males in the offspring generation . the sardinia study is a longitudinal study designed to assess the epidemiology and genetics of aging - associated conditions.30 the study enrolled 6921 volunteers from a cluster of 4 towns on the east coast of sardinia and represents a collection of large pedigrees , containing data from up to 5 generations . from each pedigree , we only considered parents and their oldest offspring from the 2 most recent generations . because this cohort included several families with only a single parent enrolled , analyses were performed on 277 mothers , 151 fathers , 168 daughters , and 136 sons . for both cohorts , families in which any member had a tg level > 400 mg / dl were excluded . only individuals with at least 2 of the 4 lipid traits available were included in the analysis . the study was approved by the institutional review boards at vanderbilt university , boston university , dartmouth college , the national institutes of health , and the local ethical committee of lanusei , sardinia , in italy . all of the included fhs and sardinia participants provided written informed consent , including consent to use of their dna data in genetic analyses . for both cohorts , false paternity was assessed by genetics data management groups and the parental information was adjusted accordingly before release of data and therefore not considered in the current study . participants of the framingham cohort are routinely followed up , permitting access to clinical phenotype data at multiple time points . we used first patient visit data for the offspring population , because at the time of the study only 1 visit was available for this generation . for the parental population of the fhs , analyses were performed on values from visit 3 only , given that it had the largest number of individuals with available lipid data . the other phenotypes relevant to our study were age , body mass index ( bmi ) , smoking status , use of lipid - lowering medications ( ever treated vs. never treated ) , as well as the menopausal status in females . these phenotypes were used as covariates for both the offspring and parental populations . for the offspring population , we used the first adult patient visit , providing a direct adult to adult comparison . in sardinia , we analyzed the same phenotypes and covariates from visit 1 in the parental and offspring population , given that both provided the largest number of patients with available lipid data . we examined the poe on the variation of fasting lipids in the offspring populations . to identify transmission effects , we performed a series of nested , sex - stratified linear regression analyses , modeling lipid traits in offspring . the models were generated by sequentially changing the variables included ; namely , all offspring covariates and corresponding parental lipid traits ( figure1 panel a ) . we report the adjusted r values throughout , which represent the proportion of variation explained by each model with all variables included in a given model . flowchart of nested models used to determine parent of origin effects on offspring lipid traits . parent of origin effects and relevant covariates ( a ) and parent of origin effects combined with snps previously associated with lipid traits ( b ) . the models assessed , also reported in figure1 panel a , were the following : the summary of all analyses modeling offspring lipid traits , including the assessment of the effect of parental covariates , is provided in the supplemental materials ( table s1 ) . to evaluate the performance of each model , we compared the adjusted r values for each lipid trait model using a likelihood ratio test . pair - wise model comparisons were carried out for nested pairs , namely , model 3 versus model 1 ( effect of offspring covariates ) and model 3 versus model 2 ( effect of parental lipid trait ) . to estimate the effects of shared environment on lipid traits , we modeled each maternal lipid trait with the corresponding paternal lipid trait under the assumption that shared environment would be revealed by large r in this regression model ( model 4 ) . the following model was used for all parents in the trio families and then separately stratified by the sex of their offspring , to mirror the analysis above : additional models for the effects of maternal and paternal covariates on the adjusted r produced by model 4 are reported in table s2 . to assess the role of early environment on lipid profiles , we compared sibling lipids in families with more than 1 offspring . specifically , we determined the variance explained in same sex versus different sex sibling pairs . to examine whether the effects of parental lipid traits are explained by genetic variants in offspring , we analyzed the effects of the 95 previously validated single - nucleotide variants ( snps ) from teslovich et al.11 on the corresponding lipid levels . to assess the dependence of offspring lipid traits on snps previously associated with each lipid trait , we performed nested , sex - stratified linear regression models and compared them to the variance explained only by the corresponding parental lipid traits ( figure1 panel b ) . in the framingham cohort , genotyping was performed using the 500k affymetrix genechip , and many of the teslovich snps were not included . we therefore used proxy variants based on high linkage disequilibrium ( ld ) in european populations ( ceu and tsi ) from phase 3 of the international hapmap project.3133 for each nongenotyped snp , we chose a variant on the same chromosome in strong ld ( r>0.75 ) , having the highest minor allele frequency ( list of snps used can be found in table s3 ) . only 63 of the 95 snps were available either through direct genotyping or as proxies . in contrast , in the sardinia cohort , genotyping information was available from 4 different illumina arrays , one of which , cardio - metabochip , included the majority of the teslovich snps ( pistis et al.34 ) . overall , in the sardinia cohort , 92 of the 95 snps reported in teslovich et al . in addition , to make all analyses directly comparable between the 2 cohorts , we also evaluated the same subset of 63 snps , original and proxies , as in the framingham cohort . to further assess the effects of using proxies , as opposed to the original teslovich snps , in sardinia we also considered models using only the original 63 teslovich snps for which we had either direct genotype data or proxies available in the framingham cohort ( table s3 ) . we only used the subset of snps previously associated with each lipid trait phenotype ( table s3 ) . because genotypes were not available for all participants , the number of observations in model 1 included in this comparison differs from the one above ( tables7 and 8 vs. tables3 and 4 , respectively ) . analyses adding offspring covariates and using the other snp sets for the sardinia cohort were also performed . effect sizes and potential redundancy among the influence of known genes and parental lipid trait measures were evaluated by comparing the results of likelihood ratio tests of model 6 versus model 1 ( effect of offspring snps ) and model 6 versus model 5 ( effect of parental lipid trait ) . all analyses were conducted using stata ( 11.1 ; statacorp lp , college station , tx ) and r software ( r foundation for statistical computing , vienna , austria ) . we examined the poe on the variation of fasting lipids in the offspring populations . to identify transmission effects , we performed a series of nested , sex - stratified linear regression analyses , modeling lipid traits in offspring . the models were generated by sequentially changing the variables included ; namely , all offspring covariates and corresponding parental lipid traits ( figure1 panel a ) . we report the adjusted r values throughout , which represent the proportion of variation explained by each model with all variables included in a given model . flowchart of nested models used to determine parent of origin effects on offspring lipid traits . parent of origin effects and relevant covariates ( a ) and parent of origin effects combined with snps previously associated with lipid traits ( b ) . the models assessed , also reported in figure1 panel a , were the following : the summary of all analyses modeling offspring lipid traits , including the assessment of the effect of parental covariates , is provided in the supplemental materials ( table s1 ) . to evaluate the performance of each model , we compared the adjusted r values for each lipid trait model using a likelihood ratio test . pair - wise model comparisons were carried out for nested pairs , namely , model 3 versus model 1 ( effect of offspring covariates ) and model 3 versus model 2 ( effect of parental lipid trait ) . to estimate the effects of shared environment on lipid traits , we modeled each maternal lipid trait with the corresponding paternal lipid trait under the assumption that shared environment would be revealed by large r in this regression model ( model 4 ) . the following model was used for all parents in the trio families and then separately stratified by the sex of their offspring , to mirror the analysis above : additional models for the effects of maternal and paternal covariates on the adjusted r produced by model 4 are reported in table s2 . to assess the role of early environment on lipid profiles specifically , we determined the variance explained in same sex versus different sex sibling pairs . to examine whether the effects of parental lipid traits are explained by genetic variants in offspring , we analyzed the effects of the 95 previously validated single - nucleotide variants ( snps ) from teslovich et al.11 on the corresponding lipid levels . to assess the dependence of offspring lipid traits on snps previously associated with each lipid trait , we performed nested , sex - stratified linear regression models and compared them to the variance explained only by the corresponding parental lipid traits ( figure1 panel b ) . in the framingham cohort , genotyping was performed using the 500k affymetrix genechip , and many of the teslovich snps were not included . we therefore used proxy variants based on high linkage disequilibrium ( ld ) in european populations ( ceu and tsi ) from phase 3 of the international hapmap project.3133 for each nongenotyped snp , we chose a variant on the same chromosome in strong ld ( r>0.75 ) , having the highest minor allele frequency ( list of snps used can be found in table s3 ) . only 63 of the 95 snps were available either through direct genotyping or as proxies . in contrast , in the sardinia cohort , genotyping information was available from 4 different illumina arrays , one of which , cardio - metabochip , included the majority of the teslovich snps ( pistis et al.34 ) . overall , in the sardinia cohort , 92 of the 95 snps reported in teslovich et al . were available and analyzed ( table s3 ) . in addition , to make all analyses directly comparable between the 2 cohorts , we also evaluated the same subset of 63 snps , original and proxies , as in the framingham cohort . to further assess the effects of using proxies , as opposed to the original teslovich snps , in sardinia we also considered models using only the original 63 teslovich snps for which we had either direct genotype data or proxies available in the framingham cohort ( table s3 ) . we only used the subset of snps previously associated with each lipid trait phenotype ( table s3 ) . because genotypes were not available for all participants , the number of observations in model 1 included in this comparison differs from the one above ( tables7 and 8 vs. tables3 and 4 , respectively ) . analyses adding offspring covariates and using the other snp sets for the sardinia cohort were also performed . effect sizes and potential redundancy among the influence of known genes and parental lipid trait measures were evaluated by comparing the results of likelihood ratio tests of model 6 versus model 1 ( effect of offspring snps ) and model 6 versus model 5 ( effect of parental lipid trait ) . all analyses were conducted using stata ( 11.1 ; statacorp lp , college station , tx ) and r software ( r foundation for statistical computing , vienna , austria ) . summary statistics of lipid traits and covariates for participants are listed in tables1 , 2 , and s4 , s5 . population characteristics of the framingham heart study participants bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . mean ( sd ) for continuous variables , n ( % total ) for categorical variables . population characteristics of the sardinia cohort bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . mean ( sd ) for continuous variables , n ( % total ) for categorical variables . of note , in the fhs , lipid trait measures from the offspring generation were ascertained at a younger age ( mean 39.0 for daughters , 39.4 for sons ) than those for parents ( mean 46.4 for mothers , 48.7 for fathers ) . comparisons between generations show that tc and ldl - c levels were higher in fathers than sons and higher in mothers than daughters ( p<0.001 for all ) , whereas hdl was higher in sons than fathers ( p<0.001 ) . all comparisons are presented in tables1 and s4 . comparisons within each generation showed that males have higher tc , tg , and ldl , but lower hdl levels , than women . the difference between sex was statistically significant for all traits ( p<0.003 ) , except for tc in the parents ( p=0.06 ) . in the sardinia cohort , lipid trait measures from offspring were ascertained at a younger age ( mean 29.3 for daughters , 28.8 for sons ) than those for their parents ( mean 55.9 for mothers , 58.4 for fathers ) ( table2 ) . comparisons between generations showed that tc , tg , and ldl levels were higher in fathers than sons and higher in mothers than daughters ( p<0.002 for all ) . hdl was significantly higher in fathers compared to sons ( p<0.001 ) ( tables1 and s5 ) . similarly , mothers had higher hdl compared to daughters , although not significantly . comparisons within generations in the sardinia cohort showed that tg were higher in fathers than mothers ( p<0.001 ) ; hdl was significantly higher in mothers compared to fathers , as well as daughters compared to sons ( p<0.001 for both ) . in the fhs , models including only parental lipid traits ( model 1 ) explained between 1% and 5% of offspring variability in 7 trait combinations and more than 5% in 9 others . the highest proportion of explained variance was 10% for mother - son hdl ( table3 ; model 1 ) . maternal lipids explained at least 5% of the variation in tc , ldl , and hdl of the offspring for both sex . in general , maternal lipid values provided more information regarding offspring values than did paternal values ( table3 ) . estimating the parent of origin effects on lipid traits in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . for comparison , the variation of offspring lipid traits explained by all offspring covariates was more than 5% in all 16 models . the highest proportion of variability explained was 15% for ldl of daughters ( model 2 ) . when both parental lipid traits and offspring covariates were used in a single model , the explained variation ranged from 9% ( father - son ldl ) to 19% ( father - son hdl ) ( model 3 ) . importantly , adding parental lipid trait values to a model containing offspring covariates significantly increased the amount of variation explained for tc , ldl , and hdl in all parent offspring pairs ( table3 , p values m3/m2 ) . the amount of variation explained in tg models for fathers - daughters was significant , whereas all other tg parent - offspring pairs were below the significance level . this indicates that the effects of parental lipid traits and offspring covariates are significant and independent of one another , with the exception of tg measures . parental covariates explained less of offspring lipid variability than either offspring covariates or parental lipids as can be seen by comparison of models 2 , 3 , s1 , and s2 ( table s6 ) . in sardinia , models of offspring lipid traits using only corresponding parental lipid traits explained between 0.01% and 5% of in 6 models and more than 5% in 9 others . one model explained less than 0.01% of the variability ( tg for fathers - daughters ) . the highest proportion of variability explained was 15% for mother - son ldl ( table4 ; model 1 ) . as in the fhs , maternal lipid traits in sardinia explained at least 5% of the corresponding variation for tc , ldl , and hdl of both sons and daughters and generally performed better than paternal models . estimating the parent of origin effects on lipid traits in the sardinia cohort the overall numbers of parents used for this analysis are lower than the numbers used in estimating parent of origin effects in table2 because , unlike the framingham heart study , the sardinia cohort is comprised of more single - parent families and , consequently , fewer complete trios . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . the variation of offspring lipid traits explained by offspring covariates alone was between 0.01% and 5% in 4 models and more than 5% in 12 models ( model 2 ) . the highest proportion of variability explained was 36% for tc of sons ( model 2 ) . when both parental lipid traits and offspring covariates were used in the same model , 1 model explained marginal variability ( < 0.01% ) , 1 model explained between 0.01% and 5% , and 14 models explained more than 5% . the highest proportion of variability explained was 39% for father - son tc ( model 3 ) . adding parental lipid trait values to a model containing offspring covariates explained tc significantly better when modeling sons levels with fathers or mothers ( table4 , p values m3/m2 ) , whereas results for daughters trended in the same direction . with the exception of fathers - sons , adding parental hdl or ldl values to model 2 maternal tg levels explained significantly more of daughters tg , but had no effect on the other parent - offspring pairs ( table4 ) . as with the framingham results , parental covariates generally explained less of the offspring lipid variability than either offspring covariates or parental lipids , as can be seen by comparison of models 2 , 3 , s1 , and s2 ( table s7 ) . in the fhs , the variation of maternal lipid traits explained by the corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc and ldl ( table5 ) the percentage of maternal lipid traits explained by corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc , ldl , and hdl ( table6 ; supplementary results ) . both results indicate that shared adult environments do not significantly impact our findings . estimating the effects of shared environment in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . estimating the effects of shared environment in the sardinia cohort hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . in the sardinia cohort , when only parents of daughters were considered , paternal lipid traits explained up to 2.5% of variation for hdl . when only parents of sons were considered , paternal lipid traits explained up to 2.5% of ldl variation ( table6 ) . these results indicate that the shared environment of parents explains very little of lipid trait variation in the unrelated parent pairs , and suggest that the effects of parental lipids on offspring are linked to factors unrelated to the shared environment . when only parents of daughters were considered , paternal lipid traits explained between 0.02% and 3% of maternal tg and ldl , respectively . when only parents of sons were considered , paternal lipid traits only explained 0.1% of maternal hdl variation , with negligible variation explained for all other lipid traits ( table5 ) . using maternal lipid traits to model corresponding paternal lipid traits , produced similar results to using paternal lipid traits to model maternal traits when covariates were included ( tables s8 and s9 ) . the role of shared early environment was also assessed by comparing variance explained for each lipid trait values in siblings of the same to those of the opposite sex . variance explained between siblings of the same sex ranged between 5% and 12% for females and between 0.5% and 6% for males , whereas between offspring of opposite sex the results were generally smaller ( 0% to 5% ) . this supports the conclusion that the results between parents and offspring are not attributable to shared environment ( table s10 ) . in the fhs , snps previously associated with each lipid trait explained a negligible amount of variability ( < 0.01% ) in 2 models , between 0.01% and 5% in 8 models , and more than 5% in 6 models , the highest being 10% for daughters tg ( table7 ; model 5 ) . in the offspring with available genotype data ( a subset of individuals from model 1 ) , parental lipid traits explained between 0.01% and 5% of variability in 8 models and more than 5% in 8 models , the highest being 10% for sons hdl with paternal measures ( table7 ; model 1 ) . adding parental lipid values to the model containing all snps produced significantly better models in explaining tc , ldl , and hdl in all parent - offspring pairs , except fathers - sons , where only the hdl model was significantly improved ( table7 , p values m6/m5 ) . conversely , adding all snps to models containing parental lipid values significantly improved hdl in the mothers - sons model and all models of tgs , with the exception of the fathers - sons comparison ( table7 , p values m6/m1 ) . comparing lipid trait relevant snps to parent of origin effects in the framingham heart study for models 5 and 6 , there are 4 original , 25 proxy for tc ; 4 original , 17 proxy for tg ; 3 original , 15 proxy for ldl ; 5 original , 25 proxy for hdl . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . in sardinia , models with the 92 snps previously associated with lipid traits ( table8 ) resulted in negligible percent variance explained ( < 0.01% ) in 8 of the 16 models ( table8 ; model 5 ) . four models explained between 0.01% and 5% , and 4 explained more than 5% , with the highest percentage being 30% for hdl in mother - son pairs ( model 5 ) . in the subset of offspring with available genotype data , the variation of offspring measures explained by parental lipid traits only was negligible for 2 models ( sons and daughters tg with paternal levels ) ( model 1 ) . parental lipid traits explained between 0.01% and 5% in 4 models , and greater than 5% in 10 models , the highest being 16% for sons tc with maternal levels ( table8 ; model 1 ) . adding parental lipid values to the model containing all snps significantly improved the models for tc , ldl , and hdl in mother - daughter and father - daughter models , for all lipid traits in mother - son models , and for tc and hdl in father - son models ( table8 , p values m6/m5 ) . adding all snps to a model containing parental lipid values produced significant results for hdl in mother - daughter models , for tc and hdl in father - daughter models , for hdl in mother - son models , and for tc , tg , and hdl in father - son models ( table8 , p values m6/m1 ) . these results were generally consistent when the alternative snp sets were used ; namely , the subset of 63 original and proxy snps from the framingham analysis , and also when all nonproxy 63 snps ( tables s11 through s16 ) . comparing lipid trait relevant snps to parent of origin effects in the sardinia cohort for models 5 and 6 , there are 51 snps for tc , 32 snps for tg , 37 snps for ldl , and 47 snps for hdl . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . summary statistics of lipid traits and covariates for participants are listed in tables1 , 2 , and s4 , s5 . population characteristics of the framingham heart study participants bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . mean ( sd ) for continuous variables , n ( % total ) for categorical variables . population characteristics of the sardinia cohort bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . mean ( sd ) for continuous variables , n ( % total ) for categorical variables . of note , in the fhs , lipid trait measures from the offspring generation were ascertained at a younger age ( mean 39.0 for daughters , 39.4 for sons ) than those for parents ( mean 46.4 for mothers , 48.7 for fathers ) . comparisons between generations show that tc and ldl - c levels were higher in fathers than sons and higher in mothers than daughters ( p<0.001 for all ) , whereas hdl was higher in sons than fathers ( p<0.001 ) . all comparisons are presented in tables1 and s4 . comparisons within each generation showed that males have higher tc , tg , and ldl , but lower hdl levels , than women . the difference between sex was statistically significant for all traits ( p<0.003 ) , except for tc in the parents ( p=0.06 ) . in the sardinia cohort , lipid trait measures from offspring were ascertained at a younger age ( mean 29.3 for daughters , 28.8 for sons ) than those for their parents ( mean 55.9 for mothers , 58.4 for fathers ) ( table2 ) . comparisons between generations showed that tc , tg , and ldl levels were higher in fathers than sons and higher in mothers than daughters ( p<0.002 for all ) . hdl was significantly higher in fathers compared to sons ( p<0.001 ) ( tables1 and s5 ) . similarly , mothers had higher hdl compared to daughters , although not significantly . comparisons within generations in the sardinia cohort showed that tg were higher in fathers than mothers ( p<0.001 ) ; hdl was significantly higher in mothers compared to fathers , as well as daughters compared to sons ( p<0.001 for both ) . in the fhs , models including only parental lipid traits ( model 1 ) explained between 1% and 5% of offspring variability in 7 trait combinations and more than 5% in 9 others . the highest proportion of explained variance was 10% for mother - son hdl ( table3 ; model 1 ) . maternal lipids explained at least 5% of the variation in tc , ldl , and hdl of the offspring for both sex . in general , maternal lipid values provided more information regarding offspring values than did paternal values ( table3 ) . estimating the parent of origin effects on lipid traits in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . for comparison , the variation of offspring lipid traits explained by all offspring covariates was more than 5% in all 16 models . the highest proportion of variability explained was 15% for ldl of daughters ( model 2 ) . when both parental lipid traits and offspring covariates were used in a single model , the explained variation ranged from 9% ( father - son ldl ) to 19% ( father - son hdl ) ( model 3 ) . importantly , adding parental lipid trait values to a model containing offspring covariates significantly increased the amount of variation explained for tc , ldl , and hdl in all parent offspring pairs ( table3 , p values m3/m2 ) . the amount of variation explained in tg models for fathers - daughters was significant , whereas all other tg parent - offspring pairs were below the significance level . this indicates that the effects of parental lipid traits and offspring covariates are significant and independent of one another , with the exception of tg measures . parental covariates explained less of offspring lipid variability than either offspring covariates or parental lipids as can be seen by comparison of models 2 , 3 , s1 , and s2 ( table s6 ) . in sardinia , models of offspring lipid traits using only corresponding parental lipid traits explained between 0.01% and 5% of in 6 models and more than 5% in 9 others . one model explained less than 0.01% of the variability ( tg for fathers - daughters ) . the highest proportion of variability explained was 15% for mother - son ldl ( table4 ; model 1 ) . as in the fhs , maternal lipid traits in sardinia explained at least 5% of the corresponding variation for tc , ldl , and hdl of both sons and daughters and generally performed better than paternal models . estimating the parent of origin effects on lipid traits in the sardinia cohort the overall numbers of parents used for this analysis are lower than the numbers used in estimating parent of origin effects in table2 because , unlike the framingham heart study , the sardinia cohort is comprised of more single - parent families and , consequently , fewer complete trios . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . the variation of offspring lipid traits explained by offspring covariates alone was between 0.01% and 5% in 4 models and more than 5% in 12 models ( model 2 ) . the highest proportion of variability explained was 36% for tc of sons ( model 2 ) . when both parental lipid traits and offspring covariates were used in the same model , 1 model explained marginal variability ( < 0.01% ) , 1 model explained between 0.01% and 5% , and 14 models explained more than 5% . the highest proportion of variability explained was 39% for father - son tc ( model 3 ) . adding parental lipid trait values to a model containing offspring covariates explained tc significantly better when modeling sons levels with fathers or mothers ( table4 , p values m3/m2 ) , whereas results for daughters trended in the same direction . with the exception of fathers - sons , adding parental hdl or ldl values to model 2 maternal tg levels explained significantly more of daughters tg , but had no effect on the other parent - offspring pairs ( table4 ) . as with the framingham results , parental covariates generally explained less of the offspring lipid variability than either offspring covariates or parental lipids , as can be seen by comparison of models 2 , 3 , s1 , and s2 ( table s7 ) . in the fhs , the variation of maternal lipid traits explained by the corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc and ldl ( table5 ) . in sardinia , the percentage of maternal lipid traits explained by corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc , ldl , and hdl ( table6 ; supplementary results ) . both results indicate that shared adult environments do not significantly impact our findings . estimating the effects of shared environment in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . estimating the effects of shared environment in the sardinia cohort hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . in the sardinia cohort , when only parents of daughters were considered , paternal lipid traits explained up to 2.5% of variation for hdl . when only parents of sons were considered , paternal lipid traits explained up to 2.5% of ldl variation ( table6 ) . these results indicate that the shared environment of parents explains very little of lipid trait variation in the unrelated parent pairs , and suggest that the effects of parental lipids on offspring are linked to factors unrelated to the shared environment . when only parents of daughters were considered , paternal lipid traits explained between 0.02% and 3% of maternal tg and ldl , respectively . when only parents of sons were considered , paternal lipid traits only explained 0.1% of maternal hdl variation , with negligible variation explained for all other lipid traits ( table5 ) . using maternal lipid traits to model corresponding paternal lipid traits , produced similar results to using paternal lipid traits to model maternal traits when covariates were included ( tables s8 and s9 ) . the role of shared early environment was also assessed by comparing variance explained for each lipid trait values in siblings of the same to those of the opposite sex . variance explained between siblings of the same sex ranged between 5% and 12% for females and between 0.5% and 6% for males , whereas between offspring of opposite sex the results were generally smaller ( 0% to 5% ) . this supports the conclusion that the results between parents and offspring are not attributable to shared environment ( table s10 ) . in the fhs , snps previously associated with each lipid trait explained a negligible amount of variability ( < 0.01% ) in 2 models , between 0.01% and 5% in 8 models , and more than 5% in 6 models , the highest being 10% for daughters tg ( table7 ; model 5 ) . in the offspring with available genotype data ( a subset of individuals from model 1 ) , parental lipid traits explained between 0.01% and 5% of variability in 8 models and more than 5% in 8 models , the highest being 10% for sons hdl with paternal measures ( table7 ; model 1 ) . adding parental lipid values to the model containing all snps produced significantly better models in explaining tc , ldl , and hdl in all parent - offspring pairs , except fathers - sons , where only the hdl model was significantly improved ( table7 , p values m6/m5 ) . conversely , adding all snps to models containing parental lipid values significantly improved hdl in the mothers - sons model and all models of tgs , with the exception of the fathers - sons comparison ( table7 , p values m6/m1 ) . comparing lipid trait relevant snps to parent of origin effects in the framingham heart study for models 5 and 6 , there are 4 original , 25 proxy for tc ; 4 original , 17 proxy for tg ; 3 original , 15 proxy for ldl ; 5 original , 25 proxy for hdl . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . in sardinia , models with the 92 snps previously associated with lipid traits ( table8 ) resulted in negligible percent variance explained ( < 0.01% ) in 8 of the 16 models ( table8 ; model 5 ) . four models explained between 0.01% and 5% , and 4 explained more than 5% , with the highest percentage being 30% for hdl in mother - son pairs ( model 5 ) . in the subset of offspring with available genotype data , the variation of offspring measures explained by parental lipid traits only was negligible for 2 models ( sons and daughters tg with paternal levels ) ( model 1 ) . parental lipid traits explained between 0.01% and 5% in 4 models , and greater than 5% in 10 models , the highest being 16% for sons tc with maternal levels ( table8 ; model 1 ) . adding parental lipid values to the model containing all snps significantly improved the models for tc , ldl , and hdl in mother - daughter and father - daughter models , for all lipid traits in mother - son models , and for tc and hdl in father - son models ( table8 , p values m6/m5 ) . adding all snps to a model containing parental lipid values produced significant results for hdl in mother - daughter models , for tc and hdl in father - daughter models , for hdl in mother - son models , and for tc , tg , and hdl in father - son models ( table8 , p values m6/m1 ) . these results were generally consistent when the alternative snp sets were used ; namely , the subset of 63 original and proxy snps from the framingham analysis , and also when all nonproxy 63 snps ( tables s11 through s16 ) . comparing lipid trait relevant snps to parent of origin effects in the sardinia cohort for models 5 and 6 , there are 51 snps for tc , 32 snps for tg , 37 snps for ldl , and 47 snps for hdl . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . we investigated how parental serum levels of tc , tg , ldl , and hdl can be used to model lipid traits of the offspring , using sex - stratified analyses . we also compared the effect of parental lipids with that of known and validated genetic variants in loci previously associated with plasma lipid levels.11 these analyses were performed in 2 well - characterized and large prospective cohorts , the fhs ( inclusive of offspring and generation 3 cohorts ) and the sardinia study . the most important finding from our study is that , in general , parental serum lipid levels explain a higher proportion of variability in the offspring than do the 95 loci described in the work by teslovich et al.11 this effect was not owing to shared environment and was independent of nongenetic factors known to modulate lipid levels . these results were generally consistent between the framingham and sardinia cohorts ( tables3 through 8) . given the independence from other variables and from the currently known genetic loci , these results suggest the presence of unknown variants or mechanisms responsible for the missing heritability of lipid traits12,13 and serve to emphasize the size of the gap in our knowledge of factors that affect lipid levels . however , because we do not yet have an understanding of these other determinants , we argue that parental lipid levels explain those of adult offspring better than do the validated variants in 95 genes . thus , knowledge of parental lipid levels provides information to predict future lipid levels in the offspring and should be used as a tool to target pediatric lipid testing . with the exception of mendelian forms of dyslipidemia , serum lipids are complex traits influenced by multiple genetic and nongenetic factors . therefore , the use of single - gene variants is of little utility in the prediction of this complex phenotype . to date , gwa studies have identified risk loci that have high statistical significance , but low biological effect sizes , and such markers are not generally practical for predictive purposes.3538 as a consequence , genetics - based predictions using multilocus modeling thus far provide marginal clinical utility in prevention because they have low predictive power.35,39 the value of a genetic test depends on several factors , including the number of genes influencing the trait , frequency of the associating allele , and strength of association between genotype and phenotype , making accurate predictions from simple models extremely difficult.35 in contrast , the results of our study show that family history and nongenetic covariates better explain lipid levels in adult offspring than does variation in the loci known to influence lipids across study populations . our findings are in agreement with what has been shown in other complex phenotypes , such as type ii diabetes , where risk scores not including genetic variant data were virtually identical to those incorporating validated genes for type ii diabetes risk.37,38 similar results were also found in a previous study where a gene - based score did not significantly improve the association between canonical risk factors and cvd.39 recently , 62 additional lipid - associated loci were identified , but their effects were small , explaining < 2% of the total phenotype variance and therefore should not substantially impact our conclusions.40 furthermore , we demonstrate the existence of parent - of - origin effects on lipid levels , which are sex - specific and likely owing to both genetic and epigenetic factors . for example , a recent gwa study has demonstrated parent - of - origin effects in the degree to which snps in 2 genes , slc2a10 and kcnk9 , affect bmi , a major factor affecting lipids . these snps showed polar overdominance , where homozygotes of either snp had the same average bmi , whereas heterozygotes differed as a function of parent of origin.41 we also found that maternal traits generally explain more of the offspring s tc , hdl , and ldl . maternal lipid traits explained at least 5% of the offspring variability in tc , hdl , and ldl of both sons and daughters in both cohorts . paternal traits were less consistent , given that their effects ranged from nonsignificant in multiple traits to relatively high in explaining the daughter s ldl and hdl ( 5% to 8% of explained variability in framingham and 7% to 11% in sardinia ) . the finding of stronger maternal influences on offspring lipid traits is consistent with epidemiological data demonstrating that maternal lifestyle and environment ( such as nutritional status , stress level , insulin resistance , diabetes , hypertension , hypercholesterolemia , obesity , and smoking ) , both at time of conception and during pregnancy , influence the offspring s phenotypes , such as adiposity , blood pressure , fatty streak formation , or diabetes.15,17,4250 this is consistent with the barker hypothesis , that is , that early exposure , both pre- and postnatal , can affect risk of adult - onset disease.51,52 interestingly , small or nonexistent parent - of - origin effects were generally observed for tg ( tables3 and 4 ) . tgs also provided the most variable results when using genetic models ( tables5 and 6 ) . tg levels have a smaller parent - of - origin effect than the other lipid traits , and a bigger part of their heritability may be determined by other factors , such as rare variants . it has been recently demonstrated that rare apoc3 mutations have a strong influence on plasma tg levels in aggregate.53,54 our study has several limitations . although we were able to identify models that account for a significant portion of lipid variation explained , we were not able to provide a mechanism for this effect . we can only speculate that our observations may forecast discovery of additional genes , gene - gene interactions , or epigenetic effects that regulate lipid levels . furthermore , in our analyses , we did not account for specific environmental variables , such as diet , alcohol , exercise , socioeconomic status , and use of specific medications . however , it is of note that the 2 cohorts we studied would be expected to have different environmental exposures and the results were still mostly concordant . this discrepancy is not likely to have influenced the results , and it would have had an attenuating effect even if it did . both cohorts are prospective studies analyzing populations of european ancestry , but framingham s residents are from multiple european origins , whereas the participants in sardinia are part of a genetic isolate . this may be the basis for the minor discrepancies we observed ( seen in tables3 through 6 ) . dietary habits were not directly quantified and , consequently , were not represented in our models in either cohort , although bmi and lipid medication covariates can be considered partial proxies for diet and lifestyle . however , the similarities of results between cohorts provide additional strength to our main claim . in conclusion , we have determined that parent - of - origin effects explain more variability in the adult offspring s lipid levels than do common variants in the loci known to modulate lipid metabolism . knowledge of the parent s lipid levels may provide an inexpensive and practical means to predict future lipid levels in their children . this work was partially funded by grant nih 2r01hl057986 - 15a1 and 5r01hl106845 - 03 ( fazio ) , nih 2t32hl007751 - 16a2 ( predazzi ) , nih p20 gm103534 ( williams ) , nih hl116263 ( linton ) , nih national institute of aging n01-ag-1 - 2109 , n01-ag-1 - 2109 , and sardinian autonomous region ( l.r . sponsors / funders had no role in the design and conduct of the study ; collection , management , analysis , and interpretation of the data ; as well as in the preparation , review , or approval of the manuscript . snps and proxies used for each lipid trait in the genetic models . for the framingham cohort , a set of both proxies and original snps was used . for the sardinia cohort , the full set of snps for each lipid trait was used , and separate analyses were carried out using the same set of original snps and proxies as in the framingham cohort , and a set of only original snps corresponding to the framingham snps . table s4 . parent of origin effects on offspring lipid traits , adjusting for parental covariates in the framingham heart study . parent of origin effects on offspring lipid traits , adjusting for parental covariates in the sardinia cohort . table s8 . estimating effects of shared environment : modeling maternal lipid traits with their paternal counterparts in the framingham heart study . table s9 . estimating effects of shared environment : modeling paternal lipid traits with their maternal counterparts in the framingham heart study . assessment of the role of shared environment by the examination of the relationships between offspring of same and opposite gender for all lipid measures . comparing phenotype relevant snps to parent of origin effects using offspring covariates in the framingham heart study . comparing phenotype relevant snps to parent of origin effects using offspring covariates in the sardinia cohort , using 95 available teslovich snps . comparing phenotype relevant snps to parent of origin effects in the sardinia cohort using the same proxy snps as in the framingham heart study . comparing phenotype relevant snps to parent of origin effects in the sardinia cohort using the same proxy snps as in the framingham heart study and offspring covariates . comparing phenotype relevant snps to parent of origin effects in the sardinia cohort using the 63 original teslovich snps corresponding to framingham proxies and original snps . comparing phenotype relevant snps to parent of origin effects in the sardinia cohort using the 63 original teslovich snps corresponding to framingham proxies and original snps and offspring covariates .

backgroundplasma lipid levels are highly heritable traits , but known genetic loci can only explain a small portion of their heritability.methods and resultsin this study , we analyzed the role of parental levels of total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , and triglycerides ( tgs ) in explaining the values of the corresponding traits in adult offspring . we also evaluated the contribution of nongenetic factors that influence lipid traits ( age , body mass index , smoking , medications , and menopause ) alone and in combination with variability at the genetic loci known to associate with tc , ldl - c , hdl - c , and tg levels . we performed comparisons among different sex - specific regression models in 416 families from the framingham heart study and 304 from the sardinia cohort . models including parental lipid levels explain significantly more of the trait variation than models without these measures , explaining up to 39% of the total trait variation . of this variation , the parent - of - origin effect explains as much as 15% and it does so in a sex - specific way . this observation is not owing to shared environment , given that spouse - pair correlations were negligible ( < 1.5% explained variation in all cases ) and is distinct from previous genetic and acquired factors that are known to influence serum lipid levels.conclusionsthese findings support the concept that unknown genetic and epigenetic contributors are responsible for most of the heritable component of the plasma lipid phenotype , and that , at present , the clinical utility of knowing age - matched parental lipid levels in assessing risk of dyslipidemia supersedes individual locus effects . our results support the clinical utility of knowing parental lipid levels in assessing future risk of dyslipidemia .

Methods Study Participants Assessment of Risk Factors Statistical Analysis POE on offspring lipid traits Estimating environmental effects Genetic contribution to POE Results Population Characteristics Parent of Origin Effects on Offspring Lipid Traits Environmental Effects Genetic Contribution to the POE Discussion Sources of Funding Disclosures Supporting Information

the original cohort involved 5209 participants , 5124 were enrolled in the second generation starting in 1971 , and 4095 in the third generation starting in 2002.27,28 our analysis included participants from the second and third generations of the fhs for whom serum tc , tg , ldl - c ( calculated using the friedewald equation : ldl - c = tc - hdl(tg/5 ) ) and hdl - c were available for both generations.29 to limit confounding of results by relatedness , we only considered the oldest offspring for each nuclear family , creating parent - offspring trios . the other phenotypes relevant to our study were age , body mass index ( bmi ) , smoking status , use of lipid - lowering medications ( ever treated vs. never treated ) , as well as the menopausal status in females . in sardinia , we analyzed the same phenotypes and covariates from visit 1 in the parental and offspring population , given that both provided the largest number of patients with available lipid data . to identify transmission effects , we performed a series of nested , sex - stratified linear regression analyses , modeling lipid traits in offspring . to estimate the effects of shared environment on lipid traits , we modeled each maternal lipid trait with the corresponding paternal lipid trait under the assumption that shared environment would be revealed by large r in this regression model ( model 4 ) . to examine whether the effects of parental lipid traits are explained by genetic variants in offspring , we analyzed the effects of the 95 previously validated single - nucleotide variants ( snps ) from teslovich et al.11 on the corresponding lipid levels . to assess the dependence of offspring lipid traits on snps previously associated with each lipid trait , we performed nested , sex - stratified linear regression models and compared them to the variance explained only by the corresponding parental lipid traits ( figure1 panel b ) . in addition , to make all analyses directly comparable between the 2 cohorts , we also evaluated the same subset of 63 snps , original and proxies , as in the framingham cohort . the original cohort involved 5209 participants , 5124 were enrolled in the second generation starting in 1971 , and 4095 in the third generation starting in 2002.27,28 our analysis included participants from the second and third generations of the fhs for whom serum tc , tg , ldl - c ( calculated using the friedewald equation : ldl - c = tc - hdl(tg/5 ) ) and hdl - c were available for both generations.29 to limit confounding of results by relatedness , we only considered the oldest offspring for each nuclear family , creating parent - offspring trios . the other phenotypes relevant to our study were age , body mass index ( bmi ) , smoking status , use of lipid - lowering medications ( ever treated vs. never treated ) , as well as the menopausal status in females . in sardinia , we analyzed the same phenotypes and covariates from visit 1 in the parental and offspring population , given that both provided the largest number of patients with available lipid data . to identify transmission effects , we performed a series of nested , sex - stratified linear regression analyses , modeling lipid traits in offspring . to estimate the effects of shared environment on lipid traits , we modeled each maternal lipid trait with the corresponding paternal lipid trait under the assumption that shared environment would be revealed by large r in this regression model ( model 4 ) . to examine whether the effects of parental lipid traits are explained by genetic variants in offspring , we analyzed the effects of the 95 previously validated single - nucleotide variants ( snps ) from teslovich et al.11 on the corresponding lipid levels . to assess the dependence of offspring lipid traits on snps previously associated with each lipid trait , we performed nested , sex - stratified linear regression models and compared them to the variance explained only by the corresponding parental lipid traits ( figure1 panel b ) . in addition , to make all analyses directly comparable between the 2 cohorts , we also evaluated the same subset of 63 snps , original and proxies , as in the framingham cohort . to identify transmission effects , we performed a series of nested , sex - stratified linear regression analyses , modeling lipid traits in offspring . to estimate the effects of shared environment on lipid traits , we modeled each maternal lipid trait with the corresponding paternal lipid trait under the assumption that shared environment would be revealed by large r in this regression model ( model 4 ) . to examine whether the effects of parental lipid traits are explained by genetic variants in offspring , we analyzed the effects of the 95 previously validated single - nucleotide variants ( snps ) from teslovich et al.11 on the corresponding lipid levels . to assess the dependence of offspring lipid traits on snps previously associated with each lipid trait , we performed nested , sex - stratified linear regression models and compared them to the variance explained only by the corresponding parental lipid traits ( figure1 panel b ) . in addition , to make all analyses directly comparable between the 2 cohorts , we also evaluated the same subset of 63 snps , original and proxies , as in the framingham cohort . population characteristics of the framingham heart study participants bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . population characteristics of the sardinia cohort bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . maternal lipids explained at least 5% of the variation in tc , ldl , and hdl of the offspring for both sex . estimating the parent of origin effects on lipid traits in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . when both parental lipid traits and offspring covariates were used in a single model , the explained variation ranged from 9% ( father - son ldl ) to 19% ( father - son hdl ) ( model 3 ) . importantly , adding parental lipid trait values to a model containing offspring covariates significantly increased the amount of variation explained for tc , ldl , and hdl in all parent offspring pairs ( table3 , p values m3/m2 ) . as in the fhs , maternal lipid traits in sardinia explained at least 5% of the corresponding variation for tc , ldl , and hdl of both sons and daughters and generally performed better than paternal models . estimating the parent of origin effects on lipid traits in the sardinia cohort the overall numbers of parents used for this analysis are lower than the numbers used in estimating parent of origin effects in table2 because , unlike the framingham heart study , the sardinia cohort is comprised of more single - parent families and , consequently , fewer complete trios . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . when both parental lipid traits and offspring covariates were used in the same model , 1 model explained marginal variability ( < 0.01% ) , 1 model explained between 0.01% and 5% , and 14 models explained more than 5% . with the exception of fathers - sons , adding parental hdl or ldl values to model 2 maternal tg levels explained significantly more of daughters tg , but had no effect on the other parent - offspring pairs ( table4 ) . in the fhs , the variation of maternal lipid traits explained by the corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc and ldl ( table5 ) the percentage of maternal lipid traits explained by corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc , ldl , and hdl ( table6 ; supplementary results ) . estimating the effects of shared environment in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . estimating the effects of shared environment in the sardinia cohort hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . these results indicate that the shared environment of parents explains very little of lipid trait variation in the unrelated parent pairs , and suggest that the effects of parental lipids on offspring are linked to factors unrelated to the shared environment . adding parental lipid values to the model containing all snps produced significantly better models in explaining tc , ldl , and hdl in all parent - offspring pairs , except fathers - sons , where only the hdl model was significantly improved ( table7 , p values m6/m5 ) . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . in sardinia , models with the 92 snps previously associated with lipid traits ( table8 ) resulted in negligible percent variance explained ( < 0.01% ) in 8 of the 16 models ( table8 ; model 5 ) . adding parental lipid values to the model containing all snps significantly improved the models for tc , ldl , and hdl in mother - daughter and father - daughter models , for all lipid traits in mother - son models , and for tc and hdl in father - son models ( table8 , p values m6/m5 ) . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . population characteristics of the framingham heart study participants bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . population characteristics of the sardinia cohort bmi indicates body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . maternal lipids explained at least 5% of the variation in tc , ldl , and hdl of the offspring for both sex . estimating the parent of origin effects on lipid traits in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . when both parental lipid traits and offspring covariates were used in a single model , the explained variation ranged from 9% ( father - son ldl ) to 19% ( father - son hdl ) ( model 3 ) . importantly , adding parental lipid trait values to a model containing offspring covariates significantly increased the amount of variation explained for tc , ldl , and hdl in all parent offspring pairs ( table3 , p values m3/m2 ) . as in the fhs , maternal lipid traits in sardinia explained at least 5% of the corresponding variation for tc , ldl , and hdl of both sons and daughters and generally performed better than paternal models . estimating the parent of origin effects on lipid traits in the sardinia cohort the overall numbers of parents used for this analysis are lower than the numbers used in estimating parent of origin effects in table2 because , unlike the framingham heart study , the sardinia cohort is comprised of more single - parent families and , consequently , fewer complete trios . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . when both parental lipid traits and offspring covariates were used in the same model , 1 model explained marginal variability ( < 0.01% ) , 1 model explained between 0.01% and 5% , and 14 models explained more than 5% . with the exception of fathers - sons , adding parental hdl or ldl values to model 2 maternal tg levels explained significantly more of daughters tg , but had no effect on the other parent - offspring pairs ( table4 ) . in the fhs , the variation of maternal lipid traits explained by the corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc and ldl ( table5 ) . in sardinia , the percentage of maternal lipid traits explained by corresponding paternal lipid traits ranged from negligible ( < 0.01% ) for tg to 1% for tc , ldl , and hdl ( table6 ; supplementary results ) . estimating the effects of shared environment in the framingham heart study hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . estimating the effects of shared environment in the sardinia cohort hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; tc , total cholesterol ; tg , triglycerides . these results indicate that the shared environment of parents explains very little of lipid trait variation in the unrelated parent pairs , and suggest that the effects of parental lipids on offspring are linked to factors unrelated to the shared environment . in the fhs , snps previously associated with each lipid trait explained a negligible amount of variability ( < 0.01% ) in 2 models , between 0.01% and 5% in 8 models , and more than 5% in 6 models , the highest being 10% for daughters tg ( table7 ; model 5 ) . adding parental lipid values to the model containing all snps produced significantly better models in explaining tc , ldl , and hdl in all parent - offspring pairs , except fathers - sons , where only the hdl model was significantly improved ( table7 , p values m6/m5 ) . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . in sardinia , models with the 92 snps previously associated with lipid traits ( table8 ) resulted in negligible percent variance explained ( < 0.01% ) in 8 of the 16 models ( table8 ; model 5 ) . adding parental lipid values to the model containing all snps significantly improved the models for tc , ldl , and hdl in mother - daughter and father - daughter models , for all lipid traits in mother - son models , and for tc and hdl in father - son models ( table8 , p values m6/m5 ) . hdl indicates high - density lipoprotein ; ldl , low - density lipoprotein ; snp , single - nucleotide polymorphism ; tc , total cholesterol ; tg , triglycerides . we investigated how parental serum levels of tc , tg , ldl , and hdl can be used to model lipid traits of the offspring , using sex - stratified analyses . we also compared the effect of parental lipids with that of known and validated genetic variants in loci previously associated with plasma lipid levels.11 these analyses were performed in 2 well - characterized and large prospective cohorts , the fhs ( inclusive of offspring and generation 3 cohorts ) and the sardinia study . the most important finding from our study is that , in general , parental serum lipid levels explain a higher proportion of variability in the offspring than do the 95 loci described in the work by teslovich et al.11 this effect was not owing to shared environment and was independent of nongenetic factors known to modulate lipid levels . given the independence from other variables and from the currently known genetic loci , these results suggest the presence of unknown variants or mechanisms responsible for the missing heritability of lipid traits12,13 and serve to emphasize the size of the gap in our knowledge of factors that affect lipid levels . however , because we do not yet have an understanding of these other determinants , we argue that parental lipid levels explain those of adult offspring better than do the validated variants in 95 genes . to date , gwa studies have identified risk loci that have high statistical significance , but low biological effect sizes , and such markers are not generally practical for predictive purposes.3538 as a consequence , genetics - based predictions using multilocus modeling thus far provide marginal clinical utility in prevention because they have low predictive power.35,39 the value of a genetic test depends on several factors , including the number of genes influencing the trait , frequency of the associating allele , and strength of association between genotype and phenotype , making accurate predictions from simple models extremely difficult.35 in contrast , the results of our study show that family history and nongenetic covariates better explain lipid levels in adult offspring than does variation in the loci known to influence lipids across study populations . our findings are in agreement with what has been shown in other complex phenotypes , such as type ii diabetes , where risk scores not including genetic variant data were virtually identical to those incorporating validated genes for type ii diabetes risk.37,38 similar results were also found in a previous study where a gene - based score did not significantly improve the association between canonical risk factors and cvd.39 recently , 62 additional lipid - associated loci were identified , but their effects were small , explaining < 2% of the total phenotype variance and therefore should not substantially impact our conclusions.40 furthermore , we demonstrate the existence of parent - of - origin effects on lipid levels , which are sex - specific and likely owing to both genetic and epigenetic factors . these snps showed polar overdominance , where homozygotes of either snp had the same average bmi , whereas heterozygotes differed as a function of parent of origin.41 we also found that maternal traits generally explain more of the offspring s tc , hdl , and ldl . maternal lipid traits explained at least 5% of the offspring variability in tc , hdl , and ldl of both sons and daughters in both cohorts . the finding of stronger maternal influences on offspring lipid traits is consistent with epidemiological data demonstrating that maternal lifestyle and environment ( such as nutritional status , stress level , insulin resistance , diabetes , hypertension , hypercholesterolemia , obesity , and smoking ) , both at time of conception and during pregnancy , influence the offspring s phenotypes , such as adiposity , blood pressure , fatty streak formation , or diabetes.15,17,4250 this is consistent with the barker hypothesis , that is , that early exposure , both pre- and postnatal , can affect risk of adult - onset disease.51,52 interestingly , small or nonexistent parent - of - origin effects were generally observed for tg ( tables3 and 4 ) . tg levels have a smaller parent - of - origin effect than the other lipid traits , and a bigger part of their heritability may be determined by other factors , such as rare variants . in conclusion , we have determined that parent - of - origin effects explain more variability in the adult offspring s lipid levels than do common variants in the loci known to modulate lipid metabolism . for the sardinia cohort , the full set of snps for each lipid trait was used , and separate analyses were carried out using the same set of original snps and proxies as in the framingham cohort , and a set of only original snps corresponding to the framingham snps . parent of origin effects on offspring lipid traits , adjusting for parental covariates in the framingham heart study . estimating effects of shared environment : modeling maternal lipid traits with their paternal counterparts in the framingham heart study . estimating effects of shared environment : modeling paternal lipid traits with their maternal counterparts in the framingham heart study . comparing phenotype relevant snps to parent of origin effects in the sardinia cohort using the same proxy snps as in the framingham heart study . comparing phenotype relevant snps to parent of origin effects in the sardinia cohort using the same proxy snps as in the framingham heart study and offspring covariates .

the issue of bank robberies , in their different aspects , is an object of attention of the banking sector and its institutions ; however , a positive trend in terms of prevention has not emerged in recent years . according to the report on robberies in bank branches in 2012 , published by the research centre for anti - crime security of the italian banking association , since 2007 bank robberies have more than halved ( approximately 63%).1 despite this positive development , the consequences for the staff involved in a criminal event such as a robbery remain of major concern to banks . risk management in robberies has gradually become one of the main issues of health and safety at work , to be assessed in accordance with the principles and rules laid down by the legislation for prevention . the spirit of the consolidated law for the protection of health and safety in the workplace ( legislative decree no 81/2008 and subsequent amendments ) is not to protect the bank s assets , but to make the employer eliminate or minimize the risks to which employees are exposed as a result of a criminal event . indeed , the legislature assigns the employer the responsibility of protecting employees , not from general risk , namely , that any person may be exposed to an event since they are part of the community , but as a specific risk , since it is connected to the type of work.2 the italian national institute for insurance against accidents at work believed that because of the goal of the legislative decree no 81/2008 and subsequent amendments , the risk of robbery should be included under occupational hazards and be properly assessed.3 as a traumatic event , a robbery may have consequences on both the physical and psychological integrity of the employees involved . compared to the past , we now see an almost total absence of ill treatment of staff by the robbers . a survey carried out by several banks pointed out that nobody needed to seek medical attention , even after suffering violence , and that , in the few cases that required it , any injuries found resolution after a few days.4 psychological consequences are , however , a different matter . according to the scientific literature on the subject , it is clear that the response to traumatic events is the result of a complex interaction of many variables , which include the type of stressful event , the individual characteristics , the subjective response , and social support to which one has access.57 people s reactions to critical events can be varied , and the psychological consequences can be just as varied.8 if , for example , the victim of the robbery is experiencing a negative emotional state ( going through mourning , separation , health problems , etc ) , it is easy to understand that there may be a greater impact from a critical event . the reactions after the traumatic event may resolve spontaneously , especially if favorable conditions are present and other traumatic events do not arise . however , in some cases , it is possible that the reactions evolve toward more complex clinical pictures . one can pass from an acute stress disorder ( ads ) to a posttraumatic stress disorder ( ptsd ) . ads occurs within 4 weeks of stress and lasts from a minimum of 2 days to a maximum of 4 weeks ; it represents the preliminary category of ptsd and is characterized by dissociative symptoms listed in the dsm - iv ( intrusion symptoms , avoidance , and hyperactivity ) . according to the dsm - iv diagnosis of ptsd , certain criteria must be met.9 first , the person must have been exposed to a traumatic event ( which has resulted in death or threat of death or serious injury or threat of physical integrity to self or others ) and the response of the subject must have been one of intense fear , horror , or feelings of helplessness . psychopathological symptoms ( intrusion , avoidance , and arousal ) must also arise , resulting in clinically significant distress or impairment in social , occupational , or otherwise important areas . finally , the disorder must last more than 1 month . depending on the duration of the disorder , we can define ptsd as : acute ptsd , if symptoms last less than 3 months ; chronic ptsd , if symptoms last longer than 3 months ; delayed - onset ptsd , if symptoms occur at least 6 months after the event.10,11 the predictors of ptsd are pre- , peri- , and posttrauma variables . pretraumatic factors are the individual characteristics of the subject : the female sex , age less than 25 years , a low level of education , having experienced traumatic events in the past , and the presence of preexisting psychopathological disorders are all factors favoring the onset of ptsd.11,12 however , several studies have confirmed a strong association of posttraumatic syndromes with poorer mental health and with psychological problems such as depression , anxiety , and personality disorders.13,14 as far as demographic data are concerned , its association appears less solid . miller - burke et al5 did not find a significant relationship of age and sex with the development of posttraumatic symptoms . accordingly , ozer et als15 meta - analysis investigating pretrauma variables concluded in pointing out the marginal effect of these specific variables in understanding the development of ptsd . peritraumatic risk factors are those closely related to the event : duration , intensity , and nature of the traumatic event . literature found a strong association between perceived life threat and posttraumatic symptomatology , both in victims of robberies and in victims of other types of assaults and aggressions.12,15,16 for instance , believing they or someone else could be harmed , feeling threatened by the robber , and seeing a weapon might increase the risk of developing ptsd.6,16 in addition , literature found relationships between ptsd and perceived helplessness , since traumatized employees seem to commonly feel helpless or fearful during the robbery . in addition , proximity to the robber ( such as seeing the robbery , being in the same room with the robber , and being able to identify the robber ) has been found to be associated with posttraumatic symptoms following robbery.12,14 finally , the posttraumatic factors are the immediate reactions after the trauma , such as the consequences of physical or psychological damage or loss.17,18 it is important to insist on the destabilizing effects that a robbery can have on a person with a nonoptimal mental health . it is noteworthy that trauma itself overwhelms the normal systems that we use to deal with unfortunate events by assigning them some meaning . the defense system , when facing the impossibility of reacting properly , implements alternative mechanisms such as avoidance , which includes avoiding any activity or thought associated with the trauma . this is an example of a mechanism that a person with suboptimal mental health would have a hard time exploiting . furthermore , in many chronic cases of ptsd , we can observe the occurrence of other conditions , which leads to diagnostic issues , due to the overlapping medical symptoms . numerous studies have shown that a history of psychiatric disorders or a preexisting tendency to anxiety or depression can all be vulnerability factors for ptsd.4,17,19 the main aim of this research is to analyze the association of pre- , peri- , and posttrauma variables , measured a few days after the robbery , and the development of ptsd symptoms . in particular , we want to test whether exposure to the robbery , the traumatic event , may contribute to the onset of typical symptoms of ptsd : intrusion , avoidance , and arousal . according to the relevant literature , we expect that the severity of posttraumatic symptoms present positive and significant association with young age , female sex , previous trauma , robberies in the past , perceived danger of life , perception of lack of aid , and proximity to the robber . in addition , we expect that posttraumatic experience , such as the development of mental health problems , can have a positive association with the severity of posttraumatic symptoms . written consent was obtained from participants for the use of their information in this medical study . the exploration of the victims reactions after the robbery was carried out in the days immediately following the robbery ( 17 days ) and featured a questionnaire administered by occupational physicians to the people involved in the robbery or to those who had explicitly requested it . the administration of the questionnaire and the results obtained were maintained with professional secrecy and general respect of privacy . the first part consists of demographics ( pretrauma variables ) and several questions ( yes / no ) describing the event in general , which represent the peritrauma variables : whether he / she was involved directly in the robbery or was just an observer.whether he / she were previously victims of a robbery.whether he / she was in the same room as the robber.whether he / she felt threatened by the robber.whether he / she imagined being potentially harmed.whether he / she felt intense fear.whether he / she had physical contact with the robber . whether he / she was involved directly in the robbery or whether he / she felt threatened by the robber . whether he / she imagined being potentially harmed . whether he / she felt intense fear . whether he / she had physical contact with the robber . the second part featured the italian version of the impact of event scale revised-6 ( ies - r of six items ) , which investigates the mental and behavioral reactions to a traumatic event and analyses the specific symptoms : intrusion , avoidance , and hyperarousal.20,21 the respondents of the questionnaire are invited to indicate on a four point scale ranging from never ( score 1 ) to often ( score 4 ) how frequently each symptom was experienced in the previous week . the ghq12 is one of the most recognized tools within the scientific community for the measurement and assessment of mental health.22,23 a four point likert - scale scoring method was used according to the alternative responses presented to participants : ( 0= less than usual ; 1= no more than usual ; 2= rather more than usual ; and 3= much more than usual ) . thus , the questionnaire gives a total score ranging from 0 to 36 points , in which a higher score indicates mental health problems . the survey , which took place between 2012 and 2014 , involved 644 employees of a banking institution on italian territory . the sample consisted of 276 females ( 43% , n=644 ) and 368 males ( 57% , n=644 ) , with a mean age of 41.49.7 years . the questionnaires were collected from various italian regions with the following geographical distribution : 271 from the north of italy , 42% ( n=644 ) , 188 from central italy , 29% ( n=644 ) , and 185 from the south of italy , 29% ( n=644 ) . there were 15 regions involved : lombardy ( 137 ) , piedmont ( 29 ) , veneto ( 30 ) , friuli - julian venetia ( 5 ) , and emilia - romagna ( 70 ) from the north of italy ; tuscany ( 57 ) , umbria ( 22 ) , marche ( 10 ) , abruzzo ( 26 ) , and latium ( 73 ) from central italy ; sicily ( 140 ) , calabria ( 8) , apulia ( 10 ) , campania ( 20 ) , basilicata ( 6 ) , and sardinia ( 1 ) to the south , and the islands of italy . the questionnaires were delivered , in respect of anonymity and privacy , to a consortium formed by occupational medicine and occupational psychology specialists , who proceeded to analyze them using appropriate statistical methods ( descriptive statistics , pearson s r correlation , and hierarchical regressions ) . statistical analyses were performed to identify the existence of relations between the variables investigated , according to the literature.5,12 in particular , hierarchical regression was used to evaluate the relationship between a set of independent variables and the dependent variable , controlling for or taking into account the impact of a different set of independent variables on the dependent variable . in the first block , the demographic variables ( sex and age ) were tested as predictors , while , in the further blocks , dimensions of peritrauma variables were tested : proximity / familiarity with the robbery as the second block and perceived fear of harm and helplessness as the third block . finally , posttrauma variables ( mental health problems measured by the ghq-12 ) were added in the fourth block . further analysis tested intrusion , avoidance , and hyperarousal . instead of using the total score of the ies - r-6 , we used its subscales . written consent was obtained from participants for the use of their information in this medical study . the exploration of the victims reactions after the robbery was carried out in the days immediately following the robbery ( 17 days ) and featured a questionnaire administered by occupational physicians to the people involved in the robbery or to those who had explicitly requested it . the administration of the questionnaire and the results obtained were maintained with professional secrecy and general respect of privacy . the first part consists of demographics ( pretrauma variables ) and several questions ( yes / no ) describing the event in general , which represent the peritrauma variables : whether he / she was involved directly in the robbery or was just an observer.whether he / she were previously victims of a robbery.whether he / she was in the same room as the robber.whether he / she felt threatened by the robber.whether he / she imagined being potentially harmed.whether he / she felt intense fear.whether he / she had physical contact with the robber . whether he / she was involved directly in the robbery or whether he / she felt threatened by the robber . whether he / she imagined being potentially harmed . whether he / she felt intense fear . whether he / she had physical contact with the robber . the second part featured the italian version of the impact of event scale revised-6 ( ies - r of six items ) , which investigates the mental and behavioral reactions to a traumatic event and analyses the specific symptoms : intrusion , avoidance , and hyperarousal.20,21 the respondents of the questionnaire are invited to indicate on a four point scale ranging from never ( score 1 ) to often ( score 4 ) how frequently each symptom was experienced in the previous week . the ghq12 is one of the most recognized tools within the scientific community for the measurement and assessment of mental health.22,23 a four point likert - scale scoring method was used according to the alternative responses presented to participants : ( 0= less than usual ; 1= no more than usual ; 2= rather more than usual ; and 3= much more than usual ) . thus , the questionnaire gives a total score ranging from 0 to 36 points , in which a higher score indicates mental health problems . the survey , which took place between 2012 and 2014 , involved 644 employees of a banking institution on italian territory . the sample consisted of 276 females ( 43% , n=644 ) and 368 males ( 57% , n=644 ) , with a mean age of 41.49.7 years . the questionnaires were collected from various italian regions with the following geographical distribution : 271 from the north of italy , 42% ( n=644 ) , 188 from central italy , 29% ( n=644 ) , and 185 from the south of italy , 29% ( n=644 ) . there were 15 regions involved : lombardy ( 137 ) , piedmont ( 29 ) , veneto ( 30 ) , friuli - julian venetia ( 5 ) , and emilia - romagna ( 70 ) from the north of italy ; tuscany ( 57 ) , umbria ( 22 ) , marche ( 10 ) , abruzzo ( 26 ) , and latium ( 73 ) from central italy ; sicily ( 140 ) , calabria ( 8) , apulia ( 10 ) , campania ( 20 ) , basilicata ( 6 ) , and sardinia ( 1 ) to the south , and the islands of italy . the questionnaires were delivered , in respect of anonymity and privacy , to a consortium formed by occupational medicine and occupational psychology specialists , who proceeded to analyze them using appropriate statistical methods ( descriptive statistics , pearson s r correlation , and hierarchical regressions ) . statistical analyses were performed to identify the existence of relations between the variables investigated , according to the literature.5,12 in particular , hierarchical regression was used to evaluate the relationship between a set of independent variables and the dependent variable , controlling for or taking into account the impact of a different set of independent variables on the dependent variable . in the first block , the demographic variables ( sex and age ) were tested as predictors , while , in the further blocks , dimensions of peritrauma variables were tested : proximity / familiarity with the robbery as the second block and perceived fear of harm and helplessness as the third block . finally , posttrauma variables ( mental health problems measured by the ghq-12 ) were added in the fourth block . further analysis tested intrusion , avoidance , and hyperarousal . instead of using the total score of the ies - r-6 , we used its subscales . different statistical analyses were conducted . first , the descriptives of the single questions related to the bank robbery were calculated : 74% ( n=478 ) of the sample was directly involved in the robbery ; 62% ( n=400 ) had already experienced a robbery in the past ; 83% ( n=534 ) were in the same room as the robber ; 55% ( n=354 ) felt threatened by the robber ; 46% ( n=299 ) thought they would be hurt ; and 58% ( n=374 ) felt very scared and helpless , while 20% ( n=130 ) had physical contact with the robber . finally , in order to show the completeness of the data collected , company records consulted indicated that only 2% ( n=10 ) of victims of robberies had been injured . as far as the first regression model is concerned , statistical analyses showed that demographics ( age and sex ) are not significant in the prediction of trauma . as far as the peritrauma variables are concerned , having already been robbed and being in the same room of the thief were not significant , whereas being directly involved in the robbery , having had thoughts of being hurt , and having perceived intense fear were significant . finally , ghq factors were associated with the development of the trauma . only loss of confidence was not significant ( table 2 ) . specifically , in the first block , demographic data were not significant . when the peritrauma variables were added in the second and third block , the model was significant , and these dimensions each accounted for the 5% increase in variance in the second ( proximity / familiarity with the robbery ) and a further 22% in the third block ( fear of harm and helplessness ) . finally , when ghq dimensions were added in the fourth block , the model was significant , and these dimensions accounted for the 28% increase in variance . overall , the final model of four blocks explains 55% of the variance for the development of posttraumatic symptoms ( table 2 ) . in the second regression analysis , we kept the same independent variables divided into four blocks , and we considered avoidance as the dependent variable . analysis showed that demographic data were not significant . adding the variables of the second block , being directly involved in the robbery was significant ; however , when adding the third and the fourth blocks of variables , a direct involvement in the robbery lost significance . the statistically significant variables in the model were thinking about being injured , having experienced intense fear , anxiety , and depression ( ghq-12 ) . overall , the final model explains 30% of the variance for the development of symptoms of avoidance ( table 3 ) . in the third regression analysis , we considered arousal as a dependent variable , in association with the same independent variables of the previous analysis . in this case , demographic data were also not significant , and statistical significance in the second and third block pertained to the following peritraumatic variables : being directly involved in the robbery , having had thoughts of being injured , and having experienced an intense fear . however , in the fourth block , having experienced an intense fear alone remained significant . in addition , anxiety , dysphoria , and loss of confidence resulted as significant and increased the explained variance of 33% over and above demographics and the peritraumatic variables . overall , the final model explains 54% of the variance for the development of typical symptoms of arousal ( table 4 ) . in the final regression analysis , we considered intrusion as the dependent variable . as with arousal , demographic data were not significant , while the significance was being directly involved in the robbery , to having had thoughts about being injured and to having felt an intense fear . adding the variables of the last block indicated that the significance of the previous blocks did not change and , in addition , anxiety resulted as significant . overall , the final model explains 48.3% of the variance for the development of typical symptoms of intrusion ( table 5 ) . the purpose of this study was to examine , among italian employees , the role of pre- , peri- , and posttrauma variables on ptsd development . the four regression models performed in this study seemed predictive . based on the 30%55% of the variance of ptsd and based on these models , ptsd subfactors were explained in our study ( intrusion , avoidance , and arousal ) . a closer analysis of the specific contribution of each pre- , peri- , and posttrauma variable in the development of ptsd symptomatology showed interesting results . first , pretrauma variables , sex and age , did not seem very connected to the development of the ptsd . indeed , the impact of demographics in all models tested was very limited and not significant . although several studies accounted for sex differences in ptsd,24 in our research , variables such as sex and age were not significant following the results of studies conducted on victims of violence ( workplace bullying).2527 furthermore , the origin of sex differences in ptsd may be closely linked to sex differences in the subjective experience and evaluation of the trauma , rather than more objective features , such as trauma type and degree of exposure.24 second , for peritrauma variables in all tested models , higher associations with ptsd symptoms were found , particularly whether he / she felt threatened by the robber , whether he / she imagined potentially being harmed , and whether he / she felt intense fear . loss of confidence was not associated with ptsd ; however , as far as intrusion and arousal were concerned , loss of confidence was linked negatively to ptsd symptoms . this may possibly be because self - confident employees might perceive the traumatic experience as more destabilizing than employees with lower positivity . however , the impact of self - confidence on ptsd seemed quite limited , especially when compared with the effects of anxiety and depression ( ghq-12 factors ) that could develop in their chronic manifestations , causing an inability to reconcile a traumatic experience with an integrated vision of oneself and the world . people affected also display repeated conscious intrusions of painful memories , followed by a strong physiological activation related to active and passive attempts to avoid the resurfacing of those memories . this cycle of intrusion and avoidance opens the door to a progressive deterioration of symptoms and disabilities in the period following the exposure to traumatic events , even more so in those who are not completely healthy from a mental standpoint . since it is so important to prevent and counteract the negative effects of robbery traumas among employees , it has become increasingly relevant to study the psychological antecedents of ptsd . our study not only confirms that a bank robbery is a traumatic event for the employee , but also highlights the importance of considering the influence of the individual characteristics of the victim and their immediate reactions on posttraumatic symptomatology.1520 while personal characteristics ( sex and age ) and the characteristics of the event itself do not seem to have a crucial role , mental health problems seem to have a fundamental role in influencing the development of posttraumatic symptoms over and above pre- and peritrauma variables . as far as peritrauma variables are concerned , intense fear , accompanied by a strong sense of powerlessness , the threat to their own and others safety felt during the robbery , and the presence of anxiety seem the main risk factors for the development of posttraumatic symptoms . this consideration reinforces our opinion , also supported by the literature of the importance of the victim s personal interpretation with respect to the degree of threat in a robbery at the moment in which it occurs : there are many factors that affect this assessment and not all have a relationship with the objective gravity of the event.18,28 the role of mental health is also fundamental . the first limitation of this study is that it can not prove causality since the study design was cross - sectional . a second limitation is the use of self - reports , which may contribute to common method bias.29 a third limitation concerns the sample , which is not representative of the italian population ; indeed regions have been exclusively used for purposes of description of the sample . unlike other demographic characteristics , such as female sex or age below 25 years , all of which were shown to be unimportant , we underline instead the relevance of preexisting psychopathological disorders as predictors of ptsd . this study definitely deserves follow - up to reach its assigned purpose , namely , to minimize the risk to the employees of a given occupation when they are exposed to a criminal event like a robbery . our findings suggest that banks should adopt corporate policies containing activities of prevention and protection toward stress and , more generally , mental health of workers . for example , the availability of a specific policy for bank robberies offers both the company and the workers important tools for well - being , including postrobbery psychological support , online / phone counseling center , training groups , etc .

in the literature , there are many studies that have investigated the psychological reactions resulting from traumatic events of varying degrees , such as wars , natural disasters , and acts of violence . few , however , are the searches performed on employees who are victims of robbery . we carried out a research to assess the psychological reactions of 644 bank employees who had been victims of robbery , especially with regard to the possible development of post - traumatic stress disorder ( ptsd ) . the aim of this study was to evaluate the variables pre- , peri- , and postrobbery trauma in relation to the development of psychopathological symptoms . the exploration of the reactions after the robbery was carried out on 644 employees of a banking institution , present throughout the national territory , through a survey , consisting of a general description of the event , the impact of event scale revised-6 scale , and the general health questionnaire-12 , during the days after the robbery . the analysis showed that the development of pretrauma variables is not significant and that peritrauma variables are partially significant . in particular , being directly involved in the robbery , the thought of being hurt , and the feeling of intense fear are associated with posttraumatic symptoms . finally , among the posttrauma variables , anxiety and depression played a major role . surprisingly , a lower level of self - confidence seems to be related negatively to the ptsd symptomatology .

Introduction Aim Methods Questionnaire Sample Analyses Results Discussion and conclusion

the issue of bank robberies , in their different aspects , is an object of attention of the banking sector and its institutions ; however , a positive trend in terms of prevention has not emerged in recent years . according to the report on robberies in bank branches in 2012 , published by the research centre for anti - crime security of the italian banking association , since 2007 bank robberies have more than halved ( approximately 63%).1 despite this positive development , the consequences for the staff involved in a criminal event such as a robbery remain of major concern to banks . the spirit of the consolidated law for the protection of health and safety in the workplace ( legislative decree no 81/2008 and subsequent amendments ) is not to protect the bank s assets , but to make the employer eliminate or minimize the risks to which employees are exposed as a result of a criminal event . indeed , the legislature assigns the employer the responsibility of protecting employees , not from general risk , namely , that any person may be exposed to an event since they are part of the community , but as a specific risk , since it is connected to the type of work.2 the italian national institute for insurance against accidents at work believed that because of the goal of the legislative decree no 81/2008 and subsequent amendments , the risk of robbery should be included under occupational hazards and be properly assessed.3 as a traumatic event , a robbery may have consequences on both the physical and psychological integrity of the employees involved . a survey carried out by several banks pointed out that nobody needed to seek medical attention , even after suffering violence , and that , in the few cases that required it , any injuries found resolution after a few days.4 psychological consequences are , however , a different matter . according to the scientific literature on the subject , it is clear that the response to traumatic events is the result of a complex interaction of many variables , which include the type of stressful event , the individual characteristics , the subjective response , and social support to which one has access.57 people s reactions to critical events can be varied , and the psychological consequences can be just as varied.8 if , for example , the victim of the robbery is experiencing a negative emotional state ( going through mourning , separation , health problems , etc ) , it is easy to understand that there may be a greater impact from a critical event . the reactions after the traumatic event may resolve spontaneously , especially if favorable conditions are present and other traumatic events do not arise . however , in some cases , it is possible that the reactions evolve toward more complex clinical pictures . one can pass from an acute stress disorder ( ads ) to a posttraumatic stress disorder ( ptsd ) . ads occurs within 4 weeks of stress and lasts from a minimum of 2 days to a maximum of 4 weeks ; it represents the preliminary category of ptsd and is characterized by dissociative symptoms listed in the dsm - iv ( intrusion symptoms , avoidance , and hyperactivity ) . according to the dsm - iv diagnosis of ptsd , certain criteria must be met.9 first , the person must have been exposed to a traumatic event ( which has resulted in death or threat of death or serious injury or threat of physical integrity to self or others ) and the response of the subject must have been one of intense fear , horror , or feelings of helplessness . depending on the duration of the disorder , we can define ptsd as : acute ptsd , if symptoms last less than 3 months ; chronic ptsd , if symptoms last longer than 3 months ; delayed - onset ptsd , if symptoms occur at least 6 months after the event.10,11 the predictors of ptsd are pre- , peri- , and posttrauma variables . pretraumatic factors are the individual characteristics of the subject : the female sex , age less than 25 years , a low level of education , having experienced traumatic events in the past , and the presence of preexisting psychopathological disorders are all factors favoring the onset of ptsd.11,12 however , several studies have confirmed a strong association of posttraumatic syndromes with poorer mental health and with psychological problems such as depression , anxiety , and personality disorders.13,14 as far as demographic data are concerned , its association appears less solid . miller - burke et al5 did not find a significant relationship of age and sex with the development of posttraumatic symptoms . accordingly , ozer et als15 meta - analysis investigating pretrauma variables concluded in pointing out the marginal effect of these specific variables in understanding the development of ptsd . peritraumatic risk factors are those closely related to the event : duration , intensity , and nature of the traumatic event . literature found a strong association between perceived life threat and posttraumatic symptomatology , both in victims of robberies and in victims of other types of assaults and aggressions.12,15,16 for instance , believing they or someone else could be harmed , feeling threatened by the robber , and seeing a weapon might increase the risk of developing ptsd.6,16 in addition , literature found relationships between ptsd and perceived helplessness , since traumatized employees seem to commonly feel helpless or fearful during the robbery . in addition , proximity to the robber ( such as seeing the robbery , being in the same room with the robber , and being able to identify the robber ) has been found to be associated with posttraumatic symptoms following robbery.12,14 finally , the posttraumatic factors are the immediate reactions after the trauma , such as the consequences of physical or psychological damage or loss.17,18 it is important to insist on the destabilizing effects that a robbery can have on a person with a nonoptimal mental health . the defense system , when facing the impossibility of reacting properly , implements alternative mechanisms such as avoidance , which includes avoiding any activity or thought associated with the trauma . numerous studies have shown that a history of psychiatric disorders or a preexisting tendency to anxiety or depression can all be vulnerability factors for ptsd.4,17,19 the main aim of this research is to analyze the association of pre- , peri- , and posttrauma variables , measured a few days after the robbery , and the development of ptsd symptoms . in particular , we want to test whether exposure to the robbery , the traumatic event , may contribute to the onset of typical symptoms of ptsd : intrusion , avoidance , and arousal . according to the relevant literature , we expect that the severity of posttraumatic symptoms present positive and significant association with young age , female sex , previous trauma , robberies in the past , perceived danger of life , perception of lack of aid , and proximity to the robber . in addition , we expect that posttraumatic experience , such as the development of mental health problems , can have a positive association with the severity of posttraumatic symptoms . the exploration of the victims reactions after the robbery was carried out in the days immediately following the robbery ( 17 days ) and featured a questionnaire administered by occupational physicians to the people involved in the robbery or to those who had explicitly requested it . the first part consists of demographics ( pretrauma variables ) and several questions ( yes / no ) describing the event in general , which represent the peritrauma variables : whether he / she was involved directly in the robbery or was just an observer.whether he / she were previously victims of a robbery.whether he / she was in the same room as the robber.whether he / she felt threatened by the robber.whether he / she imagined being potentially harmed.whether he / she felt intense fear.whether he / she had physical contact with the robber . the second part featured the italian version of the impact of event scale revised-6 ( ies - r of six items ) , which investigates the mental and behavioral reactions to a traumatic event and analyses the specific symptoms : intrusion , avoidance , and hyperarousal.20,21 the respondents of the questionnaire are invited to indicate on a four point scale ranging from never ( score 1 ) to often ( score 4 ) how frequently each symptom was experienced in the previous week . the survey , which took place between 2012 and 2014 , involved 644 employees of a banking institution on italian territory . there were 15 regions involved : lombardy ( 137 ) , piedmont ( 29 ) , veneto ( 30 ) , friuli - julian venetia ( 5 ) , and emilia - romagna ( 70 ) from the north of italy ; tuscany ( 57 ) , umbria ( 22 ) , marche ( 10 ) , abruzzo ( 26 ) , and latium ( 73 ) from central italy ; sicily ( 140 ) , calabria ( 8) , apulia ( 10 ) , campania ( 20 ) , basilicata ( 6 ) , and sardinia ( 1 ) to the south , and the islands of italy . statistical analyses were performed to identify the existence of relations between the variables investigated , according to the literature.5,12 in particular , hierarchical regression was used to evaluate the relationship between a set of independent variables and the dependent variable , controlling for or taking into account the impact of a different set of independent variables on the dependent variable . in the first block , the demographic variables ( sex and age ) were tested as predictors , while , in the further blocks , dimensions of peritrauma variables were tested : proximity / familiarity with the robbery as the second block and perceived fear of harm and helplessness as the third block . finally , posttrauma variables ( mental health problems measured by the ghq-12 ) were added in the fourth block . the exploration of the victims reactions after the robbery was carried out in the days immediately following the robbery ( 17 days ) and featured a questionnaire administered by occupational physicians to the people involved in the robbery or to those who had explicitly requested it . the first part consists of demographics ( pretrauma variables ) and several questions ( yes / no ) describing the event in general , which represent the peritrauma variables : whether he / she was involved directly in the robbery or was just an observer.whether he / she were previously victims of a robbery.whether he / she was in the same room as the robber.whether he / she felt threatened by the robber.whether he / she imagined being potentially harmed.whether he / she felt intense fear.whether he / she had physical contact with the robber . whether he / she was involved directly in the robbery or whether he / she felt threatened by the robber . the second part featured the italian version of the impact of event scale revised-6 ( ies - r of six items ) , which investigates the mental and behavioral reactions to a traumatic event and analyses the specific symptoms : intrusion , avoidance , and hyperarousal.20,21 the respondents of the questionnaire are invited to indicate on a four point scale ranging from never ( score 1 ) to often ( score 4 ) how frequently each symptom was experienced in the previous week . the survey , which took place between 2012 and 2014 , involved 644 employees of a banking institution on italian territory . there were 15 regions involved : lombardy ( 137 ) , piedmont ( 29 ) , veneto ( 30 ) , friuli - julian venetia ( 5 ) , and emilia - romagna ( 70 ) from the north of italy ; tuscany ( 57 ) , umbria ( 22 ) , marche ( 10 ) , abruzzo ( 26 ) , and latium ( 73 ) from central italy ; sicily ( 140 ) , calabria ( 8) , apulia ( 10 ) , campania ( 20 ) , basilicata ( 6 ) , and sardinia ( 1 ) to the south , and the islands of italy . statistical analyses were performed to identify the existence of relations between the variables investigated , according to the literature.5,12 in particular , hierarchical regression was used to evaluate the relationship between a set of independent variables and the dependent variable , controlling for or taking into account the impact of a different set of independent variables on the dependent variable . in the first block , the demographic variables ( sex and age ) were tested as predictors , while , in the further blocks , dimensions of peritrauma variables were tested : proximity / familiarity with the robbery as the second block and perceived fear of harm and helplessness as the third block . finally , posttrauma variables ( mental health problems measured by the ghq-12 ) were added in the fourth block . first , the descriptives of the single questions related to the bank robbery were calculated : 74% ( n=478 ) of the sample was directly involved in the robbery ; 62% ( n=400 ) had already experienced a robbery in the past ; 83% ( n=534 ) were in the same room as the robber ; 55% ( n=354 ) felt threatened by the robber ; 46% ( n=299 ) thought they would be hurt ; and 58% ( n=374 ) felt very scared and helpless , while 20% ( n=130 ) had physical contact with the robber . finally , in order to show the completeness of the data collected , company records consulted indicated that only 2% ( n=10 ) of victims of robberies had been injured . as far as the first regression model is concerned , statistical analyses showed that demographics ( age and sex ) are not significant in the prediction of trauma . as far as the peritrauma variables are concerned , having already been robbed and being in the same room of the thief were not significant , whereas being directly involved in the robbery , having had thoughts of being hurt , and having perceived intense fear were significant . finally , ghq factors were associated with the development of the trauma . when the peritrauma variables were added in the second and third block , the model was significant , and these dimensions each accounted for the 5% increase in variance in the second ( proximity / familiarity with the robbery ) and a further 22% in the third block ( fear of harm and helplessness ) . finally , when ghq dimensions were added in the fourth block , the model was significant , and these dimensions accounted for the 28% increase in variance . overall , the final model of four blocks explains 55% of the variance for the development of posttraumatic symptoms ( table 2 ) . in the second regression analysis , we kept the same independent variables divided into four blocks , and we considered avoidance as the dependent variable . analysis showed that demographic data were not significant . adding the variables of the second block , being directly involved in the robbery was significant ; however , when adding the third and the fourth blocks of variables , a direct involvement in the robbery lost significance . the statistically significant variables in the model were thinking about being injured , having experienced intense fear , anxiety , and depression ( ghq-12 ) . overall , the final model explains 30% of the variance for the development of symptoms of avoidance ( table 3 ) . in the third regression analysis , we considered arousal as a dependent variable , in association with the same independent variables of the previous analysis . in this case , demographic data were also not significant , and statistical significance in the second and third block pertained to the following peritraumatic variables : being directly involved in the robbery , having had thoughts of being injured , and having experienced an intense fear . however , in the fourth block , having experienced an intense fear alone remained significant . in addition , anxiety , dysphoria , and loss of confidence resulted as significant and increased the explained variance of 33% over and above demographics and the peritraumatic variables . overall , the final model explains 54% of the variance for the development of typical symptoms of arousal ( table 4 ) . as with arousal , demographic data were not significant , while the significance was being directly involved in the robbery , to having had thoughts about being injured and to having felt an intense fear . adding the variables of the last block indicated that the significance of the previous blocks did not change and , in addition , anxiety resulted as significant . overall , the final model explains 48.3% of the variance for the development of typical symptoms of intrusion ( table 5 ) . the purpose of this study was to examine , among italian employees , the role of pre- , peri- , and posttrauma variables on ptsd development . based on the 30%55% of the variance of ptsd and based on these models , ptsd subfactors were explained in our study ( intrusion , avoidance , and arousal ) . a closer analysis of the specific contribution of each pre- , peri- , and posttrauma variable in the development of ptsd symptomatology showed interesting results . first , pretrauma variables , sex and age , did not seem very connected to the development of the ptsd . indeed , the impact of demographics in all models tested was very limited and not significant . although several studies accounted for sex differences in ptsd,24 in our research , variables such as sex and age were not significant following the results of studies conducted on victims of violence ( workplace bullying).2527 furthermore , the origin of sex differences in ptsd may be closely linked to sex differences in the subjective experience and evaluation of the trauma , rather than more objective features , such as trauma type and degree of exposure.24 second , for peritrauma variables in all tested models , higher associations with ptsd symptoms were found , particularly whether he / she felt threatened by the robber , whether he / she imagined potentially being harmed , and whether he / she felt intense fear . loss of confidence was not associated with ptsd ; however , as far as intrusion and arousal were concerned , loss of confidence was linked negatively to ptsd symptoms . however , the impact of self - confidence on ptsd seemed quite limited , especially when compared with the effects of anxiety and depression ( ghq-12 factors ) that could develop in their chronic manifestations , causing an inability to reconcile a traumatic experience with an integrated vision of oneself and the world . this cycle of intrusion and avoidance opens the door to a progressive deterioration of symptoms and disabilities in the period following the exposure to traumatic events , even more so in those who are not completely healthy from a mental standpoint . our study not only confirms that a bank robbery is a traumatic event for the employee , but also highlights the importance of considering the influence of the individual characteristics of the victim and their immediate reactions on posttraumatic symptomatology.1520 while personal characteristics ( sex and age ) and the characteristics of the event itself do not seem to have a crucial role , mental health problems seem to have a fundamental role in influencing the development of posttraumatic symptoms over and above pre- and peritrauma variables . as far as peritrauma variables are concerned , intense fear , accompanied by a strong sense of powerlessness , the threat to their own and others safety felt during the robbery , and the presence of anxiety seem the main risk factors for the development of posttraumatic symptoms . this consideration reinforces our opinion , also supported by the literature of the importance of the victim s personal interpretation with respect to the degree of threat in a robbery at the moment in which it occurs : there are many factors that affect this assessment and not all have a relationship with the objective gravity of the event.18,28 the role of mental health is also fundamental . a second limitation is the use of self - reports , which may contribute to common method bias.29 a third limitation concerns the sample , which is not representative of the italian population ; indeed regions have been exclusively used for purposes of description of the sample . unlike other demographic characteristics , such as female sex or age below 25 years , all of which were shown to be unimportant , we underline instead the relevance of preexisting psychopathological disorders as predictors of ptsd . this study definitely deserves follow - up to reach its assigned purpose , namely , to minimize the risk to the employees of a given occupation when they are exposed to a criminal event like a robbery . for example , the availability of a specific policy for bank robberies offers both the company and the workers important tools for well - being , including postrobbery psychological support , online / phone counseling center , training groups , etc .

fish consumption reduces the risk of developing cardiovascular disease ( cvd ) and cvd mortality [ 1 , 2 ] . reduced total mortality and major coronary events , including fatal and non - fatal mi , are observed in intervention trials after intake of fish and fish oil containing the marine n-3 fatty acids eicosapentaenoic acid ( epa , 20:5 n-3 ) and docosahexaenoic acid ( dha , 22:6 n-3 ) [ 36 ] . the association between high intakes of marine n-3 fatty acids and decreased morbidity and mortality from cvd can be explained by the decrease in plasma triglycerides [ 7 , 8 ] , moderate reduction in blood pressure and reduced blood plate aggregation [ 10 , 11 ] . protection against cardiac arrhythmias has also been shown but the effect is still under discussion [ 12 , 13 ] . , the combined action of risk factors causes the gradual thickening of the arterial wall due to lipid accumulation to form an atherosclerotic plaque which can abruptly rupture , causing thrombosis . there is an association between various biomarkers of inflammation and prospective cvd risk in apparently healthy individuals as well as in patients with cvd or heart failure . systemic biomarkers of early and late atherosclerosis are of great clinical interest due to their potential for identifying high risk patients . even though there are some good candidates , only c - reactive protein ( crp ) has emerged as a leading biomarker of inflammation for clinical application [ 15 , 16 ] ; other markers with appropriate robustness , sensitivity and specificity have not yet emerged . based on the fact that endothelial dysfunction is the earliest manifestation of atherosclerosis , along with the involvement of oxidative stress and inflammation at all stages of coronary atherosclerosis development , biomarkers such as soluble intracellular adhesion molecule ( sicam-1 ) and soluble vascular adhesion molecule ( svcam-1 ) , soluble e - selectin ( se - sel ) ( for endothelial activation ) , interleukin ( il)-6 ( transcriptional driver of crp ) and monocyte chemoattractant protein ( mcp)-1 ( produced by activated vasculature ) are of particular interest ( table 1 ) . so far crp , il-6 , svcam-1 and sicam-1 provide prognostic information beyond that obtained by clinical variables after acute coronary syndromes ; these mediators seem to be powerful predictors of subsequent cardiovascular events . recently , intake of marine n-3 fatty acids has been associated with reduced plasma levels of inflammatory markers [ 1921].table 1relevant inflammatory markers and their biological functioninflammatory markersabbreviationfunctionacute - phase protein c - reactive proteincrpcrp is associated with the formation of cytokines , chemokines and the acute - phase responsecytokines interleukin-6il-6induces acute - phase response ( by inducing crp ) , anti - body secretion and differentiation interleukin-1 , interleukin-1il-1 , il-1proliferation and maturation of lymphocytes , involved in inflammation and acute - phase response interleukin-18il-18involved in the formation of th1 cellstumor necrosis factor- is a cytokinetnf-induces adhesion molecules- and cytokine expression , involved in cell deathadhesion protein soluble intercellular adhesion molecule-1sicam-1binds monocytes and lymphocytes to the endothelium soluble vascular cell adhesion molecule-1svcam-1binds monocytes and lymphocytes to the endothelium se - selectinse - selrecruits leukocytes to the inflammatory site sp - selectinsp - selrecruits leukocytes to the inflammatory site . induces monocytes and platelet interactionschemokines monocyte chemoattractant protein-1mcp-1facilitates migration of leukocytes to the intima granulocyte macrophage colony - stimulating factorgm csfgrowth and differentiation of monocytes interleukin-8il-8facilitates migration of leukocytes to the intima relevant inflammatory markers and their biological function the aim of the present paper was to review the literature in order to summarize the effects of marine n-3 fatty acids on circulating inflammatory markers among healthy subjects , subjects with high risk of developing cvd and in patients with cvd in human intervention studies . the systematic literature search was conducted in pubmed in 2009 using the following terms : eicosapentaenoic acid or docosapentaenoic acid or docosahexaenoic acid or omega-3 or fish oil or cod liver oil and inflammation . the following limitations were included in the search : added to pubmed in the last 10 years , published in the last 10 years , humans , clinical trial , english . in total , 91 articles were identified . based on these papers , we included all studies that measured the effect of marine n-3 fatty acids on circulating inflammatory markers in plasma or serum among apparently healthy individuals ( table 2 ) , individuals with high risk of developing cvd ( table 3 ) or individuals with cvd ( table 4 ) . interventions with marine n-3 fatty acids given as supplements or in the diet as fish were included . the following exclusion criteria were used : ( 1 ) interventions assessing inflammatory markers with ex vivo methods ( 2 ) , interventions with children ( 3 ) and articles describing animal or cell culture studies . after reading abstracts of the 91 papers , the literature lists of the selected papers were checked and 13 additional relevant papers were included based on the same inclusion and exclusion criteria as the literature search . however , no further papers were included from this search.table 2a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in healthy individualsstudyindividuals ( n)number of groupsdose n-3 ( g / day)durationcrpcytokinesother inflammatory markersinterventions with n-3 supplements geelen et al . 43 men and 41 women , healthy ( 5070 years)2 groups : sunflower oil or fish oilfish oil:0.7 g epa0.56 g dha0.26 g other n-3 pufa12 weeks vega - lopez et al . 80 healthy men and women ( 2055 years)4 groups : placebo , n-3 pufa , vit e or n-3 pufa / vit en-3 pufa:0.6 g epa0.9 g dha12 60 healthy men and women ( 2157 years)3 groups : olive oil , low dose fish oil or high dose fish oillow dose fish oil:0.9 g epa0.8 g dhahigh dose fish oil:3 g epa2.9 g dha12 weeks yusof et al . 20 healthy men ( 3560 years)2 groups : coconut oil or fish oilfish oil:1.8 g epa0.3 g dha8 weeks il-6 sicam-1 svcam-1 , se - sel , sp - sel fujioka et al . 59 men and 82 women , healthy ( mean age 38 years)2 groups : olive oil or fish oilfish oil:0.6 g epa0.26 g dha12 weeks tnf - r1 , tnf - r2 ciubotaru et al . 30 healthy women , postmenopausal and hormone treated ( mean age 60 years)3 groups : sunflower oil , low dose fish oil or high dose fish oillow dose fish oil:1.3 g n-3 pufahigh dose fish oil:2.56 g n-3 pufa5 weeks * il-6 * pot et al . 77 healthy elderly men and women ( 5070 years)2 groups : sunflower oil or fish oilfish oil:0.7 g epa0.56 g dha0.26 g other n-3 pufa12 weeks ( il-1 , il-1b , il-2 , il-4 , il-5 , il-6 , il-10 , il-12 , il-13 , tnf- , ifn-) mif , mcp-1 , mip-1 , rantes , ccl11 , il-8 svcam-1 and sicam-1 thies et al . 46 healthy men and women ( 5575 years)6 groups : palm and sunflower oils , ala , gla , aa , dha or fish oilfish oil:0.72 g epa0.28 g dha or dha 0.7 g12 weeks s - vcam-1 ( fish oil and ala) se - sel ( fish oil and ala) sicam-1 miles et al . 16 men < 40 years and 12 elderly men and women > 55 years2 groups : palm oil and soy bean oils or fish oilfish oil:1.2 g epa / dha12 weeks sicam-1 svcam-1 ( older) se - sel ( young ) michaeli et al . 15 healthy men ( mean age 26 years)2 groups : fish oil or not ( not - blinded)fish oil:1.1 g epa0.7 g dha34 weeks followed by lps stimulation tnf- , il-6 cazolla et al . 93 healthy young men ( 1842 years ) and 62 healthy elderly men ( 5370 years)4 groups : corn oil or 3 different doses epa oilepa oil ( epax 4510tg):1.35 g , 2.7 g or 4.05 g epa12 weeks se - sel ( 4,05 g epa , young men) sicam-1 ( 4,05 g epa , both groups ) ( tendency)interventions with n-3 diet tsitouras et al . 12 healthy elderly men and women ( 6075 years)2 groups : control diet or n-3 diet ( cross - over design)n-3 diet:720 g / week fatty fish and sardine oil ( 45 g / day epa and dha)8 weeks il-6 ( tendency ) paulo mc 275 healthy men and women ( 2040 years)4 groups : sunflower oil , fish oil , lean fish or fatty fishfish oil:0.63 g epa and 0.43 g dhalean fish:0.05 g epa and 0.21 g dhafatty fish:0.77 g epa and 1.37 g dha8 weeks sicam-1 ( lean fish)svcam-1 ( fish oil and lean fish)all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated * larger change in crp and il-6 with fish oil with low content of epa and dha compared to fish oil with high contentala -linolenic acid ( c18;3 , n-3 ) , gla -linolenic acid ( c18;3 , n-6 ) , aa arachidonic acid ( c20:4 , n-6)table 3a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with high cvd riskstudyindividuals ( n)number of groupsdose n-3 ( g / day)durationcrpcytokinesother inflammatory markersinterventions with n-3 supplements kelley et al . 34 hyperlipidemic men ( 3966 years)2 groups : olive oil or dha - oildha - oil:3 g dha3 months il-6 il-1 , il-2 , il-10 , tnf- gm - csf circulating neutrophilsmmp-2 il-8 murphy et al.74 overweight individuals , bmi > 25 and tg > 1.6 , ( 2065 years)2 groups : n-3 enriched diet or no enriched dietn-3 enriched food:1 g epa and dha6 months plat et al . 11 healthy overweight , bmi : 30 - 35 , ( mean age 59 years)2 groups : sunflower oil or fish oil ( cross - over design)fish oil:0.6 g epa0.5 g dha6 weeks sicam-1 , se - sel, mcp-1 browning et al.30 healthy overweight women , bmi > 252 groups : placebo ( la and oa ) or fish oil ( cross - over design)fish oil:1.3 g epa2.9 g dha12 weeks il-6 kabir et al . 26 women with type 2 diabetes , 4060 years2 groups : placebo ( paraffin oil ) or fish oilfish oil:1.08 g epa0.72 g dha2 months il-6 , tnf- krebs et al . 93 overweight women , bmi > 27 , fasting insulin > 7 , ( 2169 years)3 groups : placebo ( la and oa ) , fish oil + weight reduction or placebo + weight reductionfish oil:1.3 g epa2.9 g dha24 weeks il-6 , tnf- jellema et al . 11 overweight men , bmi : 30 - 352 groups : sunflower oil or fish oil ( cross - over design)fish oil:1.35 g epa and dha6 weeks il-6 , tnf- ( fasting or postprandial) stnf - r55 , stnf - r75(fasting or postprandial ) mori et al . 59 hypertensive type 2 diabetic patients ( 4075 years)3 groups : olive oil , epa or dhan-3 pufa:4 g epa ( ethyl ester)or4 g dha ( ethyl ester)6 weeks il-6 , tnf- chan et al . 48 overweight men , bmi > 29 , mean age 53 years4 groups : corn oil , atorvastatin + corn oil , fish oil , atorvastatin + fish oiln-3 pufa : omacore 4 g / day(1.8 g epa and 1.56 g dha ethyl ester)6 weeks il-6 , tnf- accinni et al . 57 dyslipidemic individuals ( 2365 years)3 groups : placebo , n-3 pufa / vite or n-3 pufa / vite and -oryzanol / niacinn-3 pufa:0.66 g epa0.44 29 individuals with type 2diabetes and 21 healthy controls4 groups : controls and patients were given n-3 pufa or not . ( not placebo controlled)n-3 pufa:1.2 g epa0.8 g dha3 weeks svcam-1 , sicam-1 , se - sel , seljeflot et al . 41 male smokers with hyperlipidemia(4557 years)4 groups : n-3 pufa and antioxidatns , n-3 pufa , antioxidants , placebon-3 pufa:4.8 g epa and dha ( ethyl ester)6 weeks se - sel , svcam-1 ( n-3 pufa)interventions with n-3 diet troseid et al . 487 elderly men ( 6476 years)4 groups : corn oil , n-3 pufa , corn oil + dietary intervention or n-3 pufa + dietary interventionn-3 pufa:0.84 g epa0.48 g dhadietary intervention : increase intake of unsaturated fat , fish , fruit and vegetables3 years il-18 ( dietary intervention , n-3 pufa) il-6 , tnf-, mcp-1 hjerkinn et al . 487 elderly men with high cvd risk ( 6476 years)4 groups : corn oil , n-3 pufa , corn oil + dietary intervention or n-3 pufa + dietary interventionn-3 pufa:0.84 g epa0.48 g dhadietary intervention : increase intake of unsaturated fat , fish , fruit and vegetables3 years sicam-1 ( n-3 pufa , dietary intervention , dietary intervention + n-3 pufa ) berstad 171 elderly men with high cvd risk ( 6575 years)4 groups : corn oil , n-3 pufa , corn oil + dietary intervention or n-3 pufa + dietary interventionn-3 pufa:0.84 g epa0.48 g dhadietary intervention : increase intake of unsaturated fat , fish , fruit and vegetables18 months sicam-1 , svcam-1 # se - sel ( dietary intervention)all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated positive correlation between change in serum n-3 pufa and svcam-1tg triglycerides , oa oleic acid ( c18:1 , n-9 ) , la linoleic acid ( c18:2 , n-6)table 4a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with cvd and cvd related diseasesindividuals ( n)number of groupsdose n-3 ( g / day)durationcrpcytokinesother inflammatory markersinterventions with n-3 supplements schiano et al . 32 men and women with pad , mean age 66 years2 groups : no change in treatment or n-3 pufa(not blinded)n-3 pufa:0.85 g epa and dha ( ethyl ester)12 weeks myeloperoxidase madsen et al . 41 men and women with previous mi , mean age 63 years2 groups : clive oil or n-3 252 men and women with previous mi , ( 2887 years)2 groups : corn oil or n-3 pufan-3 pufa:3.4 g epa and dha ( ethyl ester)12 months sicam-1 , se - sel lee et al . 77 men and women with previous mi , 40 healthy controls , mean age 57 years2 groups : no change in treatment or n-3 pufa ( not blinded)n-3 pufa : omacore 1 g / day ( ethyl ester)12 weeks il-6 sp - sel thies et al . 162 men and women awaiting carotid endarterectomy , mean age 69 years3 groups : palm and soy bean oils , sunflower oil or fish oilfish oil:1.4 g n-3 pufa7189 days ( median 42 days) sicam-1 , svcam-1 in plaques macrophage infiltration(anti - cd68 ) in plaques ( fish oil ) johansen et al . 54 men and women with chd , mean age 58 years2 groups : corn oil or n-3 pufa 5,1 g for 6 months , followed by 4 weeks where both groups received n-3 pufan-3 pufa:5.1 g epa and dha4 weeks svcam-1 , se sel ( corn oil followed by 4 weeks with n-3 pufa)interventions with n-3 diet seierstad et al . 60 men and women with chd ( 4675 years)3 groups : all groups got 700 g salmon / week with low , moderate or high content of n-3 pufan-3 content in salmon : low n-3 : 0.5 g epa + dhamoderate n-3 : 1.5 g epa + dhahigh n-3 : 2.9 g epa + dha6 weeks il-6 , ( high n-3) svcam-1 ( high n-3)all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise statedmi myocardial infarction , pad peripheral arterial disease , chd coronary heart disease a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in healthy individuals all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated * larger change in crp and il-6 with fish oil with low content of epa and dha compared to fish oil with high content ala -linolenic acid ( c18;3 , n-3 ) , gla -linolenic acid ( c18;3 , n-6 ) , aa arachidonic acid ( c20:4 , n-6 ) a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with high cvd risk all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated positive correlation between change in serum n-3 pufa and svcam-1 tg triglycerides , oa oleic acid ( c18:1 , n-9 ) , la linoleic acid ( c18:2 , n-6 ) a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with cvd and cvd related diseases all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated mi myocardial infarction , pad peripheral arterial disease , chd coronary heart disease details of the 35 studies selected in this review are given in tables 2 , 3 and 4 . twenty - nine of these are based on marine n-3 fatty acids given as supplements , whereas six studies are dietary interventions with fish . the systematic review of the literature reveals that seven out of thirteen trials reported an effect on circulating inflammatory markers [ 2234 ] in healthy individuals ( table 2 ) . six of the studies did not observe any changes in inflammatory markers [ 2224 , 26 , 29 , 33 ] ( table 2 ) . c - reactive protein ( crp ) was analyzed in seven of the trials , and in five of these no effect in crp levels was observed [ 2226 ] , while a reduction in plasma concentration of crp after intake of marine n-3 fatty acids was reported in two of the trials [ 27 , 28 ] . two of these reported a reduction in il-6 levels [ 27 , 28 ] , whereas three did not demonstrate any effect on il-6 levels [ 25 , 29 , 33 ] . table 2 also reveals that marine n-3 fatty acids reduced or had no effects on the concentration of sicam-1 in five of the trials [ 25 , 29 , 31 , 32 , 34 ] . the concentration of svcam-1 was reduced , increased or unchanged after intake of marine n-3 fatty acids among healthy individuals in these trials , whereas the level of se - sel increased or remained unchanged [ 25 , 31 , 32 , 34 ] . the effects of marine n-3 fatty acids on circulating inflammatory markers among individuals with a high risk of developing cvd are shown in table 3 [ 3549 ] . four of fifteen trials reported a reduction in the concentration of inflammatory markers after intake of marine n-3 fatty acids [ 35 , 36 , 45 , 46 ] , while nine studies did not observe any effects on inflammation [ 3744 , 48 ] . one of nine studies where crp was analyzed demonstrated a reduction in the concentration of crp after intake of marine n-3 fatty acids . three trials demonstrated a reduction in plasma cytokines or soluble adhesion molecules [ 35 , 45 , 46 ] . table 4 reviews the effects of marine n-3 fatty acids on circulating inflammatory markers among individuals with cvd or cvd related diseases [ 5056 ] . five of seven studies showed no change in the concentration of inflammatory markers after intake of marine n-3 fatty acids [ 5053 , 55 ] , whereas one study reported a reduction in the levels of il-6 and svcam-1 and one trial showed an increase in the level of se - sel and svcam -1 . intervention trials with marine n-3 fatty acids administered either from fish or fish oil supplements demonstrate different results on circulating inflammatory markers in healthy individuals , individuals with high risk of developing cvd or individuals with cvd related diseases . based on these findings , a firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can not be drawn . the majority of the trials do not show any effect , however a decreased level of circulating crp and il-6 are observed in some studies , whereas the circulating level of adhesion molecules inconsistently are elevated , decreased or not changed . first of all , a local inflammatory improvement in the arterial wall will not necessary be reflected in the circulation and may explain the lack of effect observed after intake of marine n-3 fatty acids . several of the reported trials have a relative small number of study subjects and probably low statistical power . variation in the results can also be explained by differences in the administration of the marine n-3 fatty acids ( supplement vs. diet ) as well as the doses used . furthermore , there is uncertainty regarding the background diet and to what extend the intervention changed the balance between n-3 and n-6 fatty acids or altered the intake of saturated fat in the diet . comparing the results on the effects of marine n-3 fatty acids given as supplements on serum crp in healthy individuals , the baseline level of crp seems crucial . reported a significant reduction in the level of crp in postmenopausal women who had mean baseline levels of crp above 6 mg / l . , vega - lopez et al . and madsen et al . who did not observe any changes in the crp serum levels . the baseline level of serum crp in the subjects in these studies was approximately 1 vega - lopez et al . failed to see any effect on the serum crp level using a daily dose of 0.6 g epa and 0.9 dha for 12 weeks , which is in line with the results from madsen et al . who used two different daily doses ( 3.0 g epa + 2.9 g dha or 0.9 g epa + 0.8 g dha ) . in contrast , ciubotaru et al . observed in postmenopausal women that the effect on crp is dependent on the dose of marine n-3 fatty acids . a larger decrease in crp was observed in the women who received the highest dose of marine n-3 fatty acids ( 2.56 g / d ) compared to those receiving a daily dose of 1.3 g marine n-3 fatty acids . it is interesting to note that observed decrease in circulating crp level was associated with decrease in circulating il-6 level after intake of marine n-3 fatty acids , both via supplement or diet . in this review there are relatively few studies where marine n-3 fatty acids are administered as fish . when comparing these interventions with the interventions with fish oil , there are indications for a more prominent effect on circulating inflammatory markers with fish containing diet . reductions or no change in one of the following markers ( crp , il-6 , sicam-1 , svcam-1 , il-18 , se - sel ) are observed in all the six dietary studies included in this review . a possible explanation could be that subjects participating in dietary intervention trials may change their overall dietary pattern resulting in more awareness of healthy lifestyle , which again may influence the circulating inflammatory markers . fish may also contain other bioactive compounds that may influence outcome . to further understand if marine n-3 fatty acids have an effect on inflammation in relation to atherosclerosis and the underlying molecular mechanisms responsible for their inflammatory response , new strategies should be considered . when inflammatory markers are measured in the circulation , the local effect in monocytes will not necessary be detected since the monocytes only contribute to about 5% of the total cells in plasma . the peripheral blood mononuclear cells ( pbmcs ) include monocytes and lymphocytes , which are cells central in inflammation and hence in the atherosclerosis process . the pbmcs are exposed to many of the same environmental factors as the arterial wall . alterations in gene expression levels in these cells can thus be demonstrated early in the process before signs of inflammation can be seen in vivo , and thus constitute an important source of biomarkers for progression of atherosclerotic disease . pbmcs are easily available , which makes them suitable for studying gene expression of mediators involved in the early development of atherosclerosis . a previous study recently demonstrated that 166 genes among 182 candidate cardiovascular genes were expressed in pbmcs . the ability of marine n-3 fatty acids to alter gene expression has been clearly demonstrated in vitro and may account for its potential beneficial health effects . fatty acids regulate expression of genes involved in lipid metabolism and inflammation by acting as ligands for the peroxisomal proliferator - activated receptors ( ppars ) . fatty acids are also known to reduce the activation of the transcription factor nuclear factor kappa b ( nf-b ) probably by interference with the ppars [ 60 , 61 ] or the toll - like receptors [ 62 , 63 ] . the g protein - coupled receptor 120 ( gpr120 ) has recently been characterized as an n-3 fatty acid receptor / sensor involved in the anti - inflammatory effects of n-3 fatty acids . dietary supplements containing fish oil have also been shown to affect expression of pi3k , akt , nf-b and inflammatory cytokines in mononuclear cells . to obtain a more comprehensive overview of the processes that are modulated by epa and dha , new omics technologies can be used to detect large scale changes in gene expression profiles ( transcriptomics ) . whole genome transcriptomic analysis in pbmcs will be valuable in further understanding the inflammatory role of n-3 fatty acids in humans . recently , a human intervention study among elderly who daily consumed fish oil ( containing 1.8 g epa and dha ) for 26 weeks demonstrated changes in the gene expression profiles in pbmcs . gene transcripts involved in inflammation and other processes of atherosclerosis were down - regulated among those who had consumed fish oil . the combined use of other omics technologies , such as proteomics and metabolomics ( lipidomics ) , in intervention studies will further extend the understanding of the effects of marine n-3 fatty acids on inflammation . using proteomics technology , a recent study demonstrated that daily intake of 3.5 g fish oil down - regulated a number of proteins involved in the acute phase response and lipid / lipoprotein metabolism . furthermore , lankinen et al . previously used a lipidomics approach in an intervention study where 33 individuals randomized to eat fatty fish ( 150 g four times per week ) , lean fish ( 150 g four times per week ) or no fish , in 8 weeks . they observed a reduction in the level of lipid metabolites related to inflammation and insulin - signaling among those eating fatty fish . no firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can be drawn in this review . a systematic use of transcriptome , proteome , and metabolome technologies for the construction of network based models of biological processes has emerged as an exciting research approach in molecular biology and functional genomics . in future marine n-3 supplementation and dietary intervention trials , systems biology combined with traditional circulating markers may help us to gain more insight into the underlying molecular mechanisms of marine n-3 fatty acids responsible for the inflammatory response in the progression of atherosclerotic disease . intervention trials with marine n-3 fatty acids administered either from fish or fish oil supplements demonstrate different results on circulating inflammatory markers in healthy individuals , individuals with high risk of developing cvd or individuals with cvd related diseases . based on these findings , a firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can not be drawn . the majority of the trials do not show any effect , however a decreased level of circulating crp and il-6 are observed in some studies , whereas the circulating level of adhesion molecules inconsistently are elevated , decreased or not changed . first of all , a local inflammatory improvement in the arterial wall will not necessary be reflected in the circulation and may explain the lack of effect observed after intake of marine n-3 fatty acids . several of the reported trials have a relative small number of study subjects and probably low statistical power . variation in the results can also be explained by differences in the administration of the marine n-3 fatty acids ( supplement vs. diet ) as well as the doses used . furthermore , there is uncertainty regarding the background diet and to what extend the intervention changed the balance between n-3 and n-6 fatty acids or altered the intake of saturated fat in the diet . comparing the results on the effects of marine n-3 fatty acids given as supplements on serum crp in healthy individuals , the baseline level of crp seems crucial . reported a significant reduction in the level of crp in postmenopausal women who had mean baseline levels of crp above 6 mg / l . , vega - lopez et al . and madsen et al . who did not observe any changes in the crp serum levels . the baseline level of serum crp in the subjects in these studies was approximately 1 vega - lopez et al . failed to see any effect on the serum crp level using a daily dose of 0.6 g epa and 0.9 dha for 12 weeks , which is in line with the results from madsen et al . who used two different daily doses ( 3.0 g epa + 2.9 g dha or 0.9 g epa + 0.8 g dha ) . in contrast , ciubotaru et al . observed in postmenopausal women that the effect on crp is dependent on the dose of marine n-3 fatty acids . a larger decrease in crp was observed in the women who received the highest dose of marine n-3 fatty acids ( 2.56 g / d ) compared to those receiving a daily dose of 1.3 g marine n-3 fatty acids . it is interesting to note that observed decrease in circulating crp level was associated with decrease in circulating il-6 level after intake of marine n-3 fatty acids , both via supplement or diet . in this review there are relatively few studies where marine n-3 fatty acids are administered as fish . when comparing these interventions with the interventions with fish oil , there are indications for a more prominent effect on circulating inflammatory markers with fish containing diet . reductions or no change in one of the following markers ( crp , il-6 , sicam-1 , svcam-1 , il-18 , se - sel ) are observed in all the six dietary studies included in this review . a possible explanation could be that subjects participating in dietary intervention trials may change their overall dietary pattern resulting in more awareness of healthy lifestyle , which again may influence the circulating inflammatory markers . to further understand if marine n-3 fatty acids have an effect on inflammation in relation to atherosclerosis and the underlying molecular mechanisms responsible for their inflammatory response , new strategies should be considered . when inflammatory markers are measured in the circulation , the local effect in monocytes will not necessary be detected since the monocytes only contribute to about 5% of the total cells in plasma . the peripheral blood mononuclear cells ( pbmcs ) include monocytes and lymphocytes , which are cells central in inflammation and hence in the atherosclerosis process . the pbmcs are exposed to many of the same environmental factors as the arterial wall . alterations in gene expression levels in these cells can thus be demonstrated early in the process before signs of inflammation can be seen in vivo , and thus constitute an important source of biomarkers for progression of atherosclerotic disease . pbmcs are easily available , which makes them suitable for studying gene expression of mediators involved in the early development of atherosclerosis . a previous study recently demonstrated that 166 genes among 182 candidate cardiovascular genes were expressed in pbmcs . the ability of marine n-3 fatty acids to alter gene expression has been clearly demonstrated in vitro and may account for its potential beneficial health effects . fatty acids regulate expression of genes involved in lipid metabolism and inflammation by acting as ligands for the peroxisomal proliferator - activated receptors ( ppars ) . fatty acids are also known to reduce the activation of the transcription factor nuclear factor kappa b ( nf-b ) probably by interference with the ppars [ 60 , 61 ] or the toll - like receptors [ 62 , 63 ] . the g protein - coupled receptor 120 ( gpr120 ) has recently been characterized as an n-3 fatty acid receptor / sensor involved in the anti - inflammatory effects of n-3 fatty acids . dietary supplements containing fish oil have also been shown to affect expression of pi3k , akt , nf-b and inflammatory cytokines in mononuclear cells . to obtain a more comprehensive overview of the processes that are modulated by epa and dha , new omics technologies can be used to detect large scale changes in gene expression profiles ( transcriptomics ) . whole genome transcriptomic analysis in pbmcs will be valuable in further understanding the inflammatory role of n-3 fatty acids in humans . recently , a human intervention study among elderly who daily consumed fish oil ( containing 1.8 g epa and dha ) for 26 weeks demonstrated changes in the gene expression profiles in pbmcs . gene transcripts involved in inflammation and other processes of atherosclerosis were down - regulated among those who had consumed fish oil . the combined use of other omics technologies , such as proteomics and metabolomics ( lipidomics ) , in intervention studies will further extend the understanding of the effects of marine n-3 fatty acids on inflammation . using proteomics technology , a recent study demonstrated that daily intake of 3.5 g fish oil down - regulated a number of proteins involved in the acute phase response and lipid / lipoprotein metabolism . previously used a lipidomics approach in an intervention study where 33 individuals randomized to eat fatty fish ( 150 g four times per week ) , lean fish ( 150 g four times per week ) or no fish , in 8 weeks . they observed a reduction in the level of lipid metabolites related to inflammation and insulin - signaling among those eating fatty fish . no firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can be drawn in this review . a systematic use of transcriptome , proteome , and metabolome technologies for the construction of network based models of biological processes has emerged as an exciting research approach in molecular biology and functional genomics . in future marine n-3 supplementation and dietary intervention trials , systems biology combined with traditional circulating markers may help us to gain more insight into the underlying molecular mechanisms of marine n-3 fatty acids responsible for the inflammatory response in the progression of atherosclerotic disease .

objectivethe aim of the present paper was to review the literature in order to summarize the effects of marine n-3 fatty acids on circulating inflammatory markers among healthy subjects , subjects with high risk of developing cardiovascular disease ( cvd ) and in patients with cvd in human intervention studies.methodsa systematic literature search in pubmed was performed . intervention studies describing the effects of marine n-3 fatty acids on circulating inflammatory markers in healthy subjects , subjects with high risk of cvd and patients with cvd were included . the following exclusion criteria were used : ( 1 ) interventions assessing inflammatory markers with ex vivo methods ( 2 ) interventions with children ( 3 ) articles describing animal or cell culture studies . twenty - two articles were included . additionally , 13 papers from their literature lists were included based on the same inclusion and exclusion criteria as the literature search.results and conclusionintervention studies with marine n-3 fatty acids administered from either fish or fish oil demonstrate different results on inflammatory markers . no firm conclusion can be drawn about the effect of marine n-3 fatty acids on circulating inflammatory markers in healthy individuals , individuals with high risk of developing cvd or individuals with cvd related diseases .

Introduction Methods of this review Results Discussion and conclusion Effect of marine n-3 fatty acids on circulating inflammatory markers New strategies for studying effects of marine n-3 fatty acids on inflammation

fish consumption reduces the risk of developing cardiovascular disease ( cvd ) and cvd mortality [ 1 , 2 ] . reduced total mortality and major coronary events , including fatal and non - fatal mi , are observed in intervention trials after intake of fish and fish oil containing the marine n-3 fatty acids eicosapentaenoic acid ( epa , 20:5 n-3 ) and docosahexaenoic acid ( dha , 22:6 n-3 ) [ 36 ] . the association between high intakes of marine n-3 fatty acids and decreased morbidity and mortality from cvd can be explained by the decrease in plasma triglycerides [ 7 , 8 ] , moderate reduction in blood pressure and reduced blood plate aggregation [ 10 , 11 ] . there is an association between various biomarkers of inflammation and prospective cvd risk in apparently healthy individuals as well as in patients with cvd or heart failure . based on the fact that endothelial dysfunction is the earliest manifestation of atherosclerosis , along with the involvement of oxidative stress and inflammation at all stages of coronary atherosclerosis development , biomarkers such as soluble intracellular adhesion molecule ( sicam-1 ) and soluble vascular adhesion molecule ( svcam-1 ) , soluble e - selectin ( se - sel ) ( for endothelial activation ) , interleukin ( il)-6 ( transcriptional driver of crp ) and monocyte chemoattractant protein ( mcp)-1 ( produced by activated vasculature ) are of particular interest ( table 1 ) . recently , intake of marine n-3 fatty acids has been associated with reduced plasma levels of inflammatory markers [ 1921].table 1relevant inflammatory markers and their biological functioninflammatory markersabbreviationfunctionacute - phase protein c - reactive proteincrpcrp is associated with the formation of cytokines , chemokines and the acute - phase responsecytokines interleukin-6il-6induces acute - phase response ( by inducing crp ) , anti - body secretion and differentiation interleukin-1 , interleukin-1il-1 , il-1proliferation and maturation of lymphocytes , involved in inflammation and acute - phase response interleukin-18il-18involved in the formation of th1 cellstumor necrosis factor- is a cytokinetnf-induces adhesion molecules- and cytokine expression , involved in cell deathadhesion protein soluble intercellular adhesion molecule-1sicam-1binds monocytes and lymphocytes to the endothelium soluble vascular cell adhesion molecule-1svcam-1binds monocytes and lymphocytes to the endothelium se - selectinse - selrecruits leukocytes to the inflammatory site sp - selectinsp - selrecruits leukocytes to the inflammatory site . induces monocytes and platelet interactionschemokines monocyte chemoattractant protein-1mcp-1facilitates migration of leukocytes to the intima granulocyte macrophage colony - stimulating factorgm csfgrowth and differentiation of monocytes interleukin-8il-8facilitates migration of leukocytes to the intima relevant inflammatory markers and their biological function the aim of the present paper was to review the literature in order to summarize the effects of marine n-3 fatty acids on circulating inflammatory markers among healthy subjects , subjects with high risk of developing cvd and in patients with cvd in human intervention studies . the systematic literature search was conducted in pubmed in 2009 using the following terms : eicosapentaenoic acid or docosapentaenoic acid or docosahexaenoic acid or omega-3 or fish oil or cod liver oil and inflammation . based on these papers , we included all studies that measured the effect of marine n-3 fatty acids on circulating inflammatory markers in plasma or serum among apparently healthy individuals ( table 2 ) , individuals with high risk of developing cvd ( table 3 ) or individuals with cvd ( table 4 ) . interventions with marine n-3 fatty acids given as supplements or in the diet as fish were included . the following exclusion criteria were used : ( 1 ) interventions assessing inflammatory markers with ex vivo methods ( 2 ) , interventions with children ( 3 ) and articles describing animal or cell culture studies . after reading abstracts of the 91 papers , the literature lists of the selected papers were checked and 13 additional relevant papers were included based on the same inclusion and exclusion criteria as the literature search . however , no further papers were included from this search.table 2a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in healthy individualsstudyindividuals ( n)number of groupsdose n-3 ( g / day)durationcrpcytokinesother inflammatory markersinterventions with n-3 supplements geelen et al . 12 healthy elderly men and women ( 6075 years)2 groups : control diet or n-3 diet ( cross - over design)n-3 diet:720 g / week fatty fish and sardine oil ( 45 g / day epa and dha)8 weeks il-6 ( tendency ) paulo mc 275 healthy men and women ( 2040 years)4 groups : sunflower oil , fish oil , lean fish or fatty fishfish oil:0.63 g epa and 0.43 g dhalean fish:0.05 g epa and 0.21 g dhafatty fish:0.77 g epa and 1.37 g dha8 weeks sicam-1 ( lean fish)svcam-1 ( fish oil and lean fish)all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated * larger change in crp and il-6 with fish oil with low content of epa and dha compared to fish oil with high contentala -linolenic acid ( c18;3 , n-3 ) , gla -linolenic acid ( c18;3 , n-6 ) , aa arachidonic acid ( c20:4 , n-6)table 3a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with high cvd riskstudyindividuals ( n)number of groupsdose n-3 ( g / day)durationcrpcytokinesother inflammatory markersinterventions with n-3 supplements kelley et al . 57 dyslipidemic individuals ( 2365 years)3 groups : placebo , n-3 pufa / vite or n-3 pufa / vite and -oryzanol / niacinn-3 pufa:0.66 g epa0.44 29 individuals with type 2diabetes and 21 healthy controls4 groups : controls and patients were given n-3 pufa or not . 487 elderly men with high cvd risk ( 6476 years)4 groups : corn oil , n-3 pufa , corn oil + dietary intervention or n-3 pufa + dietary interventionn-3 pufa:0.84 g epa0.48 g dhadietary intervention : increase intake of unsaturated fat , fish , fruit and vegetables3 years sicam-1 ( n-3 pufa , dietary intervention , dietary intervention + n-3 pufa ) berstad 171 elderly men with high cvd risk ( 6575 years)4 groups : corn oil , n-3 pufa , corn oil + dietary intervention or n-3 pufa + dietary interventionn-3 pufa:0.84 g epa0.48 g dhadietary intervention : increase intake of unsaturated fat , fish , fruit and vegetables18 months sicam-1 , svcam-1 # se - sel ( dietary intervention)all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated positive correlation between change in serum n-3 pufa and svcam-1tg triglycerides , oa oleic acid ( c18:1 , n-9 ) , la linoleic acid ( c18:2 , n-6)table 4a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with cvd and cvd related diseasesindividuals ( n)number of groupsdose n-3 ( g / day)durationcrpcytokinesother inflammatory markersinterventions with n-3 supplements schiano et al . 60 men and women with chd ( 4675 years)3 groups : all groups got 700 g salmon / week with low , moderate or high content of n-3 pufan-3 content in salmon : low n-3 : 0.5 g epa + dhamoderate n-3 : 1.5 g epa + dhahigh n-3 : 2.9 g epa + dha6 weeks il-6 , ( high n-3) svcam-1 ( high n-3)all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise statedmi myocardial infarction , pad peripheral arterial disease , chd coronary heart disease a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in healthy individuals all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated * larger change in crp and il-6 with fish oil with low content of epa and dha compared to fish oil with high content ala -linolenic acid ( c18;3 , n-3 ) , gla -linolenic acid ( c18;3 , n-6 ) , aa arachidonic acid ( c20:4 , n-6 ) a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with high cvd risk all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated positive correlation between change in serum n-3 pufa and svcam-1 tg triglycerides , oa oleic acid ( c18:1 , n-9 ) , la linoleic acid ( c18:2 , n-6 ) a review of marine n-3 fatty acid intervention studies and circulating inflammatory markers in individuals with cvd and cvd related diseases all the intervention studies with n-3 supplements were placebo - controlled ; double - blinded with parallel - design if not otherwise stated mi myocardial infarction , pad peripheral arterial disease , chd coronary heart disease details of the 35 studies selected in this review are given in tables 2 , 3 and 4 . twenty - nine of these are based on marine n-3 fatty acids given as supplements , whereas six studies are dietary interventions with fish . the systematic review of the literature reveals that seven out of thirteen trials reported an effect on circulating inflammatory markers [ 2234 ] in healthy individuals ( table 2 ) . six of the studies did not observe any changes in inflammatory markers [ 2224 , 26 , 29 , 33 ] ( table 2 ) . c - reactive protein ( crp ) was analyzed in seven of the trials , and in five of these no effect in crp levels was observed [ 2226 ] , while a reduction in plasma concentration of crp after intake of marine n-3 fatty acids was reported in two of the trials [ 27 , 28 ] . table 2 also reveals that marine n-3 fatty acids reduced or had no effects on the concentration of sicam-1 in five of the trials [ 25 , 29 , 31 , 32 , 34 ] . the concentration of svcam-1 was reduced , increased or unchanged after intake of marine n-3 fatty acids among healthy individuals in these trials , whereas the level of se - sel increased or remained unchanged [ 25 , 31 , 32 , 34 ] . the effects of marine n-3 fatty acids on circulating inflammatory markers among individuals with a high risk of developing cvd are shown in table 3 [ 3549 ] . four of fifteen trials reported a reduction in the concentration of inflammatory markers after intake of marine n-3 fatty acids [ 35 , 36 , 45 , 46 ] , while nine studies did not observe any effects on inflammation [ 3744 , 48 ] . one of nine studies where crp was analyzed demonstrated a reduction in the concentration of crp after intake of marine n-3 fatty acids . table 4 reviews the effects of marine n-3 fatty acids on circulating inflammatory markers among individuals with cvd or cvd related diseases [ 5056 ] . five of seven studies showed no change in the concentration of inflammatory markers after intake of marine n-3 fatty acids [ 5053 , 55 ] , whereas one study reported a reduction in the levels of il-6 and svcam-1 and one trial showed an increase in the level of se - sel and svcam -1 . intervention trials with marine n-3 fatty acids administered either from fish or fish oil supplements demonstrate different results on circulating inflammatory markers in healthy individuals , individuals with high risk of developing cvd or individuals with cvd related diseases . based on these findings , a firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can not be drawn . first of all , a local inflammatory improvement in the arterial wall will not necessary be reflected in the circulation and may explain the lack of effect observed after intake of marine n-3 fatty acids . variation in the results can also be explained by differences in the administration of the marine n-3 fatty acids ( supplement vs. diet ) as well as the doses used . comparing the results on the effects of marine n-3 fatty acids given as supplements on serum crp in healthy individuals , the baseline level of crp seems crucial . observed in postmenopausal women that the effect on crp is dependent on the dose of marine n-3 fatty acids . a larger decrease in crp was observed in the women who received the highest dose of marine n-3 fatty acids ( 2.56 g / d ) compared to those receiving a daily dose of 1.3 g marine n-3 fatty acids . it is interesting to note that observed decrease in circulating crp level was associated with decrease in circulating il-6 level after intake of marine n-3 fatty acids , both via supplement or diet . in this review there are relatively few studies where marine n-3 fatty acids are administered as fish . when comparing these interventions with the interventions with fish oil , there are indications for a more prominent effect on circulating inflammatory markers with fish containing diet . to further understand if marine n-3 fatty acids have an effect on inflammation in relation to atherosclerosis and the underlying molecular mechanisms responsible for their inflammatory response , new strategies should be considered . the pbmcs are exposed to many of the same environmental factors as the arterial wall . the ability of marine n-3 fatty acids to alter gene expression has been clearly demonstrated in vitro and may account for its potential beneficial health effects . fatty acids are also known to reduce the activation of the transcription factor nuclear factor kappa b ( nf-b ) probably by interference with the ppars [ 60 , 61 ] or the toll - like receptors [ 62 , 63 ] . the g protein - coupled receptor 120 ( gpr120 ) has recently been characterized as an n-3 fatty acid receptor / sensor involved in the anti - inflammatory effects of n-3 fatty acids . the combined use of other omics technologies , such as proteomics and metabolomics ( lipidomics ) , in intervention studies will further extend the understanding of the effects of marine n-3 fatty acids on inflammation . no firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can be drawn in this review . in future marine n-3 supplementation and dietary intervention trials , systems biology combined with traditional circulating markers may help us to gain more insight into the underlying molecular mechanisms of marine n-3 fatty acids responsible for the inflammatory response in the progression of atherosclerotic disease . intervention trials with marine n-3 fatty acids administered either from fish or fish oil supplements demonstrate different results on circulating inflammatory markers in healthy individuals , individuals with high risk of developing cvd or individuals with cvd related diseases . based on these findings , a firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can not be drawn . first of all , a local inflammatory improvement in the arterial wall will not necessary be reflected in the circulation and may explain the lack of effect observed after intake of marine n-3 fatty acids . variation in the results can also be explained by differences in the administration of the marine n-3 fatty acids ( supplement vs. diet ) as well as the doses used . comparing the results on the effects of marine n-3 fatty acids given as supplements on serum crp in healthy individuals , the baseline level of crp seems crucial . observed in postmenopausal women that the effect on crp is dependent on the dose of marine n-3 fatty acids . a larger decrease in crp was observed in the women who received the highest dose of marine n-3 fatty acids ( 2.56 g / d ) compared to those receiving a daily dose of 1.3 g marine n-3 fatty acids . it is interesting to note that observed decrease in circulating crp level was associated with decrease in circulating il-6 level after intake of marine n-3 fatty acids , both via supplement or diet . in this review there are relatively few studies where marine n-3 fatty acids are administered as fish . when comparing these interventions with the interventions with fish oil , there are indications for a more prominent effect on circulating inflammatory markers with fish containing diet . a possible explanation could be that subjects participating in dietary intervention trials may change their overall dietary pattern resulting in more awareness of healthy lifestyle , which again may influence the circulating inflammatory markers . to further understand if marine n-3 fatty acids have an effect on inflammation in relation to atherosclerosis and the underlying molecular mechanisms responsible for their inflammatory response , new strategies should be considered . the pbmcs are exposed to many of the same environmental factors as the arterial wall . the ability of marine n-3 fatty acids to alter gene expression has been clearly demonstrated in vitro and may account for its potential beneficial health effects . the g protein - coupled receptor 120 ( gpr120 ) has recently been characterized as an n-3 fatty acid receptor / sensor involved in the anti - inflammatory effects of n-3 fatty acids . recently , a human intervention study among elderly who daily consumed fish oil ( containing 1.8 g epa and dha ) for 26 weeks demonstrated changes in the gene expression profiles in pbmcs . the combined use of other omics technologies , such as proteomics and metabolomics ( lipidomics ) , in intervention studies will further extend the understanding of the effects of marine n-3 fatty acids on inflammation . no firm conclusion about the effect of marine n-3 fatty acids on circulating inflammatory markers can be drawn in this review . in future marine n-3 supplementation and dietary intervention trials , systems biology combined with traditional circulating markers may help us to gain more insight into the underlying molecular mechanisms of marine n-3 fatty acids responsible for the inflammatory response in the progression of atherosclerotic disease .

computerised prescribing is widely used in the united kingdom for repeat prescriptions in general practice . there is evidence that it has improved legibility and clarity , saved time for both medical and clerical staff , and reduced the time spent clarifying prescriptions with the dispensing pharmacist . its principal use in hospital practice has been for outpatient or inpatient discharge prescriptions , although some hospitals use fully integrated computerised prescribing systems . computer - assisted decision support in the hospital setting has already been shown to improve the use of antibiotics peri - operatively , reduce the costs of prescribing and influence clinical parameters such as limiting the emergence of antibiotic resistance . computer - assisted prescribing ( cap ) for inpatients was introduced to the intensive care unit ( icu ) of the john radcliffe hospital in may 1996 as part of the hewlett packard carevue patient information system ( hewlett packard ltd , andover , usa ) . this study evaluated the computerised prescribing system by comparing the accuracy and completeness of prescriptions , and time spent prescribing before and after the introduction of cap . carevue is a proprietary icu computer information system providing automatic charting of physiological and laboratory data . in our icu , it has been directly interfaced with the patients ' mechanical ventilators and arterial blood gas analysers , and hematology and biochemistry laboratories . in may 1996 , a prescribing module of the system software was activated and introduced clinically after a period of modification and reconfiguration . carevue is supported by mirrored primary and secondary hewlett packard 9000 series/700 servers , supporting up to 4 bedside hewlett packard 712 series diskless workstation clients running hpux 9.03 system software . the second 3-week phase was conducted 1 month after the introduction and implementation of the cap component of carevue . all medical and nursing staff received training at the bedside from the ward pharmacist and the carevue nurse specialist . each prescription chart was required to be dated and a hospital sticker attached with the patient 's address , date of birth and hospital number . the prescriber was then required to write , in the appropriate section , the drug name , the dose or amount to be diluted ( with iv infusions ) , rate of administration ( for infusions ) , volume of specified diluents required , route and frequency of administration . the prescription was then signed at the bottom with a single signature covering all drug entries . in the second phase of the study , cap was implemented following the introduction of the medication administration record ( mar ) prescribing module of hewlett packard carevue ( fig 1 ) . it is divided into the following three sections : the ` scheduled ' section contains drugs given at regular intervals ; the ` prn ' section contains drugs given as required and also iv fluids , infusions and enteral or parenteral feeds , and the ` stat ' section contains any single dose drugs or fluids . the prescribing doctor is required to complete all sections of the prescription marked with an asterisk , other sections being optional . by clicking a trackball directed pointer on the medication box a pick - list of 236 commonly prescribed medications , fluids and feeds is displayed . depending upon the drug selected , appropriate routes of administration become available in another pick - list ( eg po , iv , subcutaneous , etc ) . a preparation not included on the drug pick list may still be prescribed , but the computer can not ` advise ' on the route of administration or dose . specific doses or dose ranges are entered by typing the values into the dose box . free text entry of instructions ( either from the prescribing clinicians or nurse ) allows diluents , dilutions and infusion rates to be specified . by adding and storing the order at this point ( mandatory and requiring entry of a unique six digit alpha / numeric password ) the clinician 's name and initials are appended to the prescription . once a prescription is entered onto the system and stored , the drug , route , dose and frequency of administration can not be changed . any new entry , alteration , modification or discontinuation requires confirmation by entry of the physician 's unique password . a prescribing clinician has access to entry , storage , alteration and discontinuation of all prescriptions as well as being able to log the administration of drugs . nursing staff may also enter and discontinue prescriptions , but these are highlighted on the mar as requiring confirmation by a clinician , with no drugs administration against these prescriptions being possible until this has occurred . a prescription is not active until it has been ` signed ' or authorised by a clinician , using their password . the flowsheet is used to record the hourly rate of iv infusions and also the time of administration of scheduled ` as required ' drugs ( fig 3 ) . additional features include automatic discontinuation of drugs following the entry of a stop date on to the prescription , or the ability to highlight prescriptions which should be reviewed on a certain day . each individual drug entry for both the handwritten prescribing and cap was evaluated by the icu pharmacist according to 19 predetermined criteria ( table 1 ) over two 3-week time periods . the time taken to prescribe a single drug by hand and using the computer module was measured , and the time it took a nurse to record that the drug has been given was measured using the two prescribing modes . prescription charts were rewritten daily . each prescription chart was required to be dated and a hospital sticker attached with the patient 's address , date of birth and hospital number . the prescriber was then required to write , in the appropriate section , the drug name , the dose or amount to be diluted ( with iv infusions ) , rate of administration ( for infusions ) , volume of specified diluents required , route and frequency of administration . the prescription was then signed at the bottom with a single signature covering all drug entries . in the second phase of the study , cap was implemented following the introduction of the medication administration record ( mar ) prescribing module of hewlett packard carevue ( fig 1 ) . it is divided into the following three sections : the ` scheduled ' section contains drugs given at regular intervals ; the ` prn ' section contains drugs given as required and also iv fluids , infusions and enteral or parenteral feeds , and the ` stat ' section contains any single dose drugs or fluids . the prescribing doctor is required to complete all sections of the prescription marked with an asterisk , other sections being optional . by clicking a trackball directed pointer on the medication box a pick - list of 236 commonly prescribed medications , fluids and feeds is displayed . depending upon the drug selected , appropriate routes of administration become available in another pick - list ( eg po , iv , subcutaneous , etc ) . a preparation not included on the drug pick list may still be prescribed , but the computer can not ` advise ' on the route of administration or dose . specific doses or dose ranges are entered by typing the values into the dose box . free text entry of instructions ( either from the prescribing clinicians or nurse ) allows diluents , dilutions and infusion rates to be specified . by adding and storing the order at this point ( mandatory and requiring entry of a unique six digit alpha / numeric password ) the clinician 's name and initials are appended to the prescription . once a prescription is entered onto the system and stored , the drug , route , dose and frequency of administration can not be changed . any new entry , alteration , modification or discontinuation requires confirmation by entry of the physician 's unique password . a prescribing clinician has access to entry , storage , alteration and discontinuation of all prescriptions as well as being able to log the administration of drugs . nursing staff may also enter and discontinue prescriptions , but these are highlighted on the mar as requiring confirmation by a clinician , with no drugs administration against these prescriptions being possible until this has occurred . a prescription is not active until it has been ` signed ' or authorised by a clinician , using their password . the flowsheet is used to record the hourly rate of iv infusions and also the time of administration of scheduled ` as required ' drugs ( fig 3 ) . additional features include automatic discontinuation of drugs following the entry of a stop date on to the prescription , or the ability to highlight prescriptions which should be reviewed on a certain day . each individual drug entry for both the handwritten prescribing and cap was evaluated by the icu pharmacist according to 19 predetermined criteria ( table 1 ) over two 3-week time periods . the time taken to prescribe a single drug by hand and using the computer module was measured , and the time it took a nurse to record that the drug has been given was measured using the two prescribing modes . one hundred and twenty - eight handwritten and 110 computer - assisted patient prescription charts were monitored over the 6 weeks of the study , resulting in 1184 handwritten and 1225 computer - assisted individual drug entries being studied . there was an average of 10 individual drugs prescribed per patient per day , consisting of iv fluids , intravenous infusions , entail or parenteral feeds , ` regular ' and ` as required ' drugs . one hundred percent of the computerised patient prescriptions were signed and dated in comparison with 95% of handwritten patient prescriptions . note that with cap all individual drug entries were authorised , whereas with handwritten prescriptions one signature covered the whole prescription . only 47% of handwritten prescription charts had full patient identification ( name , date of birth and unit number , or hospital sticker ) whilst this information was available with all prescriptions on the computerised system . entries monitored in this section included all feeds , blood , blood derivatives , colloids and crystalloids . one hundred and ninety - four individual handwritten and 255 individual computerised entries were analysed in this category ( fig 4 ) . there was a decrease from 64% to 48% in the percentage of complete and correct individual entries ( see table 1 criteria ) following the introduction of cap ( fig 4 ) . the percentage of entries with a missing rate of infusion increased from 18% to 28% ( fig 4 ) . the number of drug entries remaining on the prescription chart despite having been removed from the treatment regimen more than 24 h previously also increased , from 1% to 17% ( fig 4 ) . prescriptions monitored in this section were those for sedatives , analgesics , inotropes , vasoactive agents , heparin and antiarrhythmics , administered by infusion . two hundred and eighty - four handwritten and 247 computerised drug entries were assessed in this category . the percentage of correct entries for iv infusions decreased from 47.5% to 32% with cap ( fig . thirty - one percent of cap had no prescribed diluent and 27% had no prescribed diluent volume . the percentage of prescriptions with an incorrect rate of infusion was 16% with cap and 1.4% with handwritten prescriptions . prescriptions monitored in this category included antibiotics , h2 antagonists , antihypertensives , potassium supplements and other agents . seven hundred and six handwritten and 723 computer - assisted drug entries were analysed in this category . ninety percent of entries for both handwritten and cap groups complied with the specified criteria ( dose , route , frequency ) . the results are expressed in fig 6 as a percentage of the total number of erroneous prescriptions ( approximately 10% of the total number of entries monitored ) . figure 6 shows an increased frequency of failure to discontinue drugs that the patient was no longer receiving from 9.1% of errors with handwritten prescribing to 57% of errors with cap . although this increase appears large , this represented less than 6% of the total entries in this category . time taken to prescribe one medication by computer and one medication by hand was measured . it took approximately 20 s to prescribe a single complete handwritten drug entry and 55 s to prescribe the same drug using cap . to record that a drug had been given took 2 s on the handwritten chart and 21 s with cap . the medication administration record ( mar ) computerised prescription . demographic patient data and known allergies are clearly indicated . the cross hatched areas indicate a period before the prescription was written and/or after it has been discontinued . the times under the dated vertical columns show times of drugs already given , and the blank white blocks indicate schedule administration time . stock drugs on the unit have been endorsed ` s - pharmacy ' for information . the right hand box shows part of the pick - list of 236 drugs available to the physician . the first five letters ` amiod ' have been typed into the medication box , selecting amiodarone . by moving the trackball and clicking the centre key a drug the percentage of individual drug entries for iv fluids and enteral / parenteral feeds in each category correctly prescribed , missing a rate or remaining on the prescription but not administered for over 24 h. , handwritten ; , computerised . the percentage of iv infusions in each category correctly prescribed , those remaining on the prescription but not administered for over 24 h , without a specified diluent or volume , with an incorrect rate , or prescribed in duplicate . the percentage of errors in prescription for ` regular ' and ` as required ' drugs in both categories including drug with ambiguous instructions , those remaining on the prescription chart but not administered for over 24 h , without a dose , frequency or route , spelt incorrectly , duplicated , or illegible . criteria used to examine each prescription * the prescription remained on chart despite not being needed or verbally discontinued . the drug prescribed in anticipation of need , but one hundred percent of the computerised patient prescriptions were signed and dated in comparison with 95% of handwritten patient prescriptions . note that with cap all individual drug entries were authorised , whereas with handwritten prescriptions one signature covered the whole prescription . only 47% of handwritten prescription charts had full patient identification ( name , date of birth and unit number , or hospital sticker ) whilst this information was available with all prescriptions on the computerised system . entries monitored in this section included all feeds , blood , blood derivatives , colloids and crystalloids . one hundred and ninety - four individual handwritten and 255 individual computerised entries were analysed in this category ( fig 4 ) . there was a decrease from 64% to 48% in the percentage of complete and correct individual entries ( see table 1 criteria ) following the introduction of cap ( fig 4 ) . the percentage of entries with a missing rate of infusion increased from 18% to 28% ( fig 4 ) . the number of drug entries remaining on the prescription chart despite having been removed from the treatment regimen more than 24 h previously also increased , from 1% to 17% ( fig 4 ) . prescriptions monitored in this section were those for sedatives , analgesics , inotropes , vasoactive agents , heparin and antiarrhythmics , administered by infusion . two hundred and eighty - four handwritten and 247 computerised drug entries were assessed in this category . the percentage of correct entries for iv infusions decreased from 47.5% to 32% with cap ( fig . thirty - one percent of cap had no prescribed diluent and 27% had no prescribed diluent volume . the percentage of prescriptions with an incorrect rate of infusion was 16% with cap and 1.4% with handwritten prescriptions . prescriptions monitored in this category included antibiotics , h2 antagonists , antihypertensives , potassium supplements and other agents . seven hundred and six handwritten and 723 computer - assisted drug entries were analysed in this category . ninety percent of entries for both handwritten and cap groups complied with the specified criteria ( dose , route , frequency ) . the results are expressed in fig 6 as a percentage of the total number of erroneous prescriptions ( approximately 10% of the total number of entries monitored ) . figure 6 shows an increased frequency of failure to discontinue drugs that the patient was no longer receiving from 9.1% of errors with handwritten prescribing to 57% of errors with cap . although this increase appears large , this represented less than 6% of the total entries in this category . entries monitored in this section included all feeds , blood , blood derivatives , colloids and crystalloids . one hundred and ninety - four individual handwritten and 255 individual computerised entries were analysed in this category ( fig 4 ) . there was a decrease from 64% to 48% in the percentage of complete and correct individual entries ( see table 1 criteria ) following the introduction of cap ( fig 4 ) . the percentage of entries with a missing rate of infusion increased from 18% to 28% ( fig 4 ) . the number of drug entries remaining on the prescription chart despite having been removed from the treatment regimen more than 24 h previously also increased , from 1% to 17% ( fig 4 ) . prescriptions monitored in this section were those for sedatives , analgesics , inotropes , vasoactive agents , heparin and antiarrhythmics , administered by infusion . two hundred and eighty - four handwritten and 247 computerised drug entries were assessed in this category . the percentage of correct entries for iv infusions decreased from 47.5% to 32% with cap ( fig . thirty - one percent of cap had no prescribed diluent and 27% had no prescribed diluent volume . the percentage of prescriptions with an incorrect rate of infusion was 16% with cap and 1.4% with handwritten prescriptions . prescriptions monitored in this category included antibiotics , h2 antagonists , antihypertensives , potassium supplements and other agents . seven hundred and six handwritten and 723 computer - assisted drug entries were analysed in this category . ninety percent of entries for both handwritten and cap groups complied with the specified criteria ( dose , route , frequency ) . the results are expressed in fig 6 as a percentage of the total number of erroneous prescriptions ( approximately 10% of the total number of entries monitored ) . figure 6 shows an increased frequency of failure to discontinue drugs that the patient was no longer receiving from 9.1% of errors with handwritten prescribing to 57% of errors with cap . although this increase appears large , this represented less than 6% of the total entries in this category . time taken to prescribe one medication by computer and one medication by hand was measured . it took approximately 20 s to prescribe a single complete handwritten drug entry and 55 s to prescribe the same drug using cap . to record that a drug had been given took 2 s on the handwritten chart and 21 s with cap . the medication administration record ( mar ) computerised prescription . demographic patient data and known allergies are clearly indicated . the cross hatched areas indicate a period before the prescription was written and/or after it has been discontinued . the times under the dated vertical columns show times of drugs already given , and the blank white blocks indicate schedule administration time . stock drugs on the unit have been endorsed ` s - pharmacy ' for information . the right hand box shows part of the pick - list of 236 drugs available to the physician . the first five letters ` amiod ' have been typed into the medication box , selecting amiodarone . by moving the trackball and clicking the centre key a drug is released on to the medication administration record ( mar ) . the percentage of individual drug entries for iv fluids and enteral / parenteral feeds in each category correctly prescribed , missing a rate or remaining on the prescription but not administered for over 24 h. , handwritten ; , computerised . the percentage of iv infusions in each category correctly prescribed , those remaining on the prescription but not administered for over 24 h , without a specified diluent or volume , with an incorrect rate , or prescribed in duplicate . the percentage of errors in prescription for ` regular ' and ` as required ' drugs in both categories including drug with ambiguous instructions , those remaining on the prescription chart but not administered for over 24 h , without a dose , frequency or route , spelt incorrectly , duplicated , or illegible . criteria used to examine each prescription * the prescription remained on chart despite not being needed or verbally discontinued . the drug prescribed in anticipation of need , but this study was undertaken in order to assess the accuracy , completeness and time spent prescribing with cap compared with handwritten prescriptions in a busy icu in the uk . cap provided a less accurate prescription for iv fluids and infusions , and prescriptions in all categories were more likely to remain on the chart despite having been discontinued from therapy , possibly due to a decrease in frequency of review of the chart . there was also an uncommon problem of duplicate drug entries using cap which did not occur with handwritten prescriptions . this was probably linked to the configuration of the system which segregated regularly scheduled drugs from ` stat ' and ` prn ' drugs . the three groups could not be displayed simultaneously on the bedside monitor making duplication more probable . there was no facility with the current system to highlight this , but software enhancement could address this issue . cap offered benefits in terms of legibility since almost all the drug names were preconfigured within the system and there was no variation in quality to text . cap were more complete than handwritten prescriptions in that each prescription chart uniquely and fully identified the patient ( 100% compared with 47% ) and any allergies they may have , and every individual drug entry was authorised . with intermittent and scheduled therapy the mandatory fields of drug , dose , route and frequency meant that these categories were never missing from the prescription , in contrast with handwritten prescriptions . our study confirmed previous work , that handwritten medication orders are subject to being incomplete , illegible and unsigned . since 100% of individual drug entries were authorised by password with computerised prescribing it was always possible to trace a member of staff prescribing or administering a medication . start and stop dates and changes in dose were always recorded and identifiable in comparison with handwritten prescriptions where changes in doses were less likely to be dated and signed by the prescriber . the time of initiation or discontinuation of the prescription was virtually never recorded on the handwritten charts , and discontinued prescriptions were frequently unsigned . computer - assisted prescriptions took more than twice as long to prescribe as handwritten prescriptions . administration also took the nursing staff longer to record with cap than on the handwritten charts . this was due , however , to the much greater detail entered with cap which was not part of the handwritten record , and the extra time taken to move around the computer screen and store information . our study focused on time spent prescribing drugs and logging that drugs had been given . related secondary time issues such as drug stocktaking , ordering drugs from the central pharmacy and time taken to issue discharge prescriptions were not studied . a similar study describing parenteral nutrition ( pn ) ordering in a neonatal intensive care unit looked at time spent on writing and ordering pn with manual and computer systems . time was reduced by the computerised ordering , and improvements to nutritional composition were noted in the computerised group . other investigators have demonstrated that computerised prescriptions result in total time saving advantages even when time to enter the information takes 2 - 3 times longer with computerised systems . indeed , our study focused on the task at the bedside workstation and , in this area , more time was spent with cap compared to handwritten prescribing . it may be that secondary time costs will be reduced with this system , but it was not part of this study . as a result of this evaluation a purpose - designed infusion entry component would greatly reduce prescription errors for this category of drugs . highlighting of duplicate prescriptions would prevent this error occurring , and if all sections of the prescription chart could be viewed simultaneously drug therapy could be more easily reviewed by all icu staff . a facility to make authorised changes to a prescription without the need for it being completely rewritten would be useful . a higher level of enhancement of cap could include decision support tools . these would allow preconfigured combinations or sets of drugs to be prescribed for specific clinical situations . drug interactions and incompatibilities could be flagged , much improving the safety of drug therapy . advice on cost of prescribing could be included , offering alternative cheaper options to the clinician . cap provided a less accurate prescription for iv fluids and infusions , and prescriptions in all categories were more likely to remain on the chart despite having been discontinued from therapy , possibly due to a decrease in frequency of review of the chart . there was also an uncommon problem of duplicate drug entries using cap which did not occur with handwritten prescriptions . this was probably linked to the configuration of the system which segregated regularly scheduled drugs from ` stat ' and ` prn ' drugs . the three groups could not be displayed simultaneously on the bedside monitor making duplication more probable . there was no facility with the current system to highlight this , but software enhancement could address this issue . cap offered benefits in terms of legibility since almost all the drug names were preconfigured within the system and there was no variation in quality to text . cap were more complete than handwritten prescriptions in that each prescription chart uniquely and fully identified the patient ( 100% compared with 47% ) and any allergies they may have , and every individual drug entry was authorised . with intermittent and scheduled therapy the mandatory fields of drug , dose , route and frequency meant that these categories were never missing from the prescription , in contrast with handwritten prescriptions . our study confirmed previous work , that handwritten medication orders are subject to being incomplete , illegible and unsigned . since 100% of individual drug entries were authorised by password with computerised prescribing it was always possible to trace a member of staff prescribing or administering a medication . start and stop dates and changes in dose were always recorded and identifiable in comparison with handwritten prescriptions where changes in doses were less likely to be dated and signed by the prescriber . the time of initiation or discontinuation of the prescription was virtually never recorded on the handwritten charts , and discontinued prescriptions were frequently unsigned . computer - assisted prescriptions took more than twice as long to prescribe as handwritten prescriptions . administration also took the nursing staff longer to record with cap than on the handwritten charts . this was due , however , to the much greater detail entered with cap which was not part of the handwritten record , and the extra time taken to move around the computer screen and store information . our study focused on time spent prescribing drugs and logging that drugs had been given . related secondary time issues such as drug stocktaking , ordering drugs from the central pharmacy and time taken to issue discharge prescriptions were not studied . a similar study describing parenteral nutrition ( pn ) ordering in a neonatal intensive care unit looked at time spent on writing and ordering pn with manual and computer systems . time was reduced by the computerised ordering , and improvements to nutritional composition were noted in the computerised group . other investigators have demonstrated that computerised prescriptions result in total time saving advantages even when time to enter the information takes 2 - 3 times longer with computerised systems . indeed , our study focused on the task at the bedside workstation and , in this area , more time was spent with cap compared to handwritten prescribing . it may be that secondary time costs will be reduced with this system , but it was not part of this study . as a result of this evaluation we have identified several enhancements which would improve the performance of cap . a purpose - designed infusion entry component would greatly reduce prescription errors for this category of drugs . highlighting of duplicate prescriptions would prevent this error occurring , and if all sections of the prescription chart could be viewed simultaneously drug therapy could be more easily reviewed by all icu staff . a facility to make authorised changes to a prescription without these would allow preconfigured combinations or sets of drugs to be prescribed for specific clinical situations . drug interactions and incompatibilities could be flagged , much improving the safety of drug therapy . advice on cost of prescribing could be included , offering alternative cheaper options to the clinician . the carevue mar prescribing system has become well integrated into practice in our icu . it produces a complete , fully legible and permanent prescription . it did not reduce errors in prescribing , and in fact increased such mistakes , but software enhancements to the system , and improved training of clinicians may reduce these . prescribing and recording administration of medication at the bedside takes longer with cap , but there may be secondary time savings with this system .

backgroundwe conducted a prospective comparative study to evaluate the potential benefit of computer - assisted prescribing ( cap ) . we compared the accuracy , completeness and time use of cap with that of conventional handwritten prescribing at the intensive care unit ( icu ) of the john radcliffe hospital , oxford , uk.resultstwenty-five clinicians and 2409 drug entries were evaluated for accuracy , completeness , legibility and time spent prescribing . one hundred and twenty - eight handwritten and 110 cap charts were monitored . one hundred percent of cap charts were complete compared to 47% of handwritten charts.drug prescriptions were divided into three categories : intravenous fluids , intravenous infusions and intermittent drugs . percentage of correct entries in each category were 64% , 47.5% and 90% for handwritten , compared to 48% , 32% and 90% for cap charts , respectively.the mean time taken to prescribe was 20 s for hand written prescribing and 55 s for cap.conclusionscomputer-assisted prescriptions were more complete , signed and dated than handwritten prescriptions . errors in prescribing , including failure to discontinue a drug were not reduced by cap . handwritten prescribing was quicker than cap . simple enhancements of the computer software could be introduced which might overcome these deficiencies . cap was successfully integrated into clinical practice in the icu .

Introduction Methods Handwritten prescribing Computer-assisted prescribing Criteria for evaluation Results Legibility, completeness, and authorisation of prescriptions Intravenous fluids and entail/parenteral feeds Intravenous infusions Intermittent drugs Time taken to prescribe Discussion Accuracy, completeness and `signing' or authorisation Speed of prescribing Suggested enhancements for future MAR systems Conclusions

computerised prescribing is widely used in the united kingdom for repeat prescriptions in general practice . there is evidence that it has improved legibility and clarity , saved time for both medical and clerical staff , and reduced the time spent clarifying prescriptions with the dispensing pharmacist . computer - assisted decision support in the hospital setting has already been shown to improve the use of antibiotics peri - operatively , reduce the costs of prescribing and influence clinical parameters such as limiting the emergence of antibiotic resistance . computer - assisted prescribing ( cap ) for inpatients was introduced to the intensive care unit ( icu ) of the john radcliffe hospital in may 1996 as part of the hewlett packard carevue patient information system ( hewlett packard ltd , andover , usa ) . this study evaluated the computerised prescribing system by comparing the accuracy and completeness of prescriptions , and time spent prescribing before and after the introduction of cap . in may 1996 , a prescribing module of the system software was activated and introduced clinically after a period of modification and reconfiguration . the second 3-week phase was conducted 1 month after the introduction and implementation of the cap component of carevue . all medical and nursing staff received training at the bedside from the ward pharmacist and the carevue nurse specialist . the prescriber was then required to write , in the appropriate section , the drug name , the dose or amount to be diluted ( with iv infusions ) , rate of administration ( for infusions ) , volume of specified diluents required , route and frequency of administration . the prescription was then signed at the bottom with a single signature covering all drug entries . in the second phase of the study , cap was implemented following the introduction of the medication administration record ( mar ) prescribing module of hewlett packard carevue ( fig 1 ) . it is divided into the following three sections : the ` scheduled ' section contains drugs given at regular intervals ; the ` prn ' section contains drugs given as required and also iv fluids , infusions and enteral or parenteral feeds , and the ` stat ' section contains any single dose drugs or fluids . the prescribing doctor is required to complete all sections of the prescription marked with an asterisk , other sections being optional . a preparation not included on the drug pick list may still be prescribed , but the computer can not ` advise ' on the route of administration or dose . specific doses or dose ranges are entered by typing the values into the dose box . any new entry , alteration , modification or discontinuation requires confirmation by entry of the physician 's unique password . a prescription is not active until it has been ` signed ' or authorised by a clinician , using their password . the flowsheet is used to record the hourly rate of iv infusions and also the time of administration of scheduled ` as required ' drugs ( fig 3 ) . each individual drug entry for both the handwritten prescribing and cap was evaluated by the icu pharmacist according to 19 predetermined criteria ( table 1 ) over two 3-week time periods . the time taken to prescribe a single drug by hand and using the computer module was measured , and the time it took a nurse to record that the drug has been given was measured using the two prescribing modes . prescription charts were rewritten daily . the prescriber was then required to write , in the appropriate section , the drug name , the dose or amount to be diluted ( with iv infusions ) , rate of administration ( for infusions ) , volume of specified diluents required , route and frequency of administration . the prescription was then signed at the bottom with a single signature covering all drug entries . in the second phase of the study , cap was implemented following the introduction of the medication administration record ( mar ) prescribing module of hewlett packard carevue ( fig 1 ) . it is divided into the following three sections : the ` scheduled ' section contains drugs given at regular intervals ; the ` prn ' section contains drugs given as required and also iv fluids , infusions and enteral or parenteral feeds , and the ` stat ' section contains any single dose drugs or fluids . the prescribing doctor is required to complete all sections of the prescription marked with an asterisk , other sections being optional . by clicking a trackball directed pointer on the medication box a pick - list of 236 commonly prescribed medications , fluids and feeds is displayed . a preparation not included on the drug pick list may still be prescribed , but the computer can not ` advise ' on the route of administration or dose . any new entry , alteration , modification or discontinuation requires confirmation by entry of the physician 's unique password . the flowsheet is used to record the hourly rate of iv infusions and also the time of administration of scheduled ` as required ' drugs ( fig 3 ) . each individual drug entry for both the handwritten prescribing and cap was evaluated by the icu pharmacist according to 19 predetermined criteria ( table 1 ) over two 3-week time periods . the time taken to prescribe a single drug by hand and using the computer module was measured , and the time it took a nurse to record that the drug has been given was measured using the two prescribing modes . one hundred and twenty - eight handwritten and 110 computer - assisted patient prescription charts were monitored over the 6 weeks of the study , resulting in 1184 handwritten and 1225 computer - assisted individual drug entries being studied . there was an average of 10 individual drugs prescribed per patient per day , consisting of iv fluids , intravenous infusions , entail or parenteral feeds , ` regular ' and ` as required ' drugs . one hundred percent of the computerised patient prescriptions were signed and dated in comparison with 95% of handwritten patient prescriptions . note that with cap all individual drug entries were authorised , whereas with handwritten prescriptions one signature covered the whole prescription . only 47% of handwritten prescription charts had full patient identification ( name , date of birth and unit number , or hospital sticker ) whilst this information was available with all prescriptions on the computerised system . one hundred and ninety - four individual handwritten and 255 individual computerised entries were analysed in this category ( fig 4 ) . there was a decrease from 64% to 48% in the percentage of complete and correct individual entries ( see table 1 criteria ) following the introduction of cap ( fig 4 ) . the percentage of entries with a missing rate of infusion increased from 18% to 28% ( fig 4 ) . the number of drug entries remaining on the prescription chart despite having been removed from the treatment regimen more than 24 h previously also increased , from 1% to 17% ( fig 4 ) . two hundred and eighty - four handwritten and 247 computerised drug entries were assessed in this category . the percentage of correct entries for iv infusions decreased from 47.5% to 32% with cap ( fig . thirty - one percent of cap had no prescribed diluent and 27% had no prescribed diluent volume . the percentage of prescriptions with an incorrect rate of infusion was 16% with cap and 1.4% with handwritten prescriptions . seven hundred and six handwritten and 723 computer - assisted drug entries were analysed in this category . ninety percent of entries for both handwritten and cap groups complied with the specified criteria ( dose , route , frequency ) . the results are expressed in fig 6 as a percentage of the total number of erroneous prescriptions ( approximately 10% of the total number of entries monitored ) . figure 6 shows an increased frequency of failure to discontinue drugs that the patient was no longer receiving from 9.1% of errors with handwritten prescribing to 57% of errors with cap . although this increase appears large , this represented less than 6% of the total entries in this category . time taken to prescribe one medication by computer and one medication by hand was measured . it took approximately 20 s to prescribe a single complete handwritten drug entry and 55 s to prescribe the same drug using cap . to record that a drug had been given took 2 s on the handwritten chart and 21 s with cap . the right hand box shows part of the pick - list of 236 drugs available to the physician . by moving the trackball and clicking the centre key a drug the percentage of individual drug entries for iv fluids and enteral / parenteral feeds in each category correctly prescribed , missing a rate or remaining on the prescription but not administered for over 24 h. , handwritten ; , computerised . the percentage of iv infusions in each category correctly prescribed , those remaining on the prescription but not administered for over 24 h , without a specified diluent or volume , with an incorrect rate , or prescribed in duplicate . the percentage of errors in prescription for ` regular ' and ` as required ' drugs in both categories including drug with ambiguous instructions , those remaining on the prescription chart but not administered for over 24 h , without a dose , frequency or route , spelt incorrectly , duplicated , or illegible . the drug prescribed in anticipation of need , but one hundred percent of the computerised patient prescriptions were signed and dated in comparison with 95% of handwritten patient prescriptions . note that with cap all individual drug entries were authorised , whereas with handwritten prescriptions one signature covered the whole prescription . only 47% of handwritten prescription charts had full patient identification ( name , date of birth and unit number , or hospital sticker ) whilst this information was available with all prescriptions on the computerised system . one hundred and ninety - four individual handwritten and 255 individual computerised entries were analysed in this category ( fig 4 ) . there was a decrease from 64% to 48% in the percentage of complete and correct individual entries ( see table 1 criteria ) following the introduction of cap ( fig 4 ) . the percentage of entries with a missing rate of infusion increased from 18% to 28% ( fig 4 ) . the number of drug entries remaining on the prescription chart despite having been removed from the treatment regimen more than 24 h previously also increased , from 1% to 17% ( fig 4 ) . prescriptions monitored in this section were those for sedatives , analgesics , inotropes , vasoactive agents , heparin and antiarrhythmics , administered by infusion . two hundred and eighty - four handwritten and 247 computerised drug entries were assessed in this category . the percentage of correct entries for iv infusions decreased from 47.5% to 32% with cap ( fig . thirty - one percent of cap had no prescribed diluent and 27% had no prescribed diluent volume . the percentage of prescriptions with an incorrect rate of infusion was 16% with cap and 1.4% with handwritten prescriptions . seven hundred and six handwritten and 723 computer - assisted drug entries were analysed in this category . ninety percent of entries for both handwritten and cap groups complied with the specified criteria ( dose , route , frequency ) . the results are expressed in fig 6 as a percentage of the total number of erroneous prescriptions ( approximately 10% of the total number of entries monitored ) . figure 6 shows an increased frequency of failure to discontinue drugs that the patient was no longer receiving from 9.1% of errors with handwritten prescribing to 57% of errors with cap . although this increase appears large , this represented less than 6% of the total entries in this category . one hundred and ninety - four individual handwritten and 255 individual computerised entries were analysed in this category ( fig 4 ) . there was a decrease from 64% to 48% in the percentage of complete and correct individual entries ( see table 1 criteria ) following the introduction of cap ( fig 4 ) . the percentage of entries with a missing rate of infusion increased from 18% to 28% ( fig 4 ) . the number of drug entries remaining on the prescription chart despite having been removed from the treatment regimen more than 24 h previously also increased , from 1% to 17% ( fig 4 ) . two hundred and eighty - four handwritten and 247 computerised drug entries were assessed in this category . the percentage of correct entries for iv infusions decreased from 47.5% to 32% with cap ( fig . thirty - one percent of cap had no prescribed diluent and 27% had no prescribed diluent volume . the percentage of prescriptions with an incorrect rate of infusion was 16% with cap and 1.4% with handwritten prescriptions . seven hundred and six handwritten and 723 computer - assisted drug entries were analysed in this category . ninety percent of entries for both handwritten and cap groups complied with the specified criteria ( dose , route , frequency ) . the results are expressed in fig 6 as a percentage of the total number of erroneous prescriptions ( approximately 10% of the total number of entries monitored ) . figure 6 shows an increased frequency of failure to discontinue drugs that the patient was no longer receiving from 9.1% of errors with handwritten prescribing to 57% of errors with cap . although this increase appears large , this represented less than 6% of the total entries in this category . time taken to prescribe one medication by computer and one medication by hand was measured . it took approximately 20 s to prescribe a single complete handwritten drug entry and 55 s to prescribe the same drug using cap . to record that a drug had been given took 2 s on the handwritten chart and 21 s with cap . the right hand box shows part of the pick - list of 236 drugs available to the physician . by moving the trackball and clicking the centre key a drug is released on to the medication administration record ( mar ) . the percentage of individual drug entries for iv fluids and enteral / parenteral feeds in each category correctly prescribed , missing a rate or remaining on the prescription but not administered for over 24 h. , handwritten ; , computerised . the percentage of iv infusions in each category correctly prescribed , those remaining on the prescription but not administered for over 24 h , without a specified diluent or volume , with an incorrect rate , or prescribed in duplicate . the percentage of errors in prescription for ` regular ' and ` as required ' drugs in both categories including drug with ambiguous instructions , those remaining on the prescription chart but not administered for over 24 h , without a dose , frequency or route , spelt incorrectly , duplicated , or illegible . the drug prescribed in anticipation of need , but this study was undertaken in order to assess the accuracy , completeness and time spent prescribing with cap compared with handwritten prescriptions in a busy icu in the uk . cap provided a less accurate prescription for iv fluids and infusions , and prescriptions in all categories were more likely to remain on the chart despite having been discontinued from therapy , possibly due to a decrease in frequency of review of the chart . there was also an uncommon problem of duplicate drug entries using cap which did not occur with handwritten prescriptions . this was probably linked to the configuration of the system which segregated regularly scheduled drugs from ` stat ' and ` prn ' drugs . cap were more complete than handwritten prescriptions in that each prescription chart uniquely and fully identified the patient ( 100% compared with 47% ) and any allergies they may have , and every individual drug entry was authorised . with intermittent and scheduled therapy the mandatory fields of drug , dose , route and frequency meant that these categories were never missing from the prescription , in contrast with handwritten prescriptions . since 100% of individual drug entries were authorised by password with computerised prescribing it was always possible to trace a member of staff prescribing or administering a medication . start and stop dates and changes in dose were always recorded and identifiable in comparison with handwritten prescriptions where changes in doses were less likely to be dated and signed by the prescriber . the time of initiation or discontinuation of the prescription was virtually never recorded on the handwritten charts , and discontinued prescriptions were frequently unsigned . computer - assisted prescriptions took more than twice as long to prescribe as handwritten prescriptions . administration also took the nursing staff longer to record with cap than on the handwritten charts . this was due , however , to the much greater detail entered with cap which was not part of the handwritten record , and the extra time taken to move around the computer screen and store information . our study focused on time spent prescribing drugs and logging that drugs had been given . related secondary time issues such as drug stocktaking , ordering drugs from the central pharmacy and time taken to issue discharge prescriptions were not studied . a similar study describing parenteral nutrition ( pn ) ordering in a neonatal intensive care unit looked at time spent on writing and ordering pn with manual and computer systems . time was reduced by the computerised ordering , and improvements to nutritional composition were noted in the computerised group . indeed , our study focused on the task at the bedside workstation and , in this area , more time was spent with cap compared to handwritten prescribing . highlighting of duplicate prescriptions would prevent this error occurring , and if all sections of the prescription chart could be viewed simultaneously drug therapy could be more easily reviewed by all icu staff . a higher level of enhancement of cap could include decision support tools . drug interactions and incompatibilities could be flagged , much improving the safety of drug therapy . advice on cost of prescribing could be included , offering alternative cheaper options to the clinician . cap provided a less accurate prescription for iv fluids and infusions , and prescriptions in all categories were more likely to remain on the chart despite having been discontinued from therapy , possibly due to a decrease in frequency of review of the chart . there was also an uncommon problem of duplicate drug entries using cap which did not occur with handwritten prescriptions . this was probably linked to the configuration of the system which segregated regularly scheduled drugs from ` stat ' and ` prn ' drugs . cap were more complete than handwritten prescriptions in that each prescription chart uniquely and fully identified the patient ( 100% compared with 47% ) and any allergies they may have , and every individual drug entry was authorised . with intermittent and scheduled therapy the mandatory fields of drug , dose , route and frequency meant that these categories were never missing from the prescription , in contrast with handwritten prescriptions . since 100% of individual drug entries were authorised by password with computerised prescribing it was always possible to trace a member of staff prescribing or administering a medication . start and stop dates and changes in dose were always recorded and identifiable in comparison with handwritten prescriptions where changes in doses were less likely to be dated and signed by the prescriber . the time of initiation or discontinuation of the prescription was virtually never recorded on the handwritten charts , and discontinued prescriptions were frequently unsigned . computer - assisted prescriptions took more than twice as long to prescribe as handwritten prescriptions . administration also took the nursing staff longer to record with cap than on the handwritten charts . this was due , however , to the much greater detail entered with cap which was not part of the handwritten record , and the extra time taken to move around the computer screen and store information . our study focused on time spent prescribing drugs and logging that drugs had been given . related secondary time issues such as drug stocktaking , ordering drugs from the central pharmacy and time taken to issue discharge prescriptions were not studied . a similar study describing parenteral nutrition ( pn ) ordering in a neonatal intensive care unit looked at time spent on writing and ordering pn with manual and computer systems . time was reduced by the computerised ordering , and improvements to nutritional composition were noted in the computerised group . indeed , our study focused on the task at the bedside workstation and , in this area , more time was spent with cap compared to handwritten prescribing . as a result of this evaluation we have identified several enhancements which would improve the performance of cap . a purpose - designed infusion entry component would greatly reduce prescription errors for this category of drugs . highlighting of duplicate prescriptions would prevent this error occurring , and if all sections of the prescription chart could be viewed simultaneously drug therapy could be more easily reviewed by all icu staff . drug interactions and incompatibilities could be flagged , much improving the safety of drug therapy . advice on cost of prescribing could be included , offering alternative cheaper options to the clinician . the carevue mar prescribing system has become well integrated into practice in our icu . it produces a complete , fully legible and permanent prescription . it did not reduce errors in prescribing , and in fact increased such mistakes , but software enhancements to the system , and improved training of clinicians may reduce these . prescribing and recording administration of medication at the bedside takes longer with cap , but there may be secondary time savings with this system .

uncontrolled intraoperative bleeding is a dangerous complication that can hinder both the surgeon s ability to complete the procedure and the patient s ability to recover.1 blood that remains intra - abdominally after surgery not only increases the extent and severity of postoperative intestinal adhesions , but also serves as a source of intra - abdominal infections.2 blood transfusions may become necessary in some cases ; these increase the risk of postoperative morbidity and mortality.3,4 furthermore , inadequate hemostasis increases operative time , recovery , and length of hospital stay , and constitutes a considerable economic burden.5 conventional methods of obtaining hemostasis at continuously bleeding sites include repetitive direct cauterization , placement of sutures or clips , and prolonged direct compression , but these methods can impair tissue healing and recovery through the generation of tissue damage , char , and necrosis.6,7 conventional methods might also be impractical or ineffective in regions that are hard to access or in organs and tissues located nearby that can be easily damaged ( eg , nerves or the respiratory tract ) . therefore , the development of more effective hemostatic agents is critical in order to keep surgical costs down and provide the best safety for patients . many of the commercially available hemostats have been associated with adverse safety.8 hemostats that are based on human- or animal - derived components can lead to virus and disease transmission or elicit anaphylaxis.912 additionally , multiple reports have shown granuloma formation and neurological effects associated with the use of some hemostatic agents if not removed.1319 these observations support the need to develop products that effectively achieve hemostasis in a diverse patient population and introduce minimal safety concerns . an ideal hemostatic agent has the potential to decrease the use of blood products in elective and emergency surgery and demonstrates the ability to manage bleeding from both parenchymal and small or inaccessible arterial and venous structures . such a product would also require adhesive and mechanical strength to withstand the pressure of bleeding.20 veriset hemostatic patch ( covidien , mansfield , ma , usa ) is a recent addition to the plethora of available hemostatic agents . it is composed of an absorbable backing material , oxidized cellulose , and hydrogel components . veriset hemostatic patch is provided as a ready - to - use patch that is applied polyethylene glycol - side down to the bleeding site . unlike many other hemostatic agents , which require a dry field prior to application minimal preparation is required for veriset hemostatic patch , and the device has been shown in preclinical studies to be fully absorbable ( howk , unpublished data , 2015 ) . the device lacks any human- or animal - derived components ; thus , it is unlikely to transmit viruses or elicit an immune response . veriset hemostatic patch is ce - marked for use in laparoscopic and open procedures involving solid organ or soft tissue bleeding . a previous randomized , controlled clinical study compared veriset hemostatic patch to tachosil ( nycomed austria gmbh , linz , austria ) in hepatic procedures.21 the number of subjects who achieved hemostasis within 3 minutes was significantly greater among those treated with veriset hemostatic patch than among those treated with tachosil . in addition , veriset hemostatic patch was determined to be both safe and easy to use . the aim of the present study is to provide further evidence for the safety and effectiveness of veriset hemostatic patch for controlling intraoperative bleeding in soft tissue . the clinical trial registration number for this study is nct01719172 , and it can be found at http://www.clinicaltrials.gov . this study was a prospective , multicenter , single - arm study of veriset hemostatic patch to assess its safety and effectiveness in obtaining hemostasis in patients undergoing visceral surgical procedures in soft tissue that typically result in considerable sizes of intra - abdominal or intrathoracic connective tissue wound areas . in this study , surgical procedures were included that are likely to lead to hemorrhage in soft tissue areas , which are defined as areas containing tissue that connects , supports , or surrounds other structures of the body or parts of organs . from the perspective of the surgical problem addressed ( bleeding from connective tissue in an otherwise highly vulnerable field ) , inclusion of a variety of procedures was expected to result in a tissue surface that was quite similar across procedures . during surgery , veriset hemostatic patch was applied to the target bleeding site ( tbs ) , and its effectiveness was analyzed by capturing the time required for hemostasis to be achieved . safety was determined by the number of device - related serious adverse events ( saes ) during the 30 days after surgery and the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . subjects were assessed during the procedure and then 24 hours , 7 days , 30 days , and 90 days after the operation . the clinical investigation was conducted according to good clinical practice guidelines and the declaration of helsinki , and abided by european and national regulations . they were selected from three european sites on the basis of protocol - defined inclusion and exclusion criteria . preoperative inclusion criteria required subjects be 18 years of age and already scheduled for nonemergency surgery via an open approach , in which a topical hemostatic agent can be used to control bleeding if conventional methods using energy , sutures , or clips are impractical or unsuccessful . subjects who were pregnant , breast - feeding , undergoing emergency surgery , scheduled for another planned surgery that could jeopardize study treatment , had a life expectancy of less than 6 months , or had participated in an investigational drug or device study within 30 days of enrollment that would interfere with this study were excluded from eligibility . all eligible subjects provided informed consent and agreed to comply with the treatment and evaluation schedule . enrolled subjects who did not meet intraoperative criteria were considered screen failures ; these included subjects who exhibited a local infection at the tbs , whose safety or welfare was determined to be at risk by the investigator , or who did not have a bleeding site of type 2 ( oozing / mild ) or type 3 ( moderate ) severity ( a rating system modeled after classification established by the bleeding academic research consortium).22 veriset hemostatic patch was provided in 510 cm sheets that could be cut to the appropriate size . radiopaque gauze ( covidien ) was used to assess relative intraoperative bleeding severity prior to the application of veriset hemostatic patch . the surgical procedure was performed in accordance with the appropriate practices of the institution at which the procedure occurred . after observing an active bleeding site in soft tissue and determining that conventional methods of obtaining hemostasis were either impractical ( an assessment based on the investigator s discretion ) or ineffective ( an assessment based on a failed attempt to achieve hemostasis with one or more of these methods ) , the site was selected as the tbs . veriset hemostatic patch could be cut as necessary to cover the bleeding site with 12 cm margins . multiple patches could be used , if required , with a maximum dosage of 6.43 cm / kg . hemostasis was assessed every 30 seconds until the 5-minute time point , then at 1-minute intervals for minutes 510 until hemostasis was achieved . once bleeding had appeared to stop , the tbs was monitored for 1 minute with no pressure to confirm hemostasis . if rebleeding occurred , the assessment was continued at 30-second intervals until 5 minutes , and at 1-minute intervals for minutes 510 . additional veriset hemostatic patch devices were applied if it was noticed that the initial application did not completely cover the tbs , and the timing of the procedure was continued during the subsequent applications . if bleeding persisted after 2 minutes , either continued pressure was maintained or an additional veriset hemostatic patch was applied . then , ten gauze pads ( 40 layers total ) were placed on the tbs , and gentle pressure was applied for 3 seconds . after 3 seconds , the gauze was removed , and the number of layers penetrated with blood was recorded . eligibility was based solely on the discretion of the investigator , regardless of results from the gauze method of assessment , and the tbs was assigned a bleeding severity of type 2 or type 3 . the primary effectiveness endpoint was the percent success in obtaining hemostasis within 5 minutes after the application of veriset hemostatic patch . secondary effectiveness endpoints included the percentage of subjects who achieved hemostasis within 1 minute and the median time required to achieve hemostasis . the primary safety endpoint was the number of device - related saes per subject during the 30 days after surgery . the secondary safety endpoint was the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . to determine adverse events ( aes ) , subjects were assessed at 24 hours after skin closure by laboratory tests , by testing of vital signs , and by surgical site / infection analyses . a phone call was made 7 days after the surgery to follow up on aes . subjects were assessed on the same characteristics 30 days and 90 days after surgery as during the 24-hour postoperative follow - up . sample size determination was based on the primary effectiveness endpoint , success in obtaining hemostasis within 5 minutes of veriset hemostatic patch application . the sample size was calculated by using a one - sided exact test of a binomial proportion versus an objective performance criterion value for 0.5 ( 50% ) to be the proportion of subjects obtaining hemostasis within 5 minutes . the test was performed with a true success proportion of 0.75 , an alpha of 0.025 , and a power of 80% . all statistical tests performed were one - sided at the 2.5% significance level . to analyze the effectiveness of veriset hemostatic patch in achieving hemostasis , an exact ( clopper pearson ) 95% confidence interval for the true success percentage was calculated . a one - sided exact test based on the binomial distribution was performed to test the null hypothesis that the true percentage of subjects who achieved hemostasis is less than or equal to 50% ; the alternative hypothesis was that the true percentage is greater than 50% . time to hemostasis was analyzed using the kaplan meier method to estimate the median time to hemostasis . a 95% brookmeyer crowley confidence interval for the median was computed on the basis of the sign test . to analyze the safety of veriset hemostatic patch , a 95% confidence interval for the mean number of device - related saes per subject was calculated on the basis of the t - distribution . an exact ( clopper pearson ) 95% confidence interval for the incidence of reoperation for device - related bleeding complications was calculated . paired t - tests were used to analyze changes in vital signs and other laboratory tests from baseline values . statistical analyses of data were performed by using sas version 9.1 or higher ( sas institute , inc . , cary , nc , usa ) . this study was a prospective , multicenter , single - arm study of veriset hemostatic patch to assess its safety and effectiveness in obtaining hemostasis in patients undergoing visceral surgical procedures in soft tissue that typically result in considerable sizes of intra - abdominal or intrathoracic connective tissue wound areas . in this study , surgical procedures were included that are likely to lead to hemorrhage in soft tissue areas , which are defined as areas containing tissue that connects , supports , or surrounds other structures of the body or parts of organs . from the perspective of the surgical problem addressed ( bleeding from connective tissue in an otherwise highly vulnerable field ) , inclusion of a variety of procedures was expected to result in a tissue surface that was quite similar across procedures . during surgery , veriset hemostatic patch was applied to the target bleeding site ( tbs ) , and its effectiveness was analyzed by capturing the time required for hemostasis to be achieved . safety was determined by the number of device - related serious adverse events ( saes ) during the 30 days after surgery and the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . subjects were assessed during the procedure and then 24 hours , 7 days , 30 days , and 90 days after the operation . the clinical investigation was conducted according to good clinical practice guidelines and the declaration of helsinki , and abided by european and national regulations . a total of 30 subjects participated in the study . they were selected from three european sites on the basis of protocol - defined inclusion and exclusion criteria . preoperative inclusion criteria required subjects be 18 years of age and already scheduled for nonemergency surgery via an open approach , in which a topical hemostatic agent can be used to control bleeding if conventional methods using energy , sutures , or clips are impractical or unsuccessful . subjects who were pregnant , breast - feeding , undergoing emergency surgery , scheduled for another planned surgery that could jeopardize study treatment , had a life expectancy of less than 6 months , or had participated in an investigational drug or device study within 30 days of enrollment that would interfere with this study were excluded from eligibility . all eligible subjects provided informed consent and agreed to comply with the treatment and evaluation schedule . enrolled subjects who did not meet intraoperative criteria were considered screen failures ; these included subjects who exhibited a local infection at the tbs , whose safety or welfare was determined to be at risk by the investigator , or who did not have a bleeding site of type 2 ( oozing / mild ) or type 3 ( moderate ) severity ( a rating system modeled after classification established by the bleeding academic research consortium).22 veriset hemostatic patch was provided in 510 cm sheets that could be cut to the appropriate size . radiopaque gauze ( covidien ) was used to assess relative intraoperative bleeding severity prior to the application of veriset hemostatic patch . the surgical procedure was performed in accordance with the appropriate practices of the institution at which the procedure occurred . after observing an active bleeding site in soft tissue and determining that conventional methods of obtaining hemostasis were either impractical ( an assessment based on the investigator s discretion ) or ineffective ( an assessment based on a failed attempt to achieve hemostasis with one or more of these methods ) veriset hemostatic patch could be cut as necessary to cover the bleeding site with 12 cm margins . multiple patches could be used , if required , with a maximum dosage of 6.43 cm / kg . hemostasis was assessed every 30 seconds until the 5-minute time point , then at 1-minute intervals for minutes 510 until hemostasis was achieved . once bleeding had appeared to stop , the tbs was monitored for 1 minute with no pressure to confirm hemostasis . if rebleeding occurred , the assessment was continued at 30-second intervals until 5 minutes , and at 1-minute intervals for minutes 510 . additional veriset hemostatic patch devices were applied if it was noticed that the initial application did not completely cover the tbs , and the timing of the procedure was continued during the subsequent applications . if bleeding persisted after 2 minutes , either continued pressure was maintained or an additional veriset hemostatic patch was applied . then , ten gauze pads ( 40 layers total ) were placed on the tbs , and gentle pressure was applied for 3 seconds . after 3 seconds , the gauze was removed , and the number of layers penetrated with blood was recorded . eligibility was based solely on the discretion of the investigator , regardless of results from the gauze method of assessment , and the tbs was assigned a bleeding severity of type 2 or type 3 . the primary effectiveness endpoint was the percent success in obtaining hemostasis within 5 minutes after the application of veriset hemostatic patch . secondary effectiveness endpoints included the percentage of subjects who achieved hemostasis within 1 minute and the median time required to achieve hemostasis . the primary safety endpoint was the number of device - related saes per subject during the 30 days after surgery . the secondary safety endpoint was the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . to determine adverse events ( aes ) , subjects were assessed at 24 hours after skin closure by laboratory tests , by testing of vital signs , and by surgical site / infection analyses . a phone call was made 7 days after the surgery to follow up on aes . subjects were assessed on the same characteristics 30 days and 90 days after surgery as during the 24-hour postoperative follow - up . sample size determination was based on the primary effectiveness endpoint , success in obtaining hemostasis within 5 minutes of veriset hemostatic patch application . the sample size was calculated by using a one - sided exact test of a binomial proportion versus an objective performance criterion value for 0.5 ( 50% ) to be the proportion of subjects obtaining hemostasis within 5 minutes . the test was performed with a true success proportion of 0.75 , an alpha of 0.025 , and a power of 80% . all statistical tests performed were one - sided at the 2.5% significance level . to analyze the effectiveness of veriset hemostatic patch in achieving hemostasis , an exact ( clopper pearson ) 95% confidence interval for the true success percentage was calculated . a one - sided exact test based on the binomial distribution was performed to test the null hypothesis that the true percentage of subjects who achieved hemostasis is less than or equal to 50% ; the alternative hypothesis was that the true percentage is greater than 50% . time to hemostasis was analyzed using the kaplan meier method to estimate the median time to hemostasis . crowley confidence interval for the median was computed on the basis of the sign test . to analyze the safety of veriset hemostatic patch , a 95% confidence interval for the mean number of device - related saes per subject was calculated on the basis of the t - distribution . an exact ( clopper pearson ) 95% confidence interval for the incidence of reoperation for device - related bleeding complications was calculated . paired t - tests were used to analyze changes in vital signs and other laboratory tests from baseline values . statistical analyses of data were performed by using sas version 9.1 or higher ( sas institute , inc . , cary , nc , usa ) . a total of 37 subjects consented to and were enrolled in the study , but seven did not meet preoperative or intraoperative criteria and were considered screen failures ( figure 1 ) . the remaining 30 subjects were treated with veriset hemostatic patch , but two of the 30 subjects did not complete the study . of those two subjects , one was lost to follow - up and the other died during the study from embolic complications of a pre - existing atrial thrombus that was unrelated to the test device . subjects underwent various types of surgical procedures , of which the most common were lymphadenectomy , esophagectomy , colectomy , and pancreatic bed surgery . bleeding occurred in several tissue types and from multiple sources , and the tbs ranged in size from 0.1200 cm . concerning bleeding severity , 22 ( 73.3% ) subjects had a tbs categorized as type 2 , and the remaining eight ( 26.7% ) were type 3 . there was a positive correlation between the layers of gauze penetrated with blood and the categorization of bleeding severity determined by the investigators ( table 2 ) . in 26/30 ( 86.7% ) subjects , conventional methods of obtaining hemostasis were attempted first but were deemed ineffective and , in the remaining four subjects , the investigator determined that conventional methods were impractical ( table 1 ) . in all of these subjects , veriset hemostatic patch was applied to the tbs . within 1 minute , 70.0% ( 21/30 ) of subjects had achieved hemostasis and , within 5 minutes , 96.7% ( 29/30 ) of subjects had achieved hemostasis ( table 3 ) . the median time to hemostasis was 1.0 minute ; only three subjects were still bleeding 2.0 minutes after application of the device ( figure 2 ) . the maximum amount of the device required to obtain hemostasis in a single subject was four 510 cm patches . in 27/30 ( 90.0% ) subjects , subject , the investigator opted to remove veriset hemostatic patch after 2 minutes because hemostasis had not been achieved , and an alternative topical agent was administered . in another subject , hemostasis was achieved with veriset hemostatic patch but , later in the procedure , it became detached during a gastric pull - up maneuver . , veriset hemostatic patch was later removed when the initial tbs was extracted along with an organ because of a procedural complications unrelated to the use of the device . aes were monitored at each follow - up , and device - relatedness was adjudicated by an independent medical monitor . a total of 136 aes were observed in 23/30 ( 76.7% ) subjects , and all were typical of the types of surgical procedures performed ( table 4 ) . one subject experienced 25 aes , and two subjects each experienced 13 aes ; however , most of these were only mild or moderate in severity . anemia , nausea , impaired healing , and pleural effusion were the only aes to occur in more than 10% of subjects . all but one ( 99.3% ) of the aes were determined to have no relationship to treatment with veriset hemostatic patch . in one subject with anemia , the ae was adjudicated by the independent medical monitor as having an unknown or impossible to determine relationship with the device . one subject died before the 7-day follow - up , but the cause , embolism from an atrial thrombus diagnosed prior to surgery , was unrelated to the use of veriset hemostatic patch . no device - related saes were observed for 30 days after the surgery , and no reoperations for device - related bleeding complications were performed within 5 postoperative days . the data safety monitoring board found no safety issues at the conclusion of the study . a total of 37 subjects consented to and were enrolled in the study , but seven did not meet preoperative or intraoperative criteria and were considered screen failures ( figure 1 ) . the remaining 30 subjects were treated with veriset hemostatic patch , but two of the 30 subjects did not complete the study . of those two subjects , one was lost to follow - up and the other died during the study from embolic complications of a pre - existing atrial thrombus that was unrelated to the test device . subjects underwent various types of surgical procedures , of which the most common were lymphadenectomy , esophagectomy , colectomy , and pancreatic bed surgery . bleeding occurred in several tissue types and from multiple sources , and the tbs ranged in size from 0.1200 cm . concerning bleeding severity , 22 ( 73.3% ) subjects had a tbs categorized as type 2 , and the remaining eight ( 26.7% ) were type 3 . there was a positive correlation between the layers of gauze penetrated with blood and the categorization of bleeding severity determined by the investigators ( table 2 ) . in 26/30 ( 86.7% ) subjects , conventional methods of obtaining hemostasis were attempted first but were deemed ineffective and , in the remaining four subjects , the investigator determined that conventional methods were impractical ( table 1 ) . in all of these subjects , veriset hemostatic patch was applied to the tbs . within 1 minute , 70.0% ( 21/30 ) of subjects had achieved hemostasis and , within 5 minutes , 96.7% ( 29/30 ) of subjects had achieved hemostasis ( table 3 ) . the median time to hemostasis was 1.0 minute ; only three subjects were still bleeding 2.0 minutes after application of the device ( figure 2 ) . the maximum amount of the device required to obtain hemostasis in a single subject was four 510 cm patches . in 27/30 ( 90.0% ) subjects , subject , the investigator opted to remove veriset hemostatic patch after 2 minutes because hemostasis had not been achieved , and an alternative topical agent was administered . in another subject , hemostasis was achieved with veriset hemostatic patch but , later in the procedure , it became detached during a gastric pull - up maneuver . , veriset hemostatic patch was later removed when the initial tbs was extracted along with an organ because of a procedural complications unrelated to the use of the device . aes were monitored at each follow - up , and device - relatedness was adjudicated by an independent medical monitor . a total of 136 aes were observed in 23/30 ( 76.7% ) subjects , and all were typical of the types of surgical procedures performed ( table 4 ) . one subject experienced 25 aes , and two subjects each experienced 13 aes ; however , most of these were only mild or moderate in severity . anemia , nausea , impaired healing , and pleural effusion were the only aes to occur in more than 10% of subjects . all but one ( 99.3% ) of the aes were determined to have no relationship to treatment with veriset hemostatic patch . in one subject with anemia , the ae was adjudicated by the independent medical monitor as having an unknown or impossible to determine relationship with the device . one subject died before the 7-day follow - up , but the cause , embolism from an atrial thrombus diagnosed prior to surgery , was unrelated to the use of veriset hemostatic patch . no device - related saes were observed for 30 days after the surgery , and no reoperations for device - related bleeding complications were performed within 5 postoperative days . the data safety monitoring board found no safety issues at the conclusion of the study . many different approaches to achieving hemostasis are currently available ; these include conventional methods as well as topical agents.8 available methods or devices have advantages and disadvantages that are related to their modes of action or the underlying materials used . classical surgical techniques , such as sutures , require the presence of mobile adjacent tissue , which will compress the bleeding site by mechanical force but will cause additional trauma to the target area . electrocautery is effective but causes substantial tissue degradation of an area of the bleeding site and around the focus of application . topical hemostats have been approved that are either based on coated patch application or that use blood clotting components in a semisolid , fluid - like texture . in certain situations , these existing products exhibit a supportive effect in reaching sufficient hemostasis , but there is still room for improvement in their efficacy , ease of use , and adherence to anatomical tissue . oxidized cellulose , a stand - alone hemostat , has been used for several decades in its regenerated form ; however , a recent study showed significant improvement using nonregenerated oxidized cellulose in terms of hemostatic efficiency.23 in contrast to these and other available products , veriset hemostatic patch promotes hemostasis through a dual mode of action that combines the features of two clinically used components : oxidized cellulose and polyethylene glycol hydrogel . veriset hemostatic patch promotes hemostasis by serving as a tamponade to physically stem blood flow while concentrating platelets and other clotting factors at the bleeding site to accelerate the coagulation cascade . in this study , a variety of procedure types were performed that were accompanied by soft tissue bleeding from multiple sources . veriset hemostatic patch stopped blood loss in almost all of these situations , a finding that supports its potential broad range of use . veriset hemostatic patch was deemed ineffective in only one subject whose bleeding site was determined to have an unusual geometry that did not allow sufficient contact with the device . multiple applications of veriset hemostatic patch are feasible to accommodate either especially large bleeding sites or more difficult to treat bleeding sites . several subjects in this study required multiple applications , but this did not correlate with adverse safety events . in response to uncontrolled bleeding in the operating room , surgeons typically start with traditionally available methods to achieve hemostasis , such as compression , electrocautery , or direct suture application.24 if this initial attempt is unsuccessful , either these methods are used repetitively , or more expensive ( and typically more effective ) methods are used until bleeding is halted.25 achieving hemostasis in this manner may not always be in the best interest of the patient with regard to both medical and economic outcomes . in some cases , including those in which bleeding occurs on delicate organ tissue or around nerves , it is obvious to the experienced surgeon that certain hemostats are unlikely to halt bleeding without further collateral damage . opting to use veriset hemostatic patch as an initial hemostatic agent , instead of attempting more conventional methods first , may save time by allowing quick control of bleeding with little interruption to the surgical procedure and minimal risk of complications . in the two cases in which veriset hemostatic patch either did not adhere or became detached after application , there was no visible tissue damage . this observation suggests that , unlike some conventional hemostatic methods that can cause tissue damage ( ie , electrocautery),26,27 the use of veriset hemostatic patch can be attempted with little risk to the patient . the results shown here support the use of veriset hemostatic patch across a variety of surgical procedures that can result in soft tissue bleeding . additionally , the median time to hemostasis in this study was 1 minute , the same as that observed in the randomized liver study;21 this similarity indicates that veriset hemostatic patch might offer consistent efficacy across multiple tissues . on a practical level , the device exhibits properties that render it convenient and widely applicable . further studies , particularly in the form of randomized , controlled trials , should be performed in the future to assess such characteristics as safety , efficacy , usability ( eg , adhesiveness to tissue , flexibility to underlying structures ) , and cost - effectiveness , compared to other topical hemostats on the market . while these data would be beneficial to the surgical community , our results combined with the previously published liver results , suggest that veriset hemostatic patch offers a safe hemostatic method that is effective in many surgical situations .

backgroundcontinuous bleeding after using conventional hemostatic methods involving energy , sutures , or clips , is a serious and costly surgical complication . many topical agents have been developed to promote intraoperative hemostasis , but improvement is needed in both decreasing time to hemostasis and increasing ease of use . veriset hemostatic patch is ce - marked for controlling bleeding on the liver and in soft tissue . in the current study , we aimed to gather further evidence for the safety and effectiveness of veriset hemostatic patch in soft tissue bleeding during a variety of surgical procedures.methodsthirty patients scheduled for nonemergency surgery , each with an intraoperative soft tissue bleeding site , were treated with veriset hemostatic patch . time to hemostasis was monitored , and adverse events were assessed during the 90 days after surgery.resultswhen veriset hemostatic patch was used , hemostasis occurred within 5 minutes in 29/30 ( 96.7% ) subjects and within 1 minute in 21/30 ( 70.0% ) subjects . no device - related serious adverse events were recorded during the 30 days after surgery , and no reoperations for device - related bleeding complications were performed during the 5 days after surgery.conclusionsveriset hemostatic patch is a safe and effective hemostat for controlling soft tissue bleeding during a variety of surgical procedures .

Introduction Materials and methods Study design Study participants Materials Surgical procedure Assessing intraoperative bleeding with gauze Outcome measures Postoperative visits Statistical analysis Results Surgery Effectiveness of Veriset hemostatic patch Safety of Veriset hemostatic patch Discussion Conclusion

uncontrolled intraoperative bleeding is a dangerous complication that can hinder both the surgeon s ability to complete the procedure and the patient s ability to recover.1 blood that remains intra - abdominally after surgery not only increases the extent and severity of postoperative intestinal adhesions , but also serves as a source of intra - abdominal infections.2 blood transfusions may become necessary in some cases ; these increase the risk of postoperative morbidity and mortality.3,4 furthermore , inadequate hemostasis increases operative time , recovery , and length of hospital stay , and constitutes a considerable economic burden.5 conventional methods of obtaining hemostasis at continuously bleeding sites include repetitive direct cauterization , placement of sutures or clips , and prolonged direct compression , but these methods can impair tissue healing and recovery through the generation of tissue damage , char , and necrosis.6,7 conventional methods might also be impractical or ineffective in regions that are hard to access or in organs and tissues located nearby that can be easily damaged ( eg , nerves or the respiratory tract ) . such a product would also require adhesive and mechanical strength to withstand the pressure of bleeding.20 veriset hemostatic patch ( covidien , mansfield , ma , usa ) is a recent addition to the plethora of available hemostatic agents . veriset hemostatic patch is provided as a ready - to - use patch that is applied polyethylene glycol - side down to the bleeding site . unlike many other hemostatic agents , which require a dry field prior to application minimal preparation is required for veriset hemostatic patch , and the device has been shown in preclinical studies to be fully absorbable ( howk , unpublished data , 2015 ) . veriset hemostatic patch is ce - marked for use in laparoscopic and open procedures involving solid organ or soft tissue bleeding . a previous randomized , controlled clinical study compared veriset hemostatic patch to tachosil ( nycomed austria gmbh , linz , austria ) in hepatic procedures.21 the number of subjects who achieved hemostasis within 3 minutes was significantly greater among those treated with veriset hemostatic patch than among those treated with tachosil . in addition , veriset hemostatic patch was determined to be both safe and easy to use . the aim of the present study is to provide further evidence for the safety and effectiveness of veriset hemostatic patch for controlling intraoperative bleeding in soft tissue . this study was a prospective , multicenter , single - arm study of veriset hemostatic patch to assess its safety and effectiveness in obtaining hemostasis in patients undergoing visceral surgical procedures in soft tissue that typically result in considerable sizes of intra - abdominal or intrathoracic connective tissue wound areas . in this study , surgical procedures were included that are likely to lead to hemorrhage in soft tissue areas , which are defined as areas containing tissue that connects , supports , or surrounds other structures of the body or parts of organs . from the perspective of the surgical problem addressed ( bleeding from connective tissue in an otherwise highly vulnerable field ) , inclusion of a variety of procedures was expected to result in a tissue surface that was quite similar across procedures . during surgery , veriset hemostatic patch was applied to the target bleeding site ( tbs ) , and its effectiveness was analyzed by capturing the time required for hemostasis to be achieved . safety was determined by the number of device - related serious adverse events ( saes ) during the 30 days after surgery and the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . subjects were assessed during the procedure and then 24 hours , 7 days , 30 days , and 90 days after the operation . preoperative inclusion criteria required subjects be 18 years of age and already scheduled for nonemergency surgery via an open approach , in which a topical hemostatic agent can be used to control bleeding if conventional methods using energy , sutures , or clips are impractical or unsuccessful . subjects who were pregnant , breast - feeding , undergoing emergency surgery , scheduled for another planned surgery that could jeopardize study treatment , had a life expectancy of less than 6 months , or had participated in an investigational drug or device study within 30 days of enrollment that would interfere with this study were excluded from eligibility . enrolled subjects who did not meet intraoperative criteria were considered screen failures ; these included subjects who exhibited a local infection at the tbs , whose safety or welfare was determined to be at risk by the investigator , or who did not have a bleeding site of type 2 ( oozing / mild ) or type 3 ( moderate ) severity ( a rating system modeled after classification established by the bleeding academic research consortium).22 veriset hemostatic patch was provided in 510 cm sheets that could be cut to the appropriate size . radiopaque gauze ( covidien ) was used to assess relative intraoperative bleeding severity prior to the application of veriset hemostatic patch . after observing an active bleeding site in soft tissue and determining that conventional methods of obtaining hemostasis were either impractical ( an assessment based on the investigator s discretion ) or ineffective ( an assessment based on a failed attempt to achieve hemostasis with one or more of these methods ) , the site was selected as the tbs . additional veriset hemostatic patch devices were applied if it was noticed that the initial application did not completely cover the tbs , and the timing of the procedure was continued during the subsequent applications . if bleeding persisted after 2 minutes , either continued pressure was maintained or an additional veriset hemostatic patch was applied . the primary effectiveness endpoint was the percent success in obtaining hemostasis within 5 minutes after the application of veriset hemostatic patch . the primary safety endpoint was the number of device - related saes per subject during the 30 days after surgery . the secondary safety endpoint was the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . to determine adverse events ( aes ) , subjects were assessed at 24 hours after skin closure by laboratory tests , by testing of vital signs , and by surgical site / infection analyses . subjects were assessed on the same characteristics 30 days and 90 days after surgery as during the 24-hour postoperative follow - up . sample size determination was based on the primary effectiveness endpoint , success in obtaining hemostasis within 5 minutes of veriset hemostatic patch application . to analyze the effectiveness of veriset hemostatic patch in achieving hemostasis , an exact ( clopper pearson ) 95% confidence interval for the true success percentage was calculated . time to hemostasis was analyzed using the kaplan meier method to estimate the median time to hemostasis . to analyze the safety of veriset hemostatic patch , a 95% confidence interval for the mean number of device - related saes per subject was calculated on the basis of the t - distribution . an exact ( clopper pearson ) 95% confidence interval for the incidence of reoperation for device - related bleeding complications was calculated . this study was a prospective , multicenter , single - arm study of veriset hemostatic patch to assess its safety and effectiveness in obtaining hemostasis in patients undergoing visceral surgical procedures in soft tissue that typically result in considerable sizes of intra - abdominal or intrathoracic connective tissue wound areas . in this study , surgical procedures were included that are likely to lead to hemorrhage in soft tissue areas , which are defined as areas containing tissue that connects , supports , or surrounds other structures of the body or parts of organs . from the perspective of the surgical problem addressed ( bleeding from connective tissue in an otherwise highly vulnerable field ) , inclusion of a variety of procedures was expected to result in a tissue surface that was quite similar across procedures . during surgery , veriset hemostatic patch was applied to the target bleeding site ( tbs ) , and its effectiveness was analyzed by capturing the time required for hemostasis to be achieved . safety was determined by the number of device - related serious adverse events ( saes ) during the 30 days after surgery and the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . subjects were assessed during the procedure and then 24 hours , 7 days , 30 days , and 90 days after the operation . preoperative inclusion criteria required subjects be 18 years of age and already scheduled for nonemergency surgery via an open approach , in which a topical hemostatic agent can be used to control bleeding if conventional methods using energy , sutures , or clips are impractical or unsuccessful . subjects who were pregnant , breast - feeding , undergoing emergency surgery , scheduled for another planned surgery that could jeopardize study treatment , had a life expectancy of less than 6 months , or had participated in an investigational drug or device study within 30 days of enrollment that would interfere with this study were excluded from eligibility . enrolled subjects who did not meet intraoperative criteria were considered screen failures ; these included subjects who exhibited a local infection at the tbs , whose safety or welfare was determined to be at risk by the investigator , or who did not have a bleeding site of type 2 ( oozing / mild ) or type 3 ( moderate ) severity ( a rating system modeled after classification established by the bleeding academic research consortium).22 veriset hemostatic patch was provided in 510 cm sheets that could be cut to the appropriate size . radiopaque gauze ( covidien ) was used to assess relative intraoperative bleeding severity prior to the application of veriset hemostatic patch . after observing an active bleeding site in soft tissue and determining that conventional methods of obtaining hemostasis were either impractical ( an assessment based on the investigator s discretion ) or ineffective ( an assessment based on a failed attempt to achieve hemostasis with one or more of these methods ) veriset hemostatic patch could be cut as necessary to cover the bleeding site with 12 cm margins . additional veriset hemostatic patch devices were applied if it was noticed that the initial application did not completely cover the tbs , and the timing of the procedure was continued during the subsequent applications . if bleeding persisted after 2 minutes , either continued pressure was maintained or an additional veriset hemostatic patch was applied . the primary effectiveness endpoint was the percent success in obtaining hemostasis within 5 minutes after the application of veriset hemostatic patch . the primary safety endpoint was the number of device - related saes per subject during the 30 days after surgery . the secondary safety endpoint was the incidence of reoperation for device - related bleeding complications during the 5 days after surgery . to determine adverse events ( aes ) , subjects were assessed at 24 hours after skin closure by laboratory tests , by testing of vital signs , and by surgical site / infection analyses . subjects were assessed on the same characteristics 30 days and 90 days after surgery as during the 24-hour postoperative follow - up . sample size determination was based on the primary effectiveness endpoint , success in obtaining hemostasis within 5 minutes of veriset hemostatic patch application . to analyze the effectiveness of veriset hemostatic patch in achieving hemostasis , an exact ( clopper pearson ) 95% confidence interval for the true success percentage was calculated . time to hemostasis was analyzed using the kaplan meier method to estimate the median time to hemostasis . to analyze the safety of veriset hemostatic patch , a 95% confidence interval for the mean number of device - related saes per subject was calculated on the basis of the t - distribution . an exact ( clopper pearson ) 95% confidence interval for the incidence of reoperation for device - related bleeding complications was calculated . a total of 37 subjects consented to and were enrolled in the study , but seven did not meet preoperative or intraoperative criteria and were considered screen failures ( figure 1 ) . the remaining 30 subjects were treated with veriset hemostatic patch , but two of the 30 subjects did not complete the study . subjects underwent various types of surgical procedures , of which the most common were lymphadenectomy , esophagectomy , colectomy , and pancreatic bed surgery . concerning bleeding severity , 22 ( 73.3% ) subjects had a tbs categorized as type 2 , and the remaining eight ( 26.7% ) were type 3 . in 26/30 ( 86.7% ) subjects , conventional methods of obtaining hemostasis were attempted first but were deemed ineffective and , in the remaining four subjects , the investigator determined that conventional methods were impractical ( table 1 ) . in all of these subjects , veriset hemostatic patch was applied to the tbs . within 1 minute , 70.0% ( 21/30 ) of subjects had achieved hemostasis and , within 5 minutes , 96.7% ( 29/30 ) of subjects had achieved hemostasis ( table 3 ) . the median time to hemostasis was 1.0 minute ; only three subjects were still bleeding 2.0 minutes after application of the device ( figure 2 ) . in 27/30 ( 90.0% ) subjects , subject , the investigator opted to remove veriset hemostatic patch after 2 minutes because hemostasis had not been achieved , and an alternative topical agent was administered . in another subject , hemostasis was achieved with veriset hemostatic patch but , later in the procedure , it became detached during a gastric pull - up maneuver . , veriset hemostatic patch was later removed when the initial tbs was extracted along with an organ because of a procedural complications unrelated to the use of the device . a total of 136 aes were observed in 23/30 ( 76.7% ) subjects , and all were typical of the types of surgical procedures performed ( table 4 ) . all but one ( 99.3% ) of the aes were determined to have no relationship to treatment with veriset hemostatic patch . one subject died before the 7-day follow - up , but the cause , embolism from an atrial thrombus diagnosed prior to surgery , was unrelated to the use of veriset hemostatic patch . no device - related saes were observed for 30 days after the surgery , and no reoperations for device - related bleeding complications were performed within 5 postoperative days . a total of 37 subjects consented to and were enrolled in the study , but seven did not meet preoperative or intraoperative criteria and were considered screen failures ( figure 1 ) . the remaining 30 subjects were treated with veriset hemostatic patch , but two of the 30 subjects did not complete the study . subjects underwent various types of surgical procedures , of which the most common were lymphadenectomy , esophagectomy , colectomy , and pancreatic bed surgery . concerning bleeding severity , 22 ( 73.3% ) subjects had a tbs categorized as type 2 , and the remaining eight ( 26.7% ) were type 3 . in 26/30 ( 86.7% ) subjects , conventional methods of obtaining hemostasis were attempted first but were deemed ineffective and , in the remaining four subjects , the investigator determined that conventional methods were impractical ( table 1 ) . in all of these subjects , veriset hemostatic patch was applied to the tbs . within 1 minute , 70.0% ( 21/30 ) of subjects had achieved hemostasis and , within 5 minutes , 96.7% ( 29/30 ) of subjects had achieved hemostasis ( table 3 ) . the median time to hemostasis was 1.0 minute ; only three subjects were still bleeding 2.0 minutes after application of the device ( figure 2 ) . in 27/30 ( 90.0% ) subjects , subject , the investigator opted to remove veriset hemostatic patch after 2 minutes because hemostasis had not been achieved , and an alternative topical agent was administered . in another subject , hemostasis was achieved with veriset hemostatic patch but , later in the procedure , it became detached during a gastric pull - up maneuver . , veriset hemostatic patch was later removed when the initial tbs was extracted along with an organ because of a procedural complications unrelated to the use of the device . a total of 136 aes were observed in 23/30 ( 76.7% ) subjects , and all were typical of the types of surgical procedures performed ( table 4 ) . all but one ( 99.3% ) of the aes were determined to have no relationship to treatment with veriset hemostatic patch . one subject died before the 7-day follow - up , but the cause , embolism from an atrial thrombus diagnosed prior to surgery , was unrelated to the use of veriset hemostatic patch . no device - related saes were observed for 30 days after the surgery , and no reoperations for device - related bleeding complications were performed within 5 postoperative days . in certain situations , these existing products exhibit a supportive effect in reaching sufficient hemostasis , but there is still room for improvement in their efficacy , ease of use , and adherence to anatomical tissue . veriset hemostatic patch promotes hemostasis by serving as a tamponade to physically stem blood flow while concentrating platelets and other clotting factors at the bleeding site to accelerate the coagulation cascade . in this study , a variety of procedure types were performed that were accompanied by soft tissue bleeding from multiple sources . veriset hemostatic patch stopped blood loss in almost all of these situations , a finding that supports its potential broad range of use . veriset hemostatic patch was deemed ineffective in only one subject whose bleeding site was determined to have an unusual geometry that did not allow sufficient contact with the device . multiple applications of veriset hemostatic patch are feasible to accommodate either especially large bleeding sites or more difficult to treat bleeding sites . in response to uncontrolled bleeding in the operating room , surgeons typically start with traditionally available methods to achieve hemostasis , such as compression , electrocautery , or direct suture application.24 if this initial attempt is unsuccessful , either these methods are used repetitively , or more expensive ( and typically more effective ) methods are used until bleeding is halted.25 achieving hemostasis in this manner may not always be in the best interest of the patient with regard to both medical and economic outcomes . opting to use veriset hemostatic patch as an initial hemostatic agent , instead of attempting more conventional methods first , may save time by allowing quick control of bleeding with little interruption to the surgical procedure and minimal risk of complications . in the two cases in which veriset hemostatic patch either did not adhere or became detached after application , there was no visible tissue damage . this observation suggests that , unlike some conventional hemostatic methods that can cause tissue damage ( ie , electrocautery),26,27 the use of veriset hemostatic patch can be attempted with little risk to the patient . the results shown here support the use of veriset hemostatic patch across a variety of surgical procedures that can result in soft tissue bleeding . additionally , the median time to hemostasis in this study was 1 minute , the same as that observed in the randomized liver study;21 this similarity indicates that veriset hemostatic patch might offer consistent efficacy across multiple tissues . further studies , particularly in the form of randomized , controlled trials , should be performed in the future to assess such characteristics as safety , efficacy , usability ( eg , adhesiveness to tissue , flexibility to underlying structures ) , and cost - effectiveness , compared to other topical hemostats on the market . while these data would be beneficial to the surgical community , our results combined with the previously published liver results , suggest that veriset hemostatic patch offers a safe hemostatic method that is effective in many surgical situations .

the pulmonary arterial and venous structures deliver deoxygenated blood to the lung periphery and return oxygenated blood to the systemic circulation . these highly complex branching structures support the primary function of the lung that is to bring blood into close proximity with incoming fresh gas delivered to the terminal air sacs ( alveoli ) through the process of respiration . in clinical practice , it is of great importance , for instance , to be able to characterize the vascular trees for the detection of pulmonary emboli ( localized blockages ) , detection of signs of pulmonary hypertension , and for the differentiation between vasculature and focal opacities ( for detection of lung cancer and other localized pathologies ) . the vascular trees can also serve as a roadmap for the tracking of lung tissues across lung volume changes or across time as the lung is serially monitored . recent advances in mdct scanner technology enable the scanning of the entire lung with nearly isotropic submillimeter voxel dimensions ( on the order of 0.4 mm ) . vessel segments with radii of 2 mm or less are readily detectible in those images . while detectible , manual segmentation of these complex tree structures , even if an individual were willing to take the time , has been found to present a near impossible task due to the following reasons . it is difficult to determine boundaries of a vessel consistently , especially thin segments , due to the partial volume effects and image noise . volumetric lung scans of the adult human consist of more than 500 slices and the vascular tree , in a bipodial fashion , rapidly branch as one tracks the vessels from their central to peripheral locations , with the full tree structure consisting of more than 23 generations . in addition , manual measurements of the vessels for assessment of diameters and branching angles are unreliable . the measurement of diameter requires the determination of cross - sectional planes perpendicular to the local segment centerlines . similarly , a plane that includes both parent and child segments around the branchpoint needs to be localized for branching angle measurements . both measurements are difficult to perform manually via 2d images because of problems of for - shortening in projected view . therefore , highly automated segmentation of the pulmonary vascular tree based on 3d image analysis plays an important role in detecting and characterizing the vessel structure . the segmentation results are sought as we seek to build a lung atlas in which we will establish normative values against which an individual can be compared for the detection of disease . there is great interest in identifying branchpoints of the vascular trees as a set of landmarks that may allow matching of the lung across volume changes . during a respiratory cycle , even though the registration may require separation of arterial and venous trees , segmentation of the entire vascular trees provides branchpoint information required for this purpose . eventually the arterial and venous trees can readily be separated by scanning during the infusion of iodinated contrast agent and scanning during the arterial phase of the infusion . tube enhancement filters based on a combination of the eigenvalues of a hessian matrix have been reported in [ 35 ] . reported a vessel segmentation algorithm based on a codimension two level set method . vasilevskiy and siddiqi used gradient flows implemented using a level set method for 2d and 3d vessel segmentation . a tracking direction was estimated by an eigenvector of the hessian matrix at each tracking front position . used cores [ 12 , 13 ] to track the vascular tree from a seed point . reported a method for vascular tree segmentation using correlation - based enhancement filters and a fuzzy shape representation of the data . an approach based on mathematical morphology and discrete geometry operators use of vascular tree segmentation for detection , segmentation , and analysis of pulmonary lobes and sublobes was presented in . the eigenvalues and eigenvectors of the hessian matrix are implicitly or explicitly used in some of the above algorithms and the algorithms have worked well for extracting vessels in several organ systems imaged by ct or mr . the tube enhancement filters can extract both thin segments and thick segments without using seed points . however , such filters produce disconnections around the junctions since they are based on a cylindrical vessel segment model . segmentation results obtained from vessel traversal algorithms generally have better connectivity between segments but often miss peripheral thin segments . our goal outlined in this paper has been to develop an algorithm that extracts peripheral thin segments as well as thick segments from thoracic ct images with better connectivity . the major contribution of the reported work is the development of an algorithm which extracts detailed pulmonary vascular trees by a novel integration of the tube enhancement filter and vessel traversal approaches . our approach builds on several previously developed and proven methods for vessel enhancement and vessel traversal . however , when applying these algorithms individually to extract pulmonary vessels , several additional issues need to be resolved , which is the topic of this paper . the presented method integrates existing conceptual modules in a way that the final approach is free of the inherent limitations of the individual building blocks . our integrated algorithm consists of three major steps : ( 1 ) tube enhancement based on the cylindrical shape model using an eigenvalue of the hessian matrix serves as a filter to extract vessels and to produce information that is used to determine a set of seed points in the following vessel traversal step . ( 2 ) the traversal step starts from each seed point until one of the eigenvalues of the hessian matrix changes its sign twice , signifying that the front point of a trajectory has reached a junction . ( 3 ) branchpoint analysis is accomplished by applying a thinning method which then allows for the selection of objects with many branchpoints , serving as a means of distinguishing between vascular trees and noise components . a vessel segment in a 3d image is often modeled as having a cylindrical shape with a 2d gaussian - like intensity distribution within its cross - sectional plane . a combination of the eigenvalues of the hessian matrix is often used to characterize and enhance its shape in the image . since pulmonary vessels consist of segments with a wide range of radii , a multiscale approach needs to be considered . in this vessel enhancement process , segment radius information for multiscale integration . based on the cylindrical vessel model , the eigenvalues of the hessian matrix are commonly employed recently as efficient criteria to differentiate tube structures from other image components . let three eigenvalues of the hessian matrix at a point be 1 , 2 , and 3 and let their corresponding eigenvectors be e1 , e2 , and e3 , respectively . when a point is close to the center of a segment , 1 and 2 take on large negative values whereas 3 takes on a small value . figure 1 illustrates a cylindrical vessel model , with the eigenvalues / eigenvectors at the center of the model . according to the model , the following criteria are typically used to construct a filter output function . elongation : 3 is much less than 1 and 2 ( 3/1 0 or 3/2 0 ) . symmetry : 1 and 2 are of almost the same values when a point is close enough to the center of a segment ( 2/1 1 ) . these criteria capture the characteristics of the model so as to differentiate tube structures from sheet and blob shaped structures when a point is within a vessel segment and close to its center . however , pulmonary vascular trees include many junctions , and criteria 2 and 3 above are not always satisfied around the branchpoints . 1 and 2 do not always satisfy criterion 3 above , especially in the thin segments and around the junctions . in addition , 3 sometimes takes large positive values even when a point is within a straight segment , which implies criterion 2 is not always met . figure 2(a ) shows a trajectory by a white tube starting from a point demarcated by the left most white arrow . the gray parts on the trajectory indicate the points where 3 takes on negative values . figure 2(b ) shows changes of the three eigenvalues of the hessian matrix as the front point of the trajectory advances . intensity gradually changes towards junctions when the point is in a straight segment and it may cause 3 to take on a positive value . when the point is around a junction , intensity decreases rapidly towards the segmental direction and it causes 3 to take on a negative value . for these reasons , 3 fluctuates along the trajectory . also , 2 oscillates with its phase opposite to 3 since 2 takes relatively smaller values when the point is around junctions . based on this observation , the filter output function f(x ) is defined as follows : ( 1)f(x)=max fsf22i(x ) , where f is the standard deviation of a gaussian function convoluted with an image so as to take second derivatives in the volume coordinate , and i(x ) is the intensity value at the point . thus , it may also enhance blob structures , typically image noise as well as cylindrical structures . according to our experience , in thoracic ct images , the noise will not be enhanced to the same degree as are vessels since contrast between parenchymal background and pulmonary vessels is relatively high compared to the noise . ( hounsfield units ) while lung parenchyma is typically between 800 and 900 h.u . since gaussian noise with standard deviation = 20 can be considered typical for ct images , a difference greater than 100 h.u . f(x ) takes a maximum value when f is the closest to the radius of a target segment among other f in the range s. therefore , s should include appropriate values that cover all the radii of the vessel segments in the lung . however , when s contains a wide range of values , and the filter output is calculated at a nonvessel point close to a junction or multiple thin segments are close to each other , f(x ) gets larger than it should . this is caused by a gaussian function with a large f that excessively smoothes the region . figure 3(b ) shows the result when a fixed range of s was used for all voxels in the image . the filter overly extracted nonvessel regions around the junction . in order to avoid the inappropriate enhancement , large f should be included in s only when it needs to be used for the detection of a thick segment . we applied thresholding with a fixed value to obtain thick segments , and a discrete distance transform is applied to estimate an approximate radius r. by using a distance transform value d , the radius r is estimated as r = d+1 - 1 [ voxel ] . we used 600 [ h.u . ] for both tlc and frc scans as the threshold value . using the radius information , range s is determined depending on the distance d as follows : ( 2)s={{1},0<r1,{1,2,2},1<r2,{1,2,2,22},2<r22,{1,2,2,22,4},22<r . the filter output function takes large values not only at the tube structures but also at the edges of large concave structures such as bones and the heart . in addition , the function takes a relatively long time to be calculated for a whole volume . for simplicity , a lung segmentation method based on intensity thresholding and 3d labeling is employed . obviously any existing lung segmentation algorithms such as can also be used for this purpose . they should be located in the left lung , right lung and trachea , respectively . both left and right lungs are extracted by a simple thresholding with a fixed value and 3d labeling with the two seed points in the lung . the trachea and two or three subsequent branches are also segmented by thresholding with a fixed value and 2d labeling based upon connectivity between neighboring slices and the seed point . although the segmentation results from this very simple approach often contains part of the mediastinal region , it significantly reduces the number of voxels outside of the lung which would have otherwise been processed in the filtering step . it also greatly reduces the possibility of extracting false vessels caused by structures outside of the lung region . in our experiments , it took well under a minute to perform the lung segmentation while the lung segmentation served to reduce the tube enhancement filter processing time by more than four minutes . initial segmentation can be obtained by thresholding the filter output . however , since the segmentation works on a voxel - by - voxel basis , the result contains local disconnections and small holes . these are caused mainly by image noise and the difference of the intensity distribution on a cross - sectional plane between straight segments and junctions . the cross - sectional shape of a vessel contour at a junction becomes an ellipse . it causes lower filter output and local disconnections around junctions , especially where very thin segments bifurcate from a thick segment . the vessel traversal approach is suitable for improving connectivity and for filling small holes in a segment . the connectivity improvements include the following processes : initialization including automated localization of seed points , tracking terminations at junctions , radius estimation for boundary recovery . since points near the center of a segment are generally less influenced by noise , seed points are preferentially identified at segment centers . according to the vessel model , the intensity function takes a local maximum at the center of a segment . the local maximum position xmax in the volume coordinate can then be calculated by the following equation : ( 3)xmax = x+p = x(i , e1)1e1(i , e2)2e2 , where i is a gradient vector at x , e1 and e2 are eigenvectors corresponding to the eigenvalues 1 and 2 . the operator ( , ) takes the inner product of the two vectors in the equation . i is estimated by using f which maximizes ( 1 ) in s ( i.e. , the gradient is calculated as a convolution of the first derivative of a 3d gaussian function and f ) . since estimation of xmax is sensitive to image noise , x is considered to be located close enough to the center when the following conditions are met : ( 4)px0.5 , py0.5 , pz0.5 , where px , py , and pz are the components of the vector p. nonetheless , many points in the airway wall are also included as tracking seed points if only these conditions are taken into account , because the local intensity structure on the airway wall is quite similar to that of a thin segment . many of those points in the airway wall can be differentiated since the cross - sectional shape at the points is elliptical compared to that of the thin vessel segments . since ( 3 ) is based on the cylindrical vessel model , estimation of the local maximum position is inaccurate if a point is close to a junction . in that case if the eigenvalues satisfy conditions ( 4 ) , ( 5 ) and ( 6 ) , then the point is considered to be the center of a vessel segment and is registered as a seed point for the tracking process . starting from a seed point , the front position of a trajectory advances in the continuous 3d space of the volume coordinate according to the estimated tangent direction with the step size of 0.2 voxel . at the seed point , 3 is a positive value since the seed point satisfies condition ( 6 ) . as the front position advances , 3 takes a negative value around the branchpoints and becomes positive when the front is remote from the junctions . thus , once a segment is connected to its parent segment , tracking can be terminated . this will prevent performing the tracking multiple times for a segment . at a seed point , one of them leads to a thicker segment and the other leads to a peripheral child branch . local disconnection is often observed at a junction of a thick and a thin segment . therefore , the tracking front should advance towards the thick segment in order to fill the potential gap between the two segments . since a thick segment generally has higher ct values , the intensity value at the terminated point can be the criterion for selecting the trajectory which leads to the thicker segment . after tracking in both directions , the trajectory whose intensity value at the termination point is higher than the other is selected for radius estimation and the other is discarded . after tracking , spheres with an estimated radius are drawn at each tracking front position to fill possible holes and gaps between two segments . the intensity value around a point x can be estimated using the gradient i and the hessian matrix by the following quadratic equation ; ( 7)i(x+x)i(x)+(i,x)+12(x , hx ) . we determined x as the radius of the sphere at the point when the estimated intensity value i(x + x ) reaches half value of i(x ) from background value . intensity decreases toward both the e1 and e2 directions and it tends to decrease faster along either e1 or e1 compared with any other direction since the absolute value of 1 is bigger than 2 . therefore , x is made parallel to e1 to estimate the smallest distance to the outside of the vessel from the point . then , x can be expressed as x = te1 , where t is the minimum absolute value of the following equation : ( 8)t=(i , e1)(i , e1)21(i(x)ibg)1 . the background value is determined by ibg = min i(x iopte1 ) where i = 1,2 , 3 and opt is the optimal f which maximizes the filter output function ( 1 ) at the closest voxel from x. opt at each voxel can be obtained in the vessel enhancement step and is reused to avoid expensive multiscale filtering in this step . the hessian matrix and gradient vector are calculated by convoluting the second- and first- order derivative of gaussian function with opt . the intensity value i(x ) is obtained by convoluting gaussian function with opt at x , and ibg is evaluated directly from the image using trilinear interpolation . the minimum estimated radius along a trajectory is used for drawing spheres at each positions on the trajectory . an object that contains small numbers of branchpoints can be considered as noise or part of other structures . therefore , a thinning algorithm is applied to each object in the result to obtain the number of branchpoints in the object from its graph representation . empirically determined , an object which has 100 or more branchpoints , is extracted as a vascular tree . the value 100 is chosen empirically by taking into account that the thinning algorithm may produce false branches . figure 5 shows the surface model of the tree structure and a cross - sectional image of one of the noisy phantom instances . this model was originally developed to represent an airway tree , yet it is appropriate also for use as a model for simulating a pulmonary vascular tree since the pulmonary arterial tree follows the airway tree out into the lung periphery and thus has the same general geometric relationships as the airway tree . each segment was characterized by a starting point , an end point and the associated radius . 3d gaussian spheres were moved along the linear segments to generate the tree structure in a 3d image whose dimension was 250 250 250 voxels . the radius of a segment decreases from the starting point to the end point of a segment so that it becomes the same as the radius of a child segment in order to allow smooth connection . standard deviation of the 3d gaussian spheres r changes as the radius varies . in this phantom setting , maximum r was six voxels and minimum r was approximately one voxel in the volume . we also rotated the model in 11 different angles in 3d space and generated the structure in 3d images . intensity value at the center of a segment mildly decreased from the starting point of a segment toward its end point along the tangent direction . ] at the center whereas the thinnest had a value of 700 [ h.u . the background value was 900 [ h.u . ] to simulate a typical background intensity in the parenchymal region in lung ct images . gaussian noise with a standard deviation = 20 , 30 , 40 was added to each image . after the result was obtained , a thinning method was applied to obtain its graph representation so as to evaluate how many branches were correctly extracted and how many false branches were extracted by the segmentation algorithm . both missing and extra branches were counted manually . the segmentation algorithm was applied to 44 ct scans from a total of 22 human subjects with lung volume held at 85% ( for simplicity referred to as total lung capacity or tlc ) and 20% ( referred to functional residual capacity or frc ) of the subject 's vital capacity . both tlc and frc scans subjects were healthy volunteers except for three who had mild chronic obstructive pulmonary disease ( copd ) judged both by pulmonary function tests and visual assessment of the ct images . all images were scanned by a 4-slice mdct scanner ( philips mx8000 , philips medical systems , cleveland , ohio ) . in - plane pixel sizes of the ct images ranged from 0.52 mm to 0.88 mm , slice thickness was 1.3 mm , and slice increment was 0.65 mm . approximately 550 and 500 slices per case were available for each tlc and frc scan , respectively . in order to evaluate the segmentation results quantitatively , more than 1000 points in the vessels were manually identified in each ct data set to form a validation set to assess the true positive ( tp ) rate . tp rate is defined as the ratio between the total number of points detected by the algorithm and the total number of points in the data set . we consistently chose the center point of the vessels as a member of the tp point set because the center line is more important than vessel borders in defining the vessel geometry . each lung was divided into four distinct regions in terms of radial distance from the hilum so as to avoid biased distribution of the test points . region a is the closest to the hilum and region d is close to the pleural surface . region a contains a greater number of thicker segments than the other regions . to evaluate the false positive ( fp ) rate , 150 points were randomly placed in each region , and the points within the vessels were manually eliminated such that all points represented nonvessel regions . this left approximately 120 points per region which were located in the parenchyma , airway walls and pulmonary fissures . false positive rate is defined as the ratio between the total number of points included by the segmentation result and the total number of points in the data set . we implemented this algorithm with a multithreaded process for the tube enhancement filter and the vessel traversal . since the filter works on a voxel - by - voxel basis , it can be processed in parallel . similarly , the vessel traversal from a seed point can be executed in parallel without depending on other seed point locations . other processes such as lung segmentation and connected component analysis were implemented as serial processes . we used a linux - based computer with dual xeon 3.6 ghz processors ( hyperthreading on ) and 4 gb of memory for our experiment . the processing time varies depending on the size of the lung to be processed . therefore , tlc scans take longer time than processing frc scans . it took less than one minute ( 30 to 50 seconds ) to obtain lung segmentation result . then it took about one to three minutes to complete the tube enhancement filter process . the memory requirement is on the order of three times that required to hold the original volume . in our implementation , the program required approximately 900 mb for processing a volume consisting of 512 512 595 voxels . the segmentation algorithm has one major parameter , the threshold of the filter output used to obtain the initial segmentation . when it is decreased , more vessels and noise elements are extracted , increasing the tp rate as well as the fp rate . on the contrary , less vessels ( and noise elements ) are extracted if the threshold is increased , causing a lowering of the tp and fp rates . since the threshold is a tradeoff between tp and fp rates , four thresholds were selected empirically and were tested by use of the simulation . figure 7 shows average number of false ( extra ) branches and missing branches as a function of the threshold value . when the threshold setting was 0.06 , the segmentation results were missing less than one branch per volume on average . however , the number of extra branches increased rapidly when noise levels went up . on the contrary , when the threshold was 0.09 , the results missed 3.5 to 4.5 out of 125 branches on average whereas they had less extra branches than other threshold settings . in , a noise level = 20 was used to represent typical noise in ct images . for comparison , when the threshold was 0.07 , the algorithm missed less than one branch on average for all noise levels and the results contained one or less extra branches in the case of = 20 , 30 . derived from this result , a threshold value of 0.07 was used for the following experiments using the clinical data sets . figure 8 shows volume - rendered images of the segmentation results from both tlc and frc scans of one subject . since the scale of these images is the same , they also show how the lung geometry changes between the two volumes scanned . table 1 shows a summary of the tp rate for both tlc and frc scans . tp rate for tlc scans was 99.6% from the total of 16933 validation points and 99.5% from 15,281 points in right and left lungs , respectively . similarly , tp rate for frc scans was 99.0% from 13356 points and 98.2% from 10646 points in right and left lungs , respectively . since the lung volume of frc scans is less than that of tlc scans , there were less validation points for frc data sets . also , especially in the frc scans due to increased compliance , motion artifacts may have contributed to a small increase in the error rate . table 2 shows a summary of the fp rates for both tlc and frc scans . a total of 11,925 points and 9620 points were used in right and left lung of the tlc scans , respectively , yielding fp rates of 1.21% in the right and 0.99% in the left lung . for frc scans , a total of 11752 points in right lung and 8722 points in left lung were used and fp rates of 0.96% and 0.88% in the right and left lungs were obtained . in this section , the parameters affecting segmentation results will be discussed first followed by the performance difference between the left and right lungs . we will also discuss the difference of the validation results between tlc and frc scans . the threshold for obtaining initial segmentation from the filter output influences the final result when set close to zero , more objects including noise and other structures are obtained ; less vessel segments and less noise result for larger threshold values . , four different values were used to determine an appropriate value for pulmonary vascular segmentation from clinical ct images . 0.07 was empirically determined to be the best choice and was used for in vivo human images ; 0.08 was shown to be an alternative based on the results of the simulation . when 0.08 is used for in vivo human lung images , the tp rate was 99.3% and 97.0% for tlc and frc scans , respectively . in tlc images , the tp rate decreased merely 0.3% whereas the fp rate dropped by 0.45% . the tp rate in the region a for both tlc and frc scans was lower than in the other regions . this region also contains thin segments bifurcating from the thick segments , some of which were missed by the segmentation . radii of those missing segments were typically 1 to 2 mm and they emanated from segments whose radii were 5 to 8 mm . vessel traversal was sometimes terminated prematurely before the front merged to the centerline of the thick segments . in addition , some data sets had cardiogenic motion artifacts , particularly in the left lung regions , which caused branches to be missed . anatomically , many arterial segments run along with the airway segments , and sometimes they appear attached to each other in the ct image . complete elimination of the airway wall may require a priori knowledge of the location of the airway tree . an integration of the airway tree segmentation [ 21 , 22 ] will likely solve this problem . the lung is less dense at tlc than frc and parenchymal density at tlc is more homogeneous . in frc scans , parenchymal density increases and the density of the parenchyma increases typically by 100 to 200 hounsfield units compared to that in the tlc scans . the image quality of frc scans is also degraded by motion artifacts caused largely by cardiogenic motion which is accentuated by the fact that the lung is more compliant . the motion artifacts are more prominent in the left lung both because the heart is usually leftward shifted within the thorax . the overall tp rate in the frc scans was less than that in the tlc scans by 0.6% . the pulmonary arterial tree feeds the pulmonary capillaries which in turn drain into the pulmonary veins . the segmentation results demonstrated here contain only one or two connected trees in each lung , inferring that they may be implicitly connected in some locations . the total number of generations in the segmented trees can serve as a clinically important index . it is difficult to automatically estimate the number of generations in the segmented trees because of the implicit connections . the visual evaluation of the segmentation results indicates inclusion of peripheral thin segments close to the pleural surface . the algorithm , as currently completed and described , can be useful for clinical applications which do not require arterial and venous separation such as the detection of pulmonary emboli , the evaluation of the pulmonary vasculature in pulmonary hypertension and in the characterization of pulmonary nodules . an interesting byproduct of the vascular tree segmentation is that it depicts the pulmonary fissures as regions void of the vessel segments . pulmonary fissures are visible structures in the lung , which separate each lung into lobes . lobe segmentation has required fissure detection based upon identification of the fissure itself as has been reported in [ 25 , 26 ] . rough localization of pulmonary fissures using the vascular tree segmentation results avoids detection of the fissure itself and can be performed by searching in the sparse region of the vessel segments . this helps in limiting the region of interest to search for exact pulmonary fissure locations and may help to provide a fissure definition in the cases where the actual fissure is incomplete . the main purpose of the presented paper was to report on a practical method for extraction of pulmonary vessels together with its validation on clinical ct images . development of a simple yet practical tube enhancement filter output function ( 1 ) . we have developed an algorithm to extract the pulmonary vascular trees from thoracic 3d ct images . the algorithm was applied to 44 volumetric ct scans consisting of 22 high volume scans and 22 low volume scans . it yielded 99.6% tp rate and 1.2% fp rate for the high volume scans , 98.6% tp rate and 0.9% fp rate for the low volume scans . the values for the tp and fp evaluations of these in vivo human data sets show a reliable performance and suitability of the proposed algorithm for future clinically relevant applications .

this paper describes an algorithm for extracting pulmonary vascular trees ( arteries plus veins ) from three - dimensional ( 3d ) thoracic computed tomographic ( ct ) images . the algorithm integrates tube enhancement filter and traversal approaches which are based on eigenvalues and eigenvectors of a hessian matrix to extract thin peripheral segments as well as thick vessels close to the lung hilum . the resultant algorithm was applied to a simulation data set and 44 scans from 22 human subjects imaged via multidetector - row ct ( mdct ) during breath holds at 85% and 20% of their vital capacity . a quantitative validation was performed with more than 1000 manually identified points selected from inside the vessel segments to assess true positives ( tps ) and 1000 points randomly placed outside of the vessels to evaluate false positives ( fps ) in each case . on average , for both the high and low volume lung images , 99% of the points was properly marked as vessel and 1% of the points were assessed as fps . our hybrid segmentation algorithm provides a highly reliable method of segmenting the combined pulmonary venous and arterial trees which in turn will serve as a critical starting point for further quantitative analysis tasks and aid in our overall goal of establishing a normative atlas of the human lung .

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

the pulmonary arterial and venous structures deliver deoxygenated blood to the lung periphery and return oxygenated blood to the systemic circulation . these highly complex branching structures support the primary function of the lung that is to bring blood into close proximity with incoming fresh gas delivered to the terminal air sacs ( alveoli ) through the process of respiration . in clinical practice , it is of great importance , for instance , to be able to characterize the vascular trees for the detection of pulmonary emboli ( localized blockages ) , detection of signs of pulmonary hypertension , and for the differentiation between vasculature and focal opacities ( for detection of lung cancer and other localized pathologies ) . the vascular trees can also serve as a roadmap for the tracking of lung tissues across lung volume changes or across time as the lung is serially monitored . vessel segments with radii of 2 mm or less are readily detectible in those images . while detectible , manual segmentation of these complex tree structures , even if an individual were willing to take the time , has been found to present a near impossible task due to the following reasons . it is difficult to determine boundaries of a vessel consistently , especially thin segments , due to the partial volume effects and image noise . volumetric lung scans of the adult human consist of more than 500 slices and the vascular tree , in a bipodial fashion , rapidly branch as one tracks the vessels from their central to peripheral locations , with the full tree structure consisting of more than 23 generations . in addition , manual measurements of the vessels for assessment of diameters and branching angles are unreliable . therefore , highly automated segmentation of the pulmonary vascular tree based on 3d image analysis plays an important role in detecting and characterizing the vessel structure . there is great interest in identifying branchpoints of the vascular trees as a set of landmarks that may allow matching of the lung across volume changes . during a respiratory cycle , even though the registration may require separation of arterial and venous trees , segmentation of the entire vascular trees provides branchpoint information required for this purpose . eventually the arterial and venous trees can readily be separated by scanning during the infusion of iodinated contrast agent and scanning during the arterial phase of the infusion . tube enhancement filters based on a combination of the eigenvalues of a hessian matrix have been reported in [ 35 ] . reported a vessel segmentation algorithm based on a codimension two level set method . a tracking direction was estimated by an eigenvector of the hessian matrix at each tracking front position . the eigenvalues and eigenvectors of the hessian matrix are implicitly or explicitly used in some of the above algorithms and the algorithms have worked well for extracting vessels in several organ systems imaged by ct or mr . the tube enhancement filters can extract both thin segments and thick segments without using seed points . however , such filters produce disconnections around the junctions since they are based on a cylindrical vessel segment model . our goal outlined in this paper has been to develop an algorithm that extracts peripheral thin segments as well as thick segments from thoracic ct images with better connectivity . the major contribution of the reported work is the development of an algorithm which extracts detailed pulmonary vascular trees by a novel integration of the tube enhancement filter and vessel traversal approaches . however , when applying these algorithms individually to extract pulmonary vessels , several additional issues need to be resolved , which is the topic of this paper . the presented method integrates existing conceptual modules in a way that the final approach is free of the inherent limitations of the individual building blocks . our integrated algorithm consists of three major steps : ( 1 ) tube enhancement based on the cylindrical shape model using an eigenvalue of the hessian matrix serves as a filter to extract vessels and to produce information that is used to determine a set of seed points in the following vessel traversal step . ( 2 ) the traversal step starts from each seed point until one of the eigenvalues of the hessian matrix changes its sign twice , signifying that the front point of a trajectory has reached a junction . ( 3 ) branchpoint analysis is accomplished by applying a thinning method which then allows for the selection of objects with many branchpoints , serving as a means of distinguishing between vascular trees and noise components . a combination of the eigenvalues of the hessian matrix is often used to characterize and enhance its shape in the image . based on the cylindrical vessel model , the eigenvalues of the hessian matrix are commonly employed recently as efficient criteria to differentiate tube structures from other image components . let three eigenvalues of the hessian matrix at a point be 1 , 2 , and 3 and let their corresponding eigenvectors be e1 , e2 , and e3 , respectively . when a point is close to the center of a segment , 1 and 2 take on large negative values whereas 3 takes on a small value . symmetry : 1 and 2 are of almost the same values when a point is close enough to the center of a segment ( 2/1 1 ) . these criteria capture the characteristics of the model so as to differentiate tube structures from sheet and blob shaped structures when a point is within a vessel segment and close to its center . however , pulmonary vascular trees include many junctions , and criteria 2 and 3 above are not always satisfied around the branchpoints . the gray parts on the trajectory indicate the points where 3 takes on negative values . figure 2(b ) shows changes of the three eigenvalues of the hessian matrix as the front point of the trajectory advances . based on this observation , the filter output function f(x ) is defined as follows : ( 1)f(x)=max fsf22i(x ) , where f is the standard deviation of a gaussian function convoluted with an image so as to take second derivatives in the volume coordinate , and i(x ) is the intensity value at the point . thus , it may also enhance blob structures , typically image noise as well as cylindrical structures . according to our experience , in thoracic ct images , the noise will not be enhanced to the same degree as are vessels since contrast between parenchymal background and pulmonary vessels is relatively high compared to the noise . f(x ) takes a maximum value when f is the closest to the radius of a target segment among other f in the range s. therefore , s should include appropriate values that cover all the radii of the vessel segments in the lung . however , when s contains a wide range of values , and the filter output is calculated at a nonvessel point close to a junction or multiple thin segments are close to each other , f(x ) gets larger than it should . we applied thresholding with a fixed value to obtain thick segments , and a discrete distance transform is applied to estimate an approximate radius r. by using a distance transform value d , the radius r is estimated as r = d+1 - 1 [ voxel ] . for both tlc and frc scans as the threshold value . although the segmentation results from this very simple approach often contains part of the mediastinal region , it significantly reduces the number of voxels outside of the lung which would have otherwise been processed in the filtering step . it also greatly reduces the possibility of extracting false vessels caused by structures outside of the lung region . in our experiments , it took well under a minute to perform the lung segmentation while the lung segmentation served to reduce the tube enhancement filter processing time by more than four minutes . the cross - sectional shape of a vessel contour at a junction becomes an ellipse . the vessel traversal approach is suitable for improving connectivity and for filling small holes in a segment . according to the vessel model , the intensity function takes a local maximum at the center of a segment . the operator ( , ) takes the inner product of the two vectors in the equation . i is estimated by using f which maximizes ( 1 ) in s ( i.e. , the gradient is calculated as a convolution of the first derivative of a 3d gaussian function and f ) . since estimation of xmax is sensitive to image noise , x is considered to be located close enough to the center when the following conditions are met : ( 4)px0.5 , py0.5 , pz0.5 , where px , py , and pz are the components of the vector p. nonetheless , many points in the airway wall are also included as tracking seed points if only these conditions are taken into account , because the local intensity structure on the airway wall is quite similar to that of a thin segment . many of those points in the airway wall can be differentiated since the cross - sectional shape at the points is elliptical compared to that of the thin vessel segments . since ( 3 ) is based on the cylindrical vessel model , estimation of the local maximum position is inaccurate if a point is close to a junction . in that case if the eigenvalues satisfy conditions ( 4 ) , ( 5 ) and ( 6 ) , then the point is considered to be the center of a vessel segment and is registered as a seed point for the tracking process . starting from a seed point , the front position of a trajectory advances in the continuous 3d space of the volume coordinate according to the estimated tangent direction with the step size of 0.2 voxel . at a seed point , one of them leads to a thicker segment and the other leads to a peripheral child branch . local disconnection is often observed at a junction of a thick and a thin segment . since a thick segment generally has higher ct values , the intensity value at the terminated point can be the criterion for selecting the trajectory which leads to the thicker segment . the intensity value around a point x can be estimated using the gradient i and the hessian matrix by the following quadratic equation ; ( 7)i(x+x)i(x)+(i,x)+12(x , hx ) . we determined x as the radius of the sphere at the point when the estimated intensity value i(x + x ) reaches half value of i(x ) from background value . intensity decreases toward both the e1 and e2 directions and it tends to decrease faster along either e1 or e1 compared with any other direction since the absolute value of 1 is bigger than 2 . therefore , x is made parallel to e1 to estimate the smallest distance to the outside of the vessel from the point . then , x can be expressed as x = te1 , where t is the minimum absolute value of the following equation : ( 8)t=(i , e1)(i , e1)21(i(x)ibg)1 . the background value is determined by ibg = min i(x iopte1 ) where i = 1,2 , 3 and opt is the optimal f which maximizes the filter output function ( 1 ) at the closest voxel from x. opt at each voxel can be obtained in the vessel enhancement step and is reused to avoid expensive multiscale filtering in this step . the hessian matrix and gradient vector are calculated by convoluting the second- and first- order derivative of gaussian function with opt . therefore , a thinning algorithm is applied to each object in the result to obtain the number of branchpoints in the object from its graph representation . empirically determined , an object which has 100 or more branchpoints , is extracted as a vascular tree . this model was originally developed to represent an airway tree , yet it is appropriate also for use as a model for simulating a pulmonary vascular tree since the pulmonary arterial tree follows the airway tree out into the lung periphery and thus has the same general geometric relationships as the airway tree . each segment was characterized by a starting point , an end point and the associated radius . the radius of a segment decreases from the starting point to the end point of a segment so that it becomes the same as the radius of a child segment in order to allow smooth connection . standard deviation of the 3d gaussian spheres r changes as the radius varies . intensity value at the center of a segment mildly decreased from the starting point of a segment toward its end point along the tangent direction . ] after the result was obtained , a thinning method was applied to obtain its graph representation so as to evaluate how many branches were correctly extracted and how many false branches were extracted by the segmentation algorithm . the segmentation algorithm was applied to 44 ct scans from a total of 22 human subjects with lung volume held at 85% ( for simplicity referred to as total lung capacity or tlc ) and 20% ( referred to functional residual capacity or frc ) of the subject 's vital capacity . both tlc and frc scans subjects were healthy volunteers except for three who had mild chronic obstructive pulmonary disease ( copd ) judged both by pulmonary function tests and visual assessment of the ct images . in order to evaluate the segmentation results quantitatively , more than 1000 points in the vessels were manually identified in each ct data set to form a validation set to assess the true positive ( tp ) rate . tp rate is defined as the ratio between the total number of points detected by the algorithm and the total number of points in the data set . we consistently chose the center point of the vessels as a member of the tp point set because the center line is more important than vessel borders in defining the vessel geometry . region a is the closest to the hilum and region d is close to the pleural surface . to evaluate the false positive ( fp ) rate , 150 points were randomly placed in each region , and the points within the vessels were manually eliminated such that all points represented nonvessel regions . false positive rate is defined as the ratio between the total number of points included by the segmentation result and the total number of points in the data set . we implemented this algorithm with a multithreaded process for the tube enhancement filter and the vessel traversal . similarly , the vessel traversal from a seed point can be executed in parallel without depending on other seed point locations . we used a linux - based computer with dual xeon 3.6 ghz processors ( hyperthreading on ) and 4 gb of memory for our experiment . the processing time varies depending on the size of the lung to be processed . then it took about one to three minutes to complete the tube enhancement filter process . in our implementation , the program required approximately 900 mb for processing a volume consisting of 512 512 595 voxels . the segmentation algorithm has one major parameter , the threshold of the filter output used to obtain the initial segmentation . when it is decreased , more vessels and noise elements are extracted , increasing the tp rate as well as the fp rate . on the contrary , less vessels ( and noise elements ) are extracted if the threshold is increased , causing a lowering of the tp and fp rates . figure 7 shows average number of false ( extra ) branches and missing branches as a function of the threshold value . when the threshold setting was 0.06 , the segmentation results were missing less than one branch per volume on average . on the contrary , when the threshold was 0.09 , the results missed 3.5 to 4.5 out of 125 branches on average whereas they had less extra branches than other threshold settings . for comparison , when the threshold was 0.07 , the algorithm missed less than one branch on average for all noise levels and the results contained one or less extra branches in the case of = 20 , 30 . figure 8 shows volume - rendered images of the segmentation results from both tlc and frc scans of one subject . since the scale of these images is the same , they also show how the lung geometry changes between the two volumes scanned . table 1 shows a summary of the tp rate for both tlc and frc scans . since the lung volume of frc scans is less than that of tlc scans , there were less validation points for frc data sets . also , especially in the frc scans due to increased compliance , motion artifacts may have contributed to a small increase in the error rate . table 2 shows a summary of the fp rates for both tlc and frc scans . a total of 11,925 points and 9620 points were used in right and left lung of the tlc scans , respectively , yielding fp rates of 1.21% in the right and 0.99% in the left lung . the threshold for obtaining initial segmentation from the filter output influences the final result when set close to zero , more objects including noise and other structures are obtained ; less vessel segments and less noise result for larger threshold values . , four different values were used to determine an appropriate value for pulmonary vascular segmentation from clinical ct images . 0.07 was empirically determined to be the best choice and was used for in vivo human images ; 0.08 was shown to be an alternative based on the results of the simulation . when 0.08 is used for in vivo human lung images , the tp rate was 99.3% and 97.0% for tlc and frc scans , respectively . in tlc images , the tp rate decreased merely 0.3% whereas the fp rate dropped by 0.45% . vessel traversal was sometimes terminated prematurely before the front merged to the centerline of the thick segments . complete elimination of the airway wall may require a priori knowledge of the location of the airway tree . an integration of the airway tree segmentation [ 21 , 22 ] will likely solve this problem . in frc scans , parenchymal density increases and the density of the parenchyma increases typically by 100 to 200 hounsfield units compared to that in the tlc scans . the pulmonary arterial tree feeds the pulmonary capillaries which in turn drain into the pulmonary veins . the segmentation results demonstrated here contain only one or two connected trees in each lung , inferring that they may be implicitly connected in some locations . the total number of generations in the segmented trees can serve as a clinically important index . the visual evaluation of the segmentation results indicates inclusion of peripheral thin segments close to the pleural surface . the algorithm , as currently completed and described , can be useful for clinical applications which do not require arterial and venous separation such as the detection of pulmonary emboli , the evaluation of the pulmonary vasculature in pulmonary hypertension and in the characterization of pulmonary nodules . an interesting byproduct of the vascular tree segmentation is that it depicts the pulmonary fissures as regions void of the vessel segments . pulmonary fissures are visible structures in the lung , which separate each lung into lobes . lobe segmentation has required fissure detection based upon identification of the fissure itself as has been reported in [ 25 , 26 ] . rough localization of pulmonary fissures using the vascular tree segmentation results avoids detection of the fissure itself and can be performed by searching in the sparse region of the vessel segments . the main purpose of the presented paper was to report on a practical method for extraction of pulmonary vessels together with its validation on clinical ct images . development of a simple yet practical tube enhancement filter output function ( 1 ) . we have developed an algorithm to extract the pulmonary vascular trees from thoracic 3d ct images . the algorithm was applied to 44 volumetric ct scans consisting of 22 high volume scans and 22 low volume scans . it yielded 99.6% tp rate and 1.2% fp rate for the high volume scans , 98.6% tp rate and 0.9% fp rate for the low volume scans . the values for the tp and fp evaluations of these in vivo human data sets show a reliable performance and suitability of the proposed algorithm for future clinically relevant applications .

interest in age - friendly community and aging - in - place concepts has grown over recent years as the majority of older adults wish to remain within the neighborhood in which they have become familiar and emotionally attached to . social capital is relevant to aging - in - place as it provides a framework to examine neighborhoods ' fit for older adults ; this has important implications for adapting neighborhoods to become more age - friendly and ensure opportunity to aging - in - place . hence , social capital has gained increased attention from both practice and policy makers . the research has demonstrated a positive relationship between social capital and wide range of health outcomes . in other words , individuals with high social capital however , most of the research to date has been done on middle - aged adults . it is still not clearly understood how social capital functions in relation to health as adults reach very old age . the goal of this paper was to elucidate this relationship by examining the association between five indicators of social capital with three physical health outcomes among adults aged 65 years and older . the use of social capital is increasingly recognized as a means for adapting environments for aging - in - place . as emlet and moceri argue , an elder friendly community captures the core features of social capital including civic participation , and the nature of social networks and mutuality / reciprocity [ 4 , p. 2 ] . despite the growing interest in social capital therefore , it is important to fully clarify how social capital was conceptualized in this study . four out of the five social capital indicators used in this study focused on neighborhood ( neighborhood trust , neighborhood support , neighborhood cohesion , and neighborhood participation ) , while the fifth measure ( telephone interaction ) captured community connections beyond the neighborhood . therefore , social capital in this study should be considered to be primarily a measure of the neighborhood , with the understanding that social connections can stretch beyond physical borders . putnam 's definition was used for this study : features of social organization such as networks , norms , and social trust that facilitate coordination and cooperation for mutual benefit . there were several reasons for using putnam 's definition ; primarily putnam 's conceptualization of social capital has a communitarian focus . a fundamental aspect to communitarianism is the establishment and maintenance of social norms that are fundamental to making communities stronger . strong communities enable healthier populations ; and so putnam 's definition has been widely applied in the health related literature . since health was the key focus of this study , putnam 's definition of social capital was considered appropriate here . finally , many of the indicators of social capital available in the dataset used for this study mirror those used to measure social capital as conceptualized by putnam . previous research has used a number of these same indicators to reflect social capital [ 1 , 9 ] . the five indicators used in this study ( namely , neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) have been used widely in the social capital and health literature . social capital has been shown to have significant positive associations with a vast array of health outcomes for all ages , including older adults [ 3 , 10 ] . kawachi and colleagues found in a number of studies that indicators of social capital are linked to a wide range of health conditions , such as mortality , life - expectancy , self - rated health , cancer , cardiovascular disease , obesity , diabetes , and functional limitations . social capital may be particularly important for older adults because this group is more tethered to their immediate surroundings ( and so ) the impact of the environment is likely greater [ 11 , p. 253 there is growing support for the positive impact of social capital on the health of older adults , even among the oldest - old . in their study , nyqvist and colleagues found that the structural dimension of social capital ( i.e. , social networks , social integration , and attachment ) was important for depressive symptoms among the oldest - old aged 85 and older . the authors argued that this dimension of social capital might be particularly important for this cohort due to their increasing physical frailty and reduced social networks . therefore , the examination of the relationship between structural dimensions of social capital and health , when conducted across cohorts that represent the changing lifespan of elders ( young - old ( 6574 years ) , the middle - old ( 7584 years ) , and the oldest - old ( 85 + years ) ) , can shed new light on how networks grow , develop , and adapt for this population . research on whether there are changes in the level of social capital over the lifespan is limited . research by axler and colleagues suggested that social capital was the lowest among older adults ( aged 60 years and over ) compared to younger cohorts . in terms of varying dimensions of social capital , some research has found a decrease as people live into very old age , such as size of social networks and frequency of contacts , and civic participation as older adults become less physically active . it is not clear what occurs with other aspects of social capital such as sense of belonging and trust as older adults age . place attachment , a concept closely related to sense of belonging , does seem to increase with age . however , important physical environmental factors , such as crime , may diminish the sense of belonging . furthermore , the impact of age on the relationship between social capital and health remains poorly understood . as cagney and wen point out : models of the social capital - health relationship must be attentive to age [ 11 , p. 239 , the effect of age on the relationship between social capital and health is an important public health query . by understanding the role of age in the relationship between social capital and health , interventions can be developed to optimize the fit between older adults and their community as they age in place . by partitioning social capital into five indicators , this study was focused on examining how separate dimensions of the social environment can act as determinants of health for older adults , particularly during the latter part of their lifespan . by examining these indicators separately , it is hoped that this will provide a more multifaceted understanding of how elders may feel about their sense of belonging in their neighborhoods and beyond . the relationship among five indicators of social capital ( neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) which were examined with three health outcomes : self - rated health , activities of daily living ( adl ) , and instrumental activities of daily living ( iadl ) . analyses were conducted to examine differences across three cohorts , the young - old ( 6574 years ) , the middle - old ( 7584 years ) , and the oldest - old ( 85 + years ) . in order to understand the relationship between social capital and health by age , this study was designed to investigate two questions : ( 1 ) does the level of social capital change with age ? and ( 2 ) does the relationship between social capital and health outcomes change with increasing age ? for the first research question we hypothesized that older adults will possess lower levels of certain indicators of social capital ( specifically , participation and telephone interaction ) while other indicators will increase ( specifically , trust , cohesion , and support ) . as some components of social capital are lost or gained with increasing age , we hypothesized that certain indicators of social capital will have an increased or decreased association with health as individuals progress to a later stage in their lifespan . specifically , social capital indicators , neighborhood trust , neighborhood cohesion , and neighborhood support , which mirror cognitive dimensions of social capital , will more likely be significantly associated with health among the very old ( i.e. , oldest - old cohort ) compared to younger cohorts . this assumption is based on previous research carried out with the oldest - old , where cognitive dimensions of social capital were found to become increasingly important . the other social capital indicators , neighborhood participation and telephone interaction , will be less likely associated with health outcomes into late adulthood compared to younger cohorts . as described previously , neighborhood participation and telephone interaction have been found to decrease with increased frailty in old age [ 13 , 14 ] . this study used cross - sectional data from the 2010 community health data base ( chdb ) managed by philadelphia health management corporation . the survey has been conducted biennially since 1994 in five urban and suburban counties of southeastern pennsylvania ( bucks , chester , delaware , montgomery , and philadelphia ) . persons aged 60 and older were oversampled for the analysis , representing over 700,000 older adults in the area . if a randomly selected adult respondent was unable to be interviewed because of health impairments or language barriers , the interview was conducted with an adult proxy . all respondents who had an adult proxy ( n = 14 , < 1% ) respond for them were removed from the sample as it was considered important to gain first - hand information from respondents themselves . the sample consisted of 2,344 adults ( 65 years and older ) from a five - county southeastern pennsylvania region . just over half ( 53% ) belonged to the young - old cohort , 36% belonged to the middle - old cohort , and 11% belonged to the oldest - old cohort . there were significant differences across the three cohorts across all socioeconomic indicators except by gender . the oldest - old cohort was significantly more likely to be white , have a lower level of education , be poor , and live alone . marital status varied across cohorts , where older cohorts reported higher percentages of widowed status ( table 1 ) . when comparing this complete sample of older adults to peers nationwide , the percent of older adults ( 14.5% ) was slightly higher than national figures ( 13% ) . however , it should be noted that pennsylvania ( 15.4% ) had the fourth largest proportion of older adults in 2010 . the complete sample was similar to elders nationwide in terms of age ( 74.8 years and 74.0 years , resp . ) . however , generally there were fewer females , minority , and lower level of educational attainment among elders nationwide compared to the sample examined in this study . poverty ( @ 100% fpl ) among elders nationwide ( 9.0% ) was higher than the complete sample ( 7.6% ) . however , there was considerable variation in poverty level in the complete sample by county . this was measured by a single item that asked to rate health on a 5-point likert scale , with a high number indicating better self - rated health . adl reflected eight basic activities that generally occur within the home ( i.e. , eating , dressing , grooming , walking , transferring , bathing , continence , and soiling ) . iadl measured more complex tasks needed for the adls that generally require interaction with the surrounding environment ( i.e. , talking on the phone , walking , shopping , meal preparation , housework , taking medicine , and handling money ) . for this study , adl and iadl scales were dichotomized with 0 representing no adl / iadl limitations and 1 representing 1 or more adl / iadl limitations . in this study , only 10.7% had 1 or more adl and 25.4% had 1 or more iadl , similar to national statistics from other large - scale studies from the cdc and the us census . conceptually , having 1 or more limitations with adls or iadls can have a direct impact on an older person 's ability to age in place . the five social capital indicators were originally derived from the social capital community benchmark survey . please rate how likely people in your neighborhood are willing to help their neighbors with routine activities such as picking up their trash cans , or helping to shovel snow . would you say that most people in your neighborhood are always , often , sometimes , rarely , or never willing to help their neighbors ? response categories were recoded from 1 to 4 , with 1 being rarely / never , 2 being sometimes , 3 being often , and 4 being always . neighborhood participation . this was assessed by how many local groups or organizations in your neighborhood do you currently participate in such as social , political , religious , school - related , or athletic organizations ? responses categories ranged from 0 to 12 groups . due to very small number of cases in category responses 6 and higher , this variable was top - coded so response categories ranged from 0 to 6 . neighborhood cohesion . please tell me if you strongly agree , agree , disagree , or strongly disagree with the following statement : i feel that i belong and am a part of my neighborhood . responses categories were coded from 1 to 4 with 1 being strongly disagree , 2 being disagree , 3 being agree , and 4 being strongly agree . please tell me if you strongly agree , agree , disagree , or strongly disagree with the following statement : most people in my neighborhood can be trusted . response categories were also coded from 1 to 4 with 1 being strongly disagree , 2 being disagree , 3 being agree , and 4 being strongly agree . telephone interaction . this was assessed by about how often do you talk with friends or relatives on the telephone ? response categories included several times a day , once a day , a few times a week , once a week , less often than once a week , and never . responses categories were recoded from 1 to 4 , with 1 being once a week or less , 2 being few times a week , 3 being once a day , and 4 being several times a day . demographic and socioeconomic variables entered into the analyses were age in years ranging from 65 and higher ; sex , 0 representing female and 1 representing male ; race , a dichotomized variable with 0 representing white and 1 representing minority , ( minority includes all nonwhite plus all hispanics of any race ) ; education , coded along 5 response categories : less than high school graduate ( 011 years ) , high school graduate ( 12 years ) , some college ( 1315 years ) , and postcollege ( more than 16 years ) with high school ( 16 years ) as comparison group ; poverty at 200% of the federal poverty line dichotomized into poor ( coded 1 ) and nonpoor ( coded 0 ) ; marital status , recoded into 4 dummy variables ( single , divorced , and widowed with married as comparison group ) ; and living arrangements , a dichotomized variable representing living alone ( coded 0 ) versus living with others ( coded 1 ) . poverty at 200% was used because it represented a more reasonable cut - off for poverty than 100% . the first research question was tested using kruskal - wallis rank sum tests on the five social capital indicators ( neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) with the sample ( > 65 yrs ) split along by three cohorts ( 6574 , 7584 , and 85 and older ) . cohort analysis was used to examine differences across the lifespan of older adults , where important differences may emerge for the oldest - old ( 85 and older ) , who are the most at risk . the second research question was tested using binary logistic ( adl and iadl ) and ordinal logistic regression ( self - rated health ) , analyses for each of the health outcomes as separate dependent variables for each of the three cohorts . findings suggest that age did not play a role in terms of possession of social capital except for neighborhood support ( table 1 ) . results from kruskal - wallis rank sum tests showed significant differences across cohorts for the indicator neighborhood support . the rank sum was the highest for the youngest group and became gradually lower as respondents aged ( 6574 : 1,240,000 ; 7584 : 887,372 ; 85 + : 251,588 ) , suggesting that support from neighbors as an indicator of social capital deteriorates as participants progressed to a later lifespan . significant differences were found on all four health outcomes across the three groups , with worsening health profiles as age increased . significant differences were found between the young - old and oldest - old for self - rated health . significant differences in terms of sociodemographic characteristics were also found across groups ( table 3 ) . as age increased , the sociodemographic profile was more likely to be white , less educated , poor , and living alone . tables 4 , 5 , and 6 showed the results of the logistic regressions carried out for each of the health outcomes across the three cohorts . generally the fit of these regression models was significant for health outcomes across all three groups , except for self - rated health and adl among the oldest - old . despite the limited variance explained by the models , the results showed a complex set of associations between social capital indicators and health outcomes that changed with age . all of the social capital indicators , except telephone interaction , were significantly associated with all of the health outcomes . specifically , for the health outcome self - rated health ( table 4 ) neighborhood cohesion improved health among the young - old ( or = 1.31 , p < 0.05 ) , while neighborhood trust ( or = 1.87 , p < 0.001 ) and neighborhood participation ( or = 1.14 , p < 0.05 ) improved health among the middle - old . for the middle - old , respondents who reported greater neighborhood trust ( or = 0.63 , p < 0.05 ) and neighborhood participation ( or = 0.74 , p < 0.05 ) were around 30% less likely to have an adl limitation ( table 5 ) . no significant associations were observed for social capital indicators among the young - old for adl . in terms of iadl limitations ( table 6 ) , the oldest - old group with more neighborhood cohesion were 50% less likely to have an iadl limitation ( or = 0.45 , p < 0.05 ) . on the other hand , those who were 85 and older who reported higher levels of neighborhood trust were twice as likely to report an iadl limitation ( or = 2.00 , p < 0.05 ) . social capital indicators were not observed to be significant with iadl limitations for the two other cohorts . we hypothesized that certain indicators of social capital would increase ( namely , neighborhood trust , neighborhood cohesion , and neighborhood support ) or decrease ( namely , neighborhood participation and telephone interaction ) with increasing age . the trends in the percentage breakdowns did show some support for this ( table 2 ) . for example , the oldest - old reported lower levels of both neighborhood participation and telephone interaction than their younger cohorts . also , the oldest - old also reported higher levels of trust than younger cohorts . these findings add to the limited literature on the change in social capital over the lifespan . in terms of support , our findings found the perception that neighbors are willing to help did increase to a certain age . according to our study , what is interesting to note about this cohort is that they possessed high ( or higher ) levels of neighborhood support , neighborhood cohesion , and neighborhood trust compared to the other two cohorts . this could suggest that the neighborhood , in terms of support , cohesion , and trust , may be particularly meaningful for this cohort . other indicators of social capital examined in this study ( i.e. , neighborhood participation and telephone interaction ) did not follow this trend . for example , the middle - old cohort reported the lowest frequency of telephone interactions with friends , relatives , and neighbors , although this difference was not significant . the variation in possession of different indicators of social capital by cohort could be used in terms of developing communities that maximize the potential for successful aging - in - place ; such policy implications are described further below . logistic regressions for each of the four health outcomes showed that different indicators of social capital continued to be significant predictors of health outcomes even when accounting for standard sociodemographic predictors ; however , the nature of these associations changed with increasing age . with respect to the second hypothesis , our expectation was supported in the fact that different indicators of social capital were relevant to health as age increased . we had specifically predicted that the social capital indicators , neighborhood trust , neighborhood cohesion , and neighborhood support , would more likely be associated with improved health among those of advanced age ( i.e. , the oldest - old cohort ) compared with the younger cohorts ( i.e. , young - old ) . this prediction did bear out for iadl with respect to neighborhood trust and neighborhood cohesion for the oldest - old . however , the relationship was in the opposite direction with neighborhood trust . it is likely that this finding reflects endogeneity in the relationship of neighborhood trust and reporting an iadl limitation ; elders who are among the oldest and most vulnerable must trust the people around them to even be able to seek out help for complex tasks with negotiating the outside world . we also predicted that the social capital indicators , neighborhood participation and telephone interaction , would be less likely to be significant with increasing age . the results indicated that there was no relationship to health for these variables . albeit the fact that social capital explained a very small part of the overall variance of each of the health outcomes , what is important to derive from these findings is how different aspects of the social environment matter for different health outcome at different stages of life . specifically , neighborhood trust was only significant among the middle - old cohort for self - rated health and adl , whereas neighborhood trust had an unexpected relationship with iadl limitations for the oldest - old . neighborhood cohesion was significant for the young - old for self - rated health whereas neighborhood cohesion was significant for the oldest - old in terms of iadl . neighborhood support was significant for the oldest - old in terms of self - rated health . finally , neighborhood participation was only significant for the middle - old for self - rated health and adl . this mixed pattern of associations suggests that all parts of the social environment play a role across the aging lifespan and for different indicators of health . this is a significant finding suggesting that the social environment is a critical factor to be considered when adapting settings for aging - in - place . this also highlights the importance of looking at social capital in terms of multiple indicators rather than creating a single factor . furthermore , these findings also suggest that the age structure of the community also needs to be taken into account [ 11 , p. 251 ] ; in other words , the social environment must fit the needs of the age demographics . the nature of the relationship between the five social capital indicators and the three health outcomes may provide some interesting insight into the meaning of these health outcomes in relation to the social environment . compared to adl and iadl , the relationship between social capital and self - rated health was noted across all three age groups . both self - rated health and social capital are highly subjective concepts ; and it is possible that subjective processes applied to each of these domains ( i.e. , self - rated health and social capital ) influence one another . in terms of functional health , social capital was significant only among the oldest - old for iadl whereas it was significant only among the middle - old for adl . it is important to pay attention to which dimensions of the social environment mattered : among the oldest - old , neighborhood support and neighborhood trust were critical for iadl . among the middle - old , neighborhood trust and neighborhood participation were significant . although these patterns of relationships do make sense , it is difficult to explain why social capital did not matter for either adl or iadl for the youngest - old . perhaps grandparent / family responsibilities diminish as people move from young - old to middle - old age . this may give them more time to engage with neighbors ; and this builds familiarity ( experienced as trust and sense of belonging ) with the neighborhood . as kahn and antonucci convoy model explicates , relationships change over the lifespan that can impact health and well - being . more research is needed to better understand what and how older adults interact with their neighborhoods . the associations between social capital indicators and health outcomes were in the expected direction ; in other words , high levels of social capital indicators were associated with better health outcomes . there was one exception though , and that was with the relationship between neighborhood trust and iadl limitation among the oldest - old . among this cohort , it may be that individuals who experienced high levels of iadl limitations required help from neighbors and thus develop higher levels of trust through this interaction . this interpretation does bring up an important point regarding the need for caution when making assumptions about the direction of the relationship between social capital and health . the one indicator of social capital that was not significant for any of the four health outcomes examined in this study was telephone interaction . the principal author also found similar findings in a previous study ; in that study it was noted that it may be not the frequency of contact ( as was measured in this study ) but rather the level of support provided by the network that was the critical factor for physical and mental health . lack of significant association for telephone interaction with any of the three health outcomes in this study may provide some insight into social capital : it is quality of connection rather than quantity of connections within the neighborhood that is important for social capital to capture . this finding may also pertain to other dimensions of social capital , such as neighborhood participation . in other words , the quality of neighborhood participation ( i.e. , measuring the type of activity ) rather than the number of organizations that the individual is participating with might better capture the benefits of social capital . ultimately , the findings in this study suggest that more attention needs to be paid to the perception of community and the people living in these communities . indeed , the role of neighbors is likely to increase in the future as the availability of spouses or children to provide care for aging parents is expected to diminish . this is due to reduction in fertility rates , increased divorces rates , and low rates of remarriage [ 23 , 24 ] . it is hoped that the findings of this research may build on optimal environments for aging - in - place and elder - friendly communities by furthering our understanding of what dimensions of the social environment matter for health . as emlet and moceri have argued , there is a need to further elucidate the importance of social relationships and social connectedness with aging in place and in developing elder friendly communities ( [ 4 , p. 3 ] ) . it was the aim of this research to provide a comprehensive empirical examination of the fit between the aging individual and their surrounding environment in relation to health . the dynamic between the aging individual and the community in which they live is a complex one . although previous research has described an important role of the environment in which older adults live , this study has provided rich empirical evidence for this by examining relationships between multiple indicators of social capital and health across three cohorts . it is hoped that these findings will encourage the development of programs and interventions that allow for greater exposure to these important indicators of the social environment among older adults . indeed , the results suggest that there is opportunity for intervention at numerous points throughout the end of the lifespan , and different dimensions of the social environment may be more critical at different points of the lifespan . for example , this study showed that neighborhood support was important for self - rated health among the oldest - old . a possible means of building support could be through programs that help establish connections between neighbors who have volunteered to assist with those who need assistance . although this type of intervention may appear intrusive , the need for assistance among elders is increasingly recognized and accepted by elders themselves , as well as by family who live at a distance . additionally , neighbors may not mind helping others if they understand that there is a need , and this is part of the problem they can help to address in their proverbial backyards . such awareness has grown after infamous events such as the heat waves in chicago in 1995 and in paris in 2003 where rates of death were particularly high among the frail elderly . these events highlighted the need for greater awareness of and reaching out to frail neighbors . interventions directed at the oldest - old could also incorporate neighborhood trust and neighborhood cohesion , as these were indicators of social capital found to be critical for iadl of the oldest - old . interventions focused on the oldest - old are mentioned here , yet it is important to point out that each of the indicators of social capital ( except telephone interaction ) could be incorporated into interventions directed at the young - old and middle - old as well . from a lifespan perspective , an important question to ask here is when should interventions aimed at increasing social capital be made ? for example , should an effort be made to intervene during the middle - old aged years or earlier ? ideally interventions aimed at augmenting an individual 's social capital should be implemented early in life , during youth as research has shown that children that volunteer are more likely to volunteer in adulthood and later in life ( oesterle , johnson , and mortimer , 2004 ) . perhaps interventions aimed at building social capital specifically among older adults should be done at or before the point when retirement is being considered . it is possible to conceive of a program , which at the exit job interview puts the individual , who is soon to retire , in connection with volunteer organizations in the community . in this way , the program helps build a bridge between the individual and their community , and so the older adult can start to build social capital through volunteering in the community . when considering interventions aimed at building social capital , the geographic context should also be taken into account . the sample of older adults in this study resided is urban and suburban settings of five counties of southeastern pennsylvania . philadelphia , the county with the highest proportion of older adults ( namely , 39% of sample ) , represented a highly urbanized context while the other four counties reflected mostly suburban settings . these authors describe characteristics of urbanization such as crime , minority , inequality , and population stability that may influence social capital and health . specifically , pridmore and colleagues write , poor urban settlements require special interventions to address social exclusion , disruption of social networks and trust , insecurity and violence , and high mortality and morbidity [ 27 , p. i138 ] . ultimately , the context in which the individual lives requires consideration when developing interventions aimed at building social capital . furthermore , the level at which the intervention is to be provided must also be taken into account . for example , the approach will be different if the intervention is to occur at the individual or microlevel , city or mesolevel , or the macrolevel . an important limitation of this study is that the data is based on a cross - sectional design . most likely the association between social capital and health is bidirectional , as has been demonstrated in previous research . some additional limitations to be considered when interpreting the findings : first and foremost , size of the sample for the oldest - old was fairly small ( n = 266 ) compared to the young - old ( n = 1233 ) and the middle - old ( n = 845 ) . small sample size of the oldest - old cohort may have limited the power of the statistical significance tests . this may explain the nonsignificant models for self - rated health and adl for oldest - old . those examined in this study represented those who were by default healthier and had chosen to stay aging in place . another limitation was that the definition and measurement of social capital remain elusive . as was found in this study , not all the measures used were associated with health , specifically telephone interaction . we continue to need a better understanding of how to best measure social capital . furthermore , as nyqvist and colleagues have stated : , it is generally assumed that social capital indicators measure the same thing in different groups and places [ 12 , p. 105 ] . however , research has demonstrated that there are marked differences in the questions about social capital that are considered appropriate for various groups depending , for instance , on the subjects ' age . it is also important to note that the effect sizes for the analyses were somewhat low , given the small r findings . however , because this study employed nonparametric techniques for estimation , it can be problematic to rely too heavily on these measures of linear fit . despite these concerns , the results do clearly indicate that community - level neighborhood social capital does have a relationship to health for elders , even when including important individual - level factors . this speaks to the mezzo- and macrolevel implications of these findings and warrants further investigation in research . this research clearly calls for use of longitudinal data as this could provide answers regarding causality as age increases , in the hope of developing age - sensitive interventions across the lifespan . these findings also need to be replicated with larger sample sizes , especially for the oldest - old group . furthermore , as highlighted above , the geographic context needs to be given greater focus in future research . also , in response to nyqvist and colleagues ' concerns , there is a need for social capital scale development that reflects the specific characteristics of older persons . furthermore , questions asking whether residents plan to move in the near future and why should be included in order to get a better understanding of how the social environment might impact decisions leading to moving away from one 's neighborhood . finally , based on the findings of this study certain dimensions of the social environment ( namely , trust , cohesion , support , and participation ) however , the question still remains as how does one build , for example , trust and cohesion ? successful implementation of programs that build social capital should also take into consideration individual characteristics , such as gender , race , and economic status that may influence the relationship between social capital and health . finally , impact of social capital on the individual needs to be examined in the light of lawton 's environmental docility and environmental proactivity distinction . in other words , as age progresses how do different dimensions of the social environment either benefit or impede functioning ? ultimately , there continues to be a need to refine the person - environment fit model over the lifespan .

research is needed to examine the connection between older adults and their community as they age . this is important as increasing numbers of older adults wish to age in place . regression models were examined across 3 cohorts testing relationships among social capital indicators ( neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) with health outcomes ( self - rated health , activities of daily living ( adl ) , and instrumental activities of daily living ( iadl ) ) . results showed that most social capital indicators remained significant for all health outcomes into very old age . development of tools for individual and community interventions to ensure optimal fit between the aging individual and their environment is discussed , along with recommendations for enhancing social work theory and practice .

1. Introduction 2. Methods 3. Sample 4. Measures 5. Data Analysis 6. Results 7. Discussion 8. Conclusions

interest in age - friendly community and aging - in - place concepts has grown over recent years as the majority of older adults wish to remain within the neighborhood in which they have become familiar and emotionally attached to . social capital is relevant to aging - in - place as it provides a framework to examine neighborhoods ' fit for older adults ; this has important implications for adapting neighborhoods to become more age - friendly and ensure opportunity to aging - in - place . hence , social capital has gained increased attention from both practice and policy makers . the research has demonstrated a positive relationship between social capital and wide range of health outcomes . in other words , individuals with high social capital however , most of the research to date has been done on middle - aged adults . it is still not clearly understood how social capital functions in relation to health as adults reach very old age . the goal of this paper was to elucidate this relationship by examining the association between five indicators of social capital with three physical health outcomes among adults aged 65 years and older . as emlet and moceri argue , an elder friendly community captures the core features of social capital including civic participation , and the nature of social networks and mutuality / reciprocity [ 4 , p. 2 ] . despite the growing interest in social capital therefore , it is important to fully clarify how social capital was conceptualized in this study . four out of the five social capital indicators used in this study focused on neighborhood ( neighborhood trust , neighborhood support , neighborhood cohesion , and neighborhood participation ) , while the fifth measure ( telephone interaction ) captured community connections beyond the neighborhood . putnam 's definition was used for this study : features of social organization such as networks , norms , and social trust that facilitate coordination and cooperation for mutual benefit . the five indicators used in this study ( namely , neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) have been used widely in the social capital and health literature . social capital has been shown to have significant positive associations with a vast array of health outcomes for all ages , including older adults [ 3 , 10 ] . kawachi and colleagues found in a number of studies that indicators of social capital are linked to a wide range of health conditions , such as mortality , life - expectancy , self - rated health , cancer , cardiovascular disease , obesity , diabetes , and functional limitations . social capital may be particularly important for older adults because this group is more tethered to their immediate surroundings ( and so ) the impact of the environment is likely greater [ 11 , p. 253 there is growing support for the positive impact of social capital on the health of older adults , even among the oldest - old . the authors argued that this dimension of social capital might be particularly important for this cohort due to their increasing physical frailty and reduced social networks . therefore , the examination of the relationship between structural dimensions of social capital and health , when conducted across cohorts that represent the changing lifespan of elders ( young - old ( 6574 years ) , the middle - old ( 7584 years ) , and the oldest - old ( 85 + years ) ) , can shed new light on how networks grow , develop , and adapt for this population . research on whether there are changes in the level of social capital over the lifespan is limited . research by axler and colleagues suggested that social capital was the lowest among older adults ( aged 60 years and over ) compared to younger cohorts . in terms of varying dimensions of social capital , some research has found a decrease as people live into very old age , such as size of social networks and frequency of contacts , and civic participation as older adults become less physically active . it is not clear what occurs with other aspects of social capital such as sense of belonging and trust as older adults age . as cagney and wen point out : models of the social capital - health relationship must be attentive to age [ 11 , p. 239 , the effect of age on the relationship between social capital and health is an important public health query . by understanding the role of age in the relationship between social capital and health , interventions can be developed to optimize the fit between older adults and their community as they age in place . by partitioning social capital into five indicators , this study was focused on examining how separate dimensions of the social environment can act as determinants of health for older adults , particularly during the latter part of their lifespan . the relationship among five indicators of social capital ( neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) which were examined with three health outcomes : self - rated health , activities of daily living ( adl ) , and instrumental activities of daily living ( iadl ) . analyses were conducted to examine differences across three cohorts , the young - old ( 6574 years ) , the middle - old ( 7584 years ) , and the oldest - old ( 85 + years ) . and ( 2 ) does the relationship between social capital and health outcomes change with increasing age ? for the first research question we hypothesized that older adults will possess lower levels of certain indicators of social capital ( specifically , participation and telephone interaction ) while other indicators will increase ( specifically , trust , cohesion , and support ) . as some components of social capital are lost or gained with increasing age , we hypothesized that certain indicators of social capital will have an increased or decreased association with health as individuals progress to a later stage in their lifespan . specifically , social capital indicators , neighborhood trust , neighborhood cohesion , and neighborhood support , which mirror cognitive dimensions of social capital , will more likely be significantly associated with health among the very old ( i.e. the other social capital indicators , neighborhood participation and telephone interaction , will be less likely associated with health outcomes into late adulthood compared to younger cohorts . as described previously , neighborhood participation and telephone interaction have been found to decrease with increased frailty in old age [ 13 , 14 ] . just over half ( 53% ) belonged to the young - old cohort , 36% belonged to the middle - old cohort , and 11% belonged to the oldest - old cohort . when comparing this complete sample of older adults to peers nationwide , the percent of older adults ( 14.5% ) was slightly higher than national figures ( 13% ) . however , it should be noted that pennsylvania ( 15.4% ) had the fourth largest proportion of older adults in 2010 . however , generally there were fewer females , minority , and lower level of educational attainment among elders nationwide compared to the sample examined in this study . this was measured by a single item that asked to rate health on a 5-point likert scale , with a high number indicating better self - rated health . conceptually , having 1 or more limitations with adls or iadls can have a direct impact on an older person 's ability to age in place . the five social capital indicators were originally derived from the social capital community benchmark survey . would you say that most people in your neighborhood are always , often , sometimes , rarely , or never willing to help their neighbors ? response categories were recoded from 1 to 4 , with 1 being rarely / never , 2 being sometimes , 3 being often , and 4 being always . neighborhood cohesion . responses categories were coded from 1 to 4 with 1 being strongly disagree , 2 being disagree , 3 being agree , and 4 being strongly agree . demographic and socioeconomic variables entered into the analyses were age in years ranging from 65 and higher ; sex , 0 representing female and 1 representing male ; race , a dichotomized variable with 0 representing white and 1 representing minority , ( minority includes all nonwhite plus all hispanics of any race ) ; education , coded along 5 response categories : less than high school graduate ( 011 years ) , high school graduate ( 12 years ) , some college ( 1315 years ) , and postcollege ( more than 16 years ) with high school ( 16 years ) as comparison group ; poverty at 200% of the federal poverty line dichotomized into poor ( coded 1 ) and nonpoor ( coded 0 ) ; marital status , recoded into 4 dummy variables ( single , divorced , and widowed with married as comparison group ) ; and living arrangements , a dichotomized variable representing living alone ( coded 0 ) versus living with others ( coded 1 ) . the first research question was tested using kruskal - wallis rank sum tests on the five social capital indicators ( neighborhood trust , neighborhood support , neighborhood cohesion , neighborhood participation , and telephone interaction ) with the sample ( > 65 yrs ) split along by three cohorts ( 6574 , 7584 , and 85 and older ) . cohort analysis was used to examine differences across the lifespan of older adults , where important differences may emerge for the oldest - old ( 85 and older ) , who are the most at risk . the second research question was tested using binary logistic ( adl and iadl ) and ordinal logistic regression ( self - rated health ) , analyses for each of the health outcomes as separate dependent variables for each of the three cohorts . findings suggest that age did not play a role in terms of possession of social capital except for neighborhood support ( table 1 ) . the rank sum was the highest for the youngest group and became gradually lower as respondents aged ( 6574 : 1,240,000 ; 7584 : 887,372 ; 85 + : 251,588 ) , suggesting that support from neighbors as an indicator of social capital deteriorates as participants progressed to a later lifespan . significant differences were found between the young - old and oldest - old for self - rated health . tables 4 , 5 , and 6 showed the results of the logistic regressions carried out for each of the health outcomes across the three cohorts . generally the fit of these regression models was significant for health outcomes across all three groups , except for self - rated health and adl among the oldest - old . despite the limited variance explained by the models , the results showed a complex set of associations between social capital indicators and health outcomes that changed with age . all of the social capital indicators , except telephone interaction , were significantly associated with all of the health outcomes . specifically , for the health outcome self - rated health ( table 4 ) neighborhood cohesion improved health among the young - old ( or = 1.31 , p < 0.05 ) , while neighborhood trust ( or = 1.87 , p < 0.001 ) and neighborhood participation ( or = 1.14 , p < 0.05 ) improved health among the middle - old . for the middle - old , respondents who reported greater neighborhood trust ( or = 0.63 , p < 0.05 ) and neighborhood participation ( or = 0.74 , p < 0.05 ) were around 30% less likely to have an adl limitation ( table 5 ) . no significant associations were observed for social capital indicators among the young - old for adl . in terms of iadl limitations ( table 6 ) , the oldest - old group with more neighborhood cohesion were 50% less likely to have an iadl limitation ( or = 0.45 , p < 0.05 ) . social capital indicators were not observed to be significant with iadl limitations for the two other cohorts . we hypothesized that certain indicators of social capital would increase ( namely , neighborhood trust , neighborhood cohesion , and neighborhood support ) or decrease ( namely , neighborhood participation and telephone interaction ) with increasing age . for example , the oldest - old reported lower levels of both neighborhood participation and telephone interaction than their younger cohorts . according to our study , what is interesting to note about this cohort is that they possessed high ( or higher ) levels of neighborhood support , neighborhood cohesion , and neighborhood trust compared to the other two cohorts . this could suggest that the neighborhood , in terms of support , cohesion , and trust , may be particularly meaningful for this cohort . , neighborhood participation and telephone interaction ) did not follow this trend . logistic regressions for each of the four health outcomes showed that different indicators of social capital continued to be significant predictors of health outcomes even when accounting for standard sociodemographic predictors ; however , the nature of these associations changed with increasing age . with respect to the second hypothesis , our expectation was supported in the fact that different indicators of social capital were relevant to health as age increased . we had specifically predicted that the social capital indicators , neighborhood trust , neighborhood cohesion , and neighborhood support , would more likely be associated with improved health among those of advanced age ( i.e. this prediction did bear out for iadl with respect to neighborhood trust and neighborhood cohesion for the oldest - old . we also predicted that the social capital indicators , neighborhood participation and telephone interaction , would be less likely to be significant with increasing age . albeit the fact that social capital explained a very small part of the overall variance of each of the health outcomes , what is important to derive from these findings is how different aspects of the social environment matter for different health outcome at different stages of life . specifically , neighborhood trust was only significant among the middle - old cohort for self - rated health and adl , whereas neighborhood trust had an unexpected relationship with iadl limitations for the oldest - old . neighborhood cohesion was significant for the young - old for self - rated health whereas neighborhood cohesion was significant for the oldest - old in terms of iadl . neighborhood support was significant for the oldest - old in terms of self - rated health . finally , neighborhood participation was only significant for the middle - old for self - rated health and adl . this is a significant finding suggesting that the social environment is a critical factor to be considered when adapting settings for aging - in - place . this also highlights the importance of looking at social capital in terms of multiple indicators rather than creating a single factor . the nature of the relationship between the five social capital indicators and the three health outcomes may provide some interesting insight into the meaning of these health outcomes in relation to the social environment . compared to adl and iadl , the relationship between social capital and self - rated health was noted across all three age groups . both self - rated health and social capital are highly subjective concepts ; and it is possible that subjective processes applied to each of these domains ( i.e. , self - rated health and social capital ) influence one another . in terms of functional health , social capital was significant only among the oldest - old for iadl whereas it was significant only among the middle - old for adl . it is important to pay attention to which dimensions of the social environment mattered : among the oldest - old , neighborhood support and neighborhood trust were critical for iadl . among the middle - old , neighborhood trust and neighborhood participation were significant . this may give them more time to engage with neighbors ; and this builds familiarity ( experienced as trust and sense of belonging ) with the neighborhood . more research is needed to better understand what and how older adults interact with their neighborhoods . the associations between social capital indicators and health outcomes were in the expected direction ; in other words , high levels of social capital indicators were associated with better health outcomes . there was one exception though , and that was with the relationship between neighborhood trust and iadl limitation among the oldest - old . this interpretation does bring up an important point regarding the need for caution when making assumptions about the direction of the relationship between social capital and health . the one indicator of social capital that was not significant for any of the four health outcomes examined in this study was telephone interaction . lack of significant association for telephone interaction with any of the three health outcomes in this study may provide some insight into social capital : it is quality of connection rather than quantity of connections within the neighborhood that is important for social capital to capture . this finding may also pertain to other dimensions of social capital , such as neighborhood participation . , measuring the type of activity ) rather than the number of organizations that the individual is participating with might better capture the benefits of social capital . this is due to reduction in fertility rates , increased divorces rates , and low rates of remarriage [ 23 , 24 ] . as emlet and moceri have argued , there is a need to further elucidate the importance of social relationships and social connectedness with aging in place and in developing elder friendly communities ( [ 4 , p. 3 ] ) . it was the aim of this research to provide a comprehensive empirical examination of the fit between the aging individual and their surrounding environment in relation to health . the dynamic between the aging individual and the community in which they live is a complex one . although previous research has described an important role of the environment in which older adults live , this study has provided rich empirical evidence for this by examining relationships between multiple indicators of social capital and health across three cohorts . it is hoped that these findings will encourage the development of programs and interventions that allow for greater exposure to these important indicators of the social environment among older adults . for example , this study showed that neighborhood support was important for self - rated health among the oldest - old . additionally , neighbors may not mind helping others if they understand that there is a need , and this is part of the problem they can help to address in their proverbial backyards . interventions directed at the oldest - old could also incorporate neighborhood trust and neighborhood cohesion , as these were indicators of social capital found to be critical for iadl of the oldest - old . interventions focused on the oldest - old are mentioned here , yet it is important to point out that each of the indicators of social capital ( except telephone interaction ) could be incorporated into interventions directed at the young - old and middle - old as well . ideally interventions aimed at augmenting an individual 's social capital should be implemented early in life , during youth as research has shown that children that volunteer are more likely to volunteer in adulthood and later in life ( oesterle , johnson , and mortimer , 2004 ) . perhaps interventions aimed at building social capital specifically among older adults should be done at or before the point when retirement is being considered . in this way , the program helps build a bridge between the individual and their community , and so the older adult can start to build social capital through volunteering in the community . when considering interventions aimed at building social capital , the geographic context should also be taken into account . the sample of older adults in this study resided is urban and suburban settings of five counties of southeastern pennsylvania . philadelphia , the county with the highest proportion of older adults ( namely , 39% of sample ) , represented a highly urbanized context while the other four counties reflected mostly suburban settings . these authors describe characteristics of urbanization such as crime , minority , inequality , and population stability that may influence social capital and health . specifically , pridmore and colleagues write , poor urban settlements require special interventions to address social exclusion , disruption of social networks and trust , insecurity and violence , and high mortality and morbidity [ 27 , p. i138 ] . ultimately , the context in which the individual lives requires consideration when developing interventions aimed at building social capital . this may explain the nonsignificant models for self - rated health and adl for oldest - old . as was found in this study , not all the measures used were associated with health , specifically telephone interaction . furthermore , as nyqvist and colleagues have stated : , it is generally assumed that social capital indicators measure the same thing in different groups and places [ 12 , p. 105 ] . however , research has demonstrated that there are marked differences in the questions about social capital that are considered appropriate for various groups depending , for instance , on the subjects ' age . also , in response to nyqvist and colleagues ' concerns , there is a need for social capital scale development that reflects the specific characteristics of older persons . finally , based on the findings of this study certain dimensions of the social environment ( namely , trust , cohesion , support , and participation ) however , the question still remains as how does one build , for example , trust and cohesion ? successful implementation of programs that build social capital should also take into consideration individual characteristics , such as gender , race , and economic status that may influence the relationship between social capital and health .

hepatitis b and c viruses ( hbv and hcv ) are the leading causes of chronic liver disease worldwide ( el - serag , 2012 ) . with 400 million individuals chronically infected with hbv or hcv and at risk for severe liver disease , these viruses are a major global health burden ( el - serag , 2012 , wedemeyer et al . , 2015 ) . although hbv and hcv differ in their genomic organization and life cycles , these viruses exclusively infect human hepatocytes ( baumert et al . , 2014 ) , suggesting that liver - specific factors are important for the life cycle of both viruses . hcv requires a number of host proteins , including cluster of differentiation 81 ( cd81 ) , scavenger receptor bi ( sr - bi ) , claudin-1 ( cldn1 ) , occludin ( ocln ) , and accessory factors , such as epidermal growth factor receptor ( egfr ) , to enter hepatocytes ( baumert et al . , 2011 , martin and uprichard , 2013 , zeisel et al . , 2013b , zona et al . , 2013 ) some of these host factors interact directly with hcv glycoproteins , whereas others contribute to hcv internalization by promoting co - receptor associations and inducing intracellular signaling pathways ( kim et al . 2011 ) . while key host factors mediating hcv entry are well characterized ( zeisel et al . , the sodium taurocholate co - transporting polypeptide ( ntcp ) , a bile acid transporter expressed at the hepatocyte basolateral membrane ( claro da silva et al . , 2013 ) , was identified previously as a receptor for hbv and hepatitis d virus ( hdv ) ( ni et al . , 2014 , yan et al . , 2012 ) . hdv can use hbv envelope proteins to assemble infectious particles and is widely used as a surrogate model to study hbv entry ( sureau , 2010 ) . exogenous ntcp expression in ntcp - lacking human hepatoma cell lines ( such as huh7 and hepg2 ) renders these cells susceptible to hbv / hdv entry ( ni et al . , 2014 , yan et al . , 2012 ) . the natural ligands of ntcp ( i.e. , conjugated and hydrophilic bile acids ) compete with hbv / hdv for ntcp binding and inhibit viral infection ( yan et al . , 2014 ) . in contrast , bile acids have been shown to enhance hcv replication ( chang and george , 2007 , chhatwal et al . , 2012 ) , although the mechanisms are not yet defined . furthermore , whether bile acid transporters such as ntcp play a role in infection of alternative hepatotropic viruses , such as hcv , is unclear . in this study , we tested the role of ntcp in hcv infection , and we identified the mechanisms by which a solute carrier affects infectivity of three major hepatotropic viruses . to investigate the effect of ntcp expression on hcv infection , we transduced the huh7.5.1 cell line ( zhong et al . , 2005 ) huh7.5.1-ntcp cells expressed significantly higher ntcp mrna and protein levels ( figure 1a ) and surface levels of ntcp ( figure s1a ) than parental huh7.5.1 cells . to confirm that ntcp is functional as a viral host factor in these cells , huh7.5.1-ntcp cells supported hdv infection , as demonstrated by the presence of hdv rna and delta antigen ( hdag ) 7 days post - inoculation ( figure 1b ) . moreover , hdv infection was inhibited by the hbv pres1-derived peptide ( figure 1b ) , which binds to ntcp and prevents hbv / hdv entry ( schieck et al . , 2013 ) . to assess if ntcp expression affects hcv infection , we used cell culture - derived hcv ( hcvcc ) and lentiviral particles pseudotyped with hcv e1e2 glycoproteins ( hcvpp ) of different genotypes . expression of ntcp significantly enhanced hcvcc ( jc1 and jcr2a ) infection and hcvpp ( genotypes 1b , 2a , 3a , and 4 ) entry compared to the parental cells ( normalized as 100% ) ( figures 1c and 1d ) . interestingly , entry of vesicular stomatitis virus pseudoparticles ( vsvpp ) and murine leukemia virus pseudoparticles ( mlvpp ) was not significantly affected by ntcp expression under these conditions ( figure 1d ) . the growth of huh7.5.1 and huh7.5.1-ntcp cells after seeding was similar , indicating that our observations were unrelated to effects on cell proliferation ( figure s1b ) . furthermore , the effect of ntcp on hcv entry / infection was independent of the infectivity levels of different hcvpp and hcvcc strains ( figures s1c and s1d ) . since hcv can infect cells by direct cell - to - cell transmission ( meredith et al . , 2013 , 2008 ) , we evaluated the role of ntcp in this mode of viral dissemination . hcv - replicating huh7.5.1 cells were co - cultured with gfp - expressing huh7.5 cells ( lupberger et al . , 2011 , xiao et al . , 2014 ) transduced or not to express ntcp ( figure 1e ) . in this assay , the presence of an anti - envelope e2 antibody inhibits cell - free infection , allowing the specific assessment of cell - to - cell transmission . ntcp expression significantly increased the percentage of gfp - positive hcv - infected cells , suggesting that ntcp also plays a role in cell - to - cell spread ( figures 1f and 1 g ) . we confirmed the role of ntcp in hcv infection by silencing ntcp expression in huh7.5.1-ntcp cells using a small interfering rna ( sirna ) targeting ntcp , prior to infection with hcvcc . following ntcp silencing in huh7.5.1-ntcp cells , total protein expression was decreased by 80% , which was reflected by decreased surface expression ( figure 2a ) . this corresponded to a significant decrease in hcvcc infection ( figure 2b ) . to rule out potential off - target effects , we tested multiple ntcp - targeting small hairpin rna ( shrna ) sequences in huh7.5.1-ntcp cells . we confirmed that a decrease in ntcp expression ( by shntcp2 and shntcp3 ) ( figure 2c ) correlated with a decrease in hcvcc infection ( figure 2d ) . furthermore , silencing ntcp expression with shntcp2 decreased protein expression by > 80% ( figure 2e ) , which significantly decreased entry of hcvpp into huh7.5.1-ntcp cells ( figure 2f ) , confirming the relevance of ntcp for hcv entry . to assess whether ntcp interacts directly with hcv glycoproteins , we measured binding of recombinant soluble hcv e2 ( se2 ) to cells . we observed no significant difference in se2 binding to ntcp - expressing or parental cells ( figure 3a ) . however , se2 treatment of huh7.5.1 cells inhibited hcvcc infection ( figure 3b ) , confirming the functionality of the se2 protein . furthermore , ntcp expression did not affect the expression of canonical hcv entry factors in huh7.5.1-ntcp cells compared to the parental cells ( figure 3c ) . we next tested the effect of the hbv pres1-derived peptide , which is known to bind to ntcp and inhibit hbv / hdv entry ( ni et al . , 2014 ) . although pres1 inhibited hdv infection of huh7.5.1-ntcp cells after 1 hr pre - treatment , it had no effect on hcvpp entry under these conditions ( figure 3d ) , indicating that the hbv pres1-binding domain of ntcp is not required to promote hcv entry . we next evaluated whether the bile acid transporter function of ntcp is important for hcv entry . , 2014 , slijepcevic et al . , 2015 ) , which inhibits bile acid uptake in vitro ( ic50 , 4 nm ) ( ni et al . , 2014 ) , for 24 , 48 , and 72 hr prior to the addition of hdv , hcvpp , or vsvpp ( figures 3e3 g ) . pres1 treatment ( 200 nm ) significantly inhibited hcvpp entry in a time - dependent manner , with a maximal inhibition of 70% after 72 hr ( figure 3e ) . under these conditions , vsvpp entry also was reduced by pres1 , although to a lesser extent ( figure 3f ) , suggesting a general effect on viral infection . in contrast , hdv infection was impaired by pres1 regardless of the treatment duration ( figure 3 g ) . hcvcc infection also was decreased by 72-hr pres1 treatment ( figure 3h ) , confirming that our observations are relevant for the infectious virus and the full hcv life cycle . taken together , our data suggest that long - term pres1 inhibition of ntcp - mediated bile acid transport perturbs cellular physiology to modulate hcv entry . since pres1 only affected hcv entry after long - term treatment ( figure 3 ) , we hypothesized that the bile acid transport activity of ntcp induces metabolic or transcriptional changes that regulate virus infection . to test this hypothesis we performed genome - wide microarray analyses of huh7.5.1-ntcp cells that were treated with pres1 or a scrambled peptide ( 200 nm ) for 48 hr . gene set enrichment analysis ( gsea ) indicated that pres1 treatment induces the expression of genes involved in bile acid and fatty acid metabolism , as previously described ( oehler et al . , 2014 ) . unexpectedly , pres1 treatment also induced the expression of genes involved in innate immunity , such as ifn responses and the jak / stat - signaling pathway ( table 1 ; figure 4a ) . interestingly , ifitm2 and ifitm3 , which encode two restriction factors targeting hcv entry ( narayana et al . , 2015 ) , were among the pres1-induced genes ( figure 4b ) . to confirm that ifitms function as restriction factors in huh7.5.1-ntcp cells indeed , ifitm2 and ifitm3 overexpression in huh7.5.1-ntcp cells inhibited hcvcc infection ( figure 4d ) . however , as endogenous ifitm2 expression in huh7.5.1 and huh7.5.1-ntcp cells was low and at the limit of detection , we focused on ifitm3 in further functional studies . notably , ifitm3 expression was decreased at the protein level by 60% in huh7.5.1-ntcp cells compared to parental cells ( figure 4e ) , suggesting that ntcp modulates ifitm3 expression . to confirm that the changes in gene expression were directly related to ntcp and not to off - target effects of pres1 , we selected ifitm3 and two other genes involved in ifn responses ( parp9 and cxcl10 ) which were induced by pres1 treatment ( figure 4b ) , and we compared their expression levels in huh7.5.1 and huh7.5.1-ntcp cells . as expected , huh7.5.1-ntcp cells had decreased mrna expression of ifitm3 , parp9 , and cxcl10 compared to parental cells ( figure 4f ) , confirming the specific role of ntcp in the suppression of these genes . these data support previous findings that pres1 binds with high specificity to ntcp without off - target effects ( bogomolov et al . , 2016 ) . the gene expression analyses implied that ntcp facilitates hcv entry by altering the expression of interferon - stimulated genes ( isgs ) . given that the physiological function of ntcp is to transport bile acids , and bile acids are known to affect isg expression in hepatocytes ( graf et al . , 2010 , podevin et al . , 1999 ) , we hypothesized that bile acid transport by ntcp regulates the expression of isgs and viral infection . since ifitm3 functions as an hcv restriction factor in our model system ( figure 4d ) , we selected ifitm3 as a representative isg for functional studies to probe the link between ntcp and the ifn response . to ensure that ifn responses are indeed functional in huh7.5.1-ntcp cells , we treated cells with poly(i : c ) and ifn2 , and we evaluated isg induction by the expression of ifitm3 . poly(i : c ) stimulation of huh7.5.1-ntcp cells significantly increased ifitm3 expression ( figure s2a ) . moreover , stat1 phosphorylation ( figure s2b ) and ifitm3 mrna expression ( figure s2c ) were markedly induced after ifn2 treatment . this induction was repressed by a specific antibody targeting the type i ifn receptor ( ifnar ) ( figures s2b and s2c ) . the mere presence of ntcp decreased mrna expression of ifitm3 by 50% compared to parental cells ( figure 5a ) . the addition of bile acid ( 100 m sodium taurocholate ) to huh7.5.1-ntcp cells decreased ifitm3 mrna expression even further , whereas blocking bile acid transport with the pres1 peptide restored ifitm3 mrna expression to the levels observed in huh7.5.1 cells ( figure 5a ) . these findings suggest that the effect of ntcp on isg expression and hcv infection is dependent on bile acid . indeed , the addition of supplementary bile acid in the cell culture medium dose - dependently increased hcvcc infection in huh7.5.1-ntcp cells , but not in parental cells ( figure 5b ) . we next evaluated the effect of pres1 treatment on ifitm3 protein expression and hcv infection in the presence of bile acids . reflecting our observations of the mrna level ( figure 5a ) , ifitm3 protein expression was decreased in huh7.5.1-ntcp cells compared to huh7.5.1 cells in the presence of bile acid ( figure 5c ) . however , treatment of huh7.5.1-ntcp cells with pres1 ( to block bile acid uptake ) under these conditions induced a 2-fold increase in ifitm3 protein expression , effectively restoring it to the level observed in huh7.5.1 cells ( figure 5c ) . furthermore , treatment of huh7.5.1-ntcp cells with the pres1 peptide in the presence of bile acid inhibited hcvcc infection ( figure 5d ) . we did not observe these effects in huh7.5.1 cells ( figures 5c and 5d ) , confirming that the bile acid - mediated effect is dependent on ntcp . interestingly , when we silenced ifitm3 expression ( figure 5e ) in the presence of bile acid , we observed an increase in hcv infection ( figure 5f ) . however , this effect was no longer observed in the presence of pres1 to block bile acid uptake into the cells or in the absence of bile acid ( figure 5f ) . these results suggest that bile acid - mediated suppression of other isgs ( which may otherwise compensate the absence of an individual isg ) is necessary to observe a functional effect of ifitm3 silencing on hcv infection . differences in bile acid levels in cell culture medium also may explain why ifitm3 was not identified as an hcv restriction factor in previous screens ( brass et al . , we investigated the role of ntcp during hcv entry into primary human hepatocytes ( phhs ) . following transfection of sintcp , the expression of ntcp protein was reduced by 50% ( figure 6a ) . the decrease in ntcp expression correlated with a significant decrease in the entry of hcvpp genotype 1b ( figure 6b ) . we next evaluated whether ntcp - mediated bile acid transport affects isg expression in phhs . we treated phhs with bile acid in the presence or absence of pres1 and performed genome - wide microarray analyses . gsea showed that bile acid treatment of phhs suppressed the expression of genes involved in the ifn response ( normalized enrichment score [ nes ] 2.11 ; p value < 0.001 ; false discovery rate [ fdr ] < 0.001 ) ( figure 6c ) as well as other immune - related pathways . however , treatment of phhs with pres1 under these conditions induced the expression of genes involved in the ifn response ( nes 1.5 ; p value = 0.014 ; fdr = 0.035 ) to restore their expression levels to normal conditions ( i.e. , phhs in the absence of bile acid ) ( figure 6d ) . these findings are consistent with our microarray analyses in huh7.5.1-ntcp cells ( figure 4 ) , where expression of genes involved in ifn responses was regulated by treatment with pres1 . interestingly , ifitm1 , ifitm2 , and ifitm3 were among the genes suppressed by bile acid treatment of phhs ( figure 6c ) , and their expression could be rescued by the addition of pres1 ( figure 6d ) . moreover , the effects we observed in phhs were dependent on the presence of bile acid , as treatment with pres1 in the absence of bile acid did not have a major impact on the expression of these genes ( data not shown ) . we next evaluated the functional effect of these changes in isg expression on hcvpp entry into phhs . treatment of phhs with bile acid increased hcvpp genotype 1b entry , and the addition of pres1 under these conditions restored the level of infection to that observed in the absence of bile acid ( figure 6e ) . interestingly , the concentration of bile acid required to see a robust effect for hcvpp entry into phhs was higher than for hcvcc infection of huh7.5.1-ntcp cells ( figure 5b ) , which may reflect different bile acid uptake efficiency in phhs or that the effect is more potent when the full hcv life cycle is measured and isgs targeting different steps of the viral infection cycle are involved . finally , to confirm that the activity of pres1 is linked to ifn responses , we tested the effect of pres1 in the presence of an antibody blocking the type i ifn receptor . in the presence of this antibody , pres1 no longer inhibited hcvpp entry into phhs ( figure 6f ) , suggesting that the inhibitory effect of pres1 against hcv entry is indeed mediated by the ifn signal transduction cascade and resulting ifn responses in phhs . . exogenous expression of ntcp in huh7.5.1 hepatoma cells increased hcv infection , whereas silencing of ntcp expression reduced hcv entry ( figures 1 and 2 ) . using microarray analyses in cell lines ( figure 4 ) and phhs ( figure 6 ) , we discovered that ntcp - mediated bile acid transport regulates innate antiviral responses , thereby inhibiting hcv infection and uncovering a role for ntcp as a regulator of antiviral immunity . innate antiviral responses mediated by isgs have been shown to target multiple steps during viral infection , including entry ( liu et al . , 2011 , smith et al . , 2014 ) . ifitm1 , ifitm2 , and ifitm3 , which belong to a group of five ifn - induced transmembrane proteins ( smith et al . , 2014 ) , have been shown to exert broad antiviral activity against a range of viruses , including vsv , hiv , dengue virus , influenza virus , and zika virus ( savidis et al . , 2016 , smith et al . , 2014 ) . interestingly , ifitm2 and ifitm3 were recently reported to restrict hcv infection at a late entry step by targeting hcv for lysosomal degradation following endocytosis ( narayana et al . , 2015 ) . ifitm1 was shown to interact with hcv co - receptors at tight junctions to disrupt the hcv entry process by alternative mechanisms ( wilkins et al . given that bile acids have been shown to modulate cellular antiviral responses ( graf et al . , 2010 , podevin et al . , 1999 ) , we hypothesized that ntcp affects the induction of isg expression via its bile acid transport activity . indeed , the expression of isgs ( including ifitm1 , ifitm2 , and ifitm3 ) in phhs was suppressed by the addition of bile acid ( figure 6c ) . however , expression of these isgs in phhs was restored by addition of the pres1 peptide , which blocks ntcp - mediated bile acid uptake ( figure 6d ) . furthermore , modulation of the expression of ifitm3 by bile acid or pres1 treatment had a clear functional effect on hcv infection ( figures 5 and 6 ) . our findings are consistent with a previous report showing that bile acids affect hcv replication ( chhatwal et al . , 2012 ) , probably by similar mechanisms as those we describe here . in this study , we selected ifitm3 as a representative isg for functional characterization , but the expression of other isgs is also affected by bile acids ( figure 6 ) , likely contributing further to the overall effect on hcv infection . since isgs broadly restrict viral infection , this is consistent with the time - dependent effect of pres1 on vsvpp entry that we observed ( figure 3 ) . our data suggest that ntcp facilitates hcv infection by modulating bile acid transport and isg expression . isgs also restrict hcv cell - to - cell transmission ( meredith et al . , 2014 ) , consistent with our finding that ntcp contributes to hcv cell - to - cell spread ( figures 1e1 g ) . it should be noted that ntcp expression did not appear to affect hcv entry in a recent huh7 cell line model ( meredith et al . , 2016 ) . huh7.5.1 cells are derived from huh7.5 cells , which differ from huh7 cells by a single point mutation in the retinoic acid - inducible gene - i ( bartenschlager and pietschmann , 2005 , zhong et al . , 2005 ) . moreover , differences in bile acid concentrations in cell culture medium may be responsible for differences in the effect of ntcp on cell entry of hcv ( figure 5c ) . however , our loss - of - function studies and microarray analyses in phhs ( figure 6 ) clearly demonstrate the impact of ntcp on hcv infection in primary cells with physiological innate immune responses . for hbv / hdv infection , viral entry requires direct interaction with ntcp ( ni et al . , 2014 , yan et al . , 2012 ) . furthermore , hbv binding to ntcp may interfere with the physiological function of ntcp ( i.e. , bile acid uptake ) , and ntcp ligands can abrogate hbv / hdv infection ( oehler et al . we confirmed the inhibitory effect of the hbv pres1-derived peptide on hdv infection following 1-hr treatment ( figure 3d ) , but we did not see a corresponding effect on hcv infection under these conditions . furthermore , the effect of ntcp on hcv infection does not appear to involve hcv e2 or binding factors cd81 and sr - bi ( pileri et al . ntcp expression did not modulate the expression of canonical entry factors cldn1 and ocln or egfr either ( figure 3c ) . other mechanisms may contribute to the effects of ntcp on viral entry and infection that we observed . indeed , modulation of ntcp bile acid transport activity by pres1 affected ifn responses , but also bile acid metabolism and cholesterol homeostasis in huh7.5.1-ntcp cells as well as other pathways ( table 1 ) . in particular , modulation of bile acid metabolism and cholesterol homeostasis by pres1 would alter cellular cholesterol pools , which has been shown to affect the activation of type i ifn responses ( york et al . , 2015 ) and may potentially contribute to the effect on hcv infection that we observed . furthermore , modulation of signaling and transcription factor targets ( table 1 ) may have additional effects on viral replication and translation . our results demonstrate that ntcp , acting by distinct mechanisms , is relevant for the three major viruses causing chronic hepatitis and liver disease . targeting viral cell entry with receptor antagonists , antibodies , peptides , and receptor kinase inhibitors has provided perspectives to prevent and treat chronic hepatitis b and c infections ( colpitts et al . , 2015 ) . 2013 ) , has been shown to protect against hbv infection ( petersen et al . , 2008 ) , to modulate viral spread in animal models ( volz et al . , 2013 ) , and to decrease hdv viral load in patients in a phase ii clinical trial ( bogomolov et al . , these data suggest that myrcludex b may inhibit hbv infection by a dual mode of action , by interfering with viral binding and potentially increasing isg expression . ntcp - targeting agents in clinical development also may inhibit hcv infection , which is of interest particularly for patients with hbv / hdv / hcv co - infections . ntcp is a member of the solute carrier ( slc ) family of proteins , a group of membrane proteins having crucial roles in many physiological functions ( csar - razquin et al . , 2015 ) . we uncover ntcp as a mediator of innate antiviral responses in hepatocytes and establish a role for ntcp in the entry process of multiple viruses . our data uncover an important role of slcs in virus - host interactions by linking their function to the regulation of innate immune responses . moreover , bile acid transport through slcs profoundly affects liver gene expression ( table 1 ) . overall , we have identified ntcp as a regulator of innate immune responses in the human liver . these findings improve the understanding of virus - host interactions in the human liver , and they may open perspectives for the development of broad antiviral therapies targeting hepatotropic viruses that cause chronic liver disease and cancer . the sources for 293 t and huh7.5.1 cells have been described ( lupberger et al . , 2011 ) . huh7.5.1 cells were seeded in six - well plates at 50% confluency 1 day prior to transduction with human ntcp - expressing vsvpp ( genecopoeia ) . cells were incubated in dmem ( gibco ) containing 10% fetal bovine serum ( dutscher ) . after 3 days , the cells were expanded and selected for ntcp expression with 1.8 g / ml puromycin . liver tissue from patients undergoing surgical resection was obtained with informed consent from all patients . the respective protocols were approved by the ethics committee of the university of strasbourg hospitals ( cpp 10 - 17 ) . phhs were isolated from liver resection tissue and cultured in william s e medium ( sigma ) ( krieger et al . , 2010 , lupberger et al . , 2011 ) . lentiviral pseudoparticles expressing hcv envelope glycoproteins ( hcvpp strains hcv - j [ 1b ] , jfh1 [ 2a ] , nih s52 [ 3a ] , and ukn4.21.16 ) , vsvpp , mlvpp , as well as cell culture - derived hcvcc ( luc - jc1 and luc - jcr2a ) were generated as described ( fofana et al . huh7.5.1 cells and phhs were infected as described ( krieger et al . , 2010 , lupberger et al . , 2011 ) . pseudoparticle entry and hcvcc infection were assessed by measuring luciferase activity 72 hr post - infection ( krieger et al . , 2010 , lupberger et al . , 2011 ) . hdv production and infection have been described ( verrier et al . , 2016 ) huh7.5.1-ntcp cells were treated with pres1 or a control peptide ( 200 nm ) for 48 hr . alternatively , phhs were treated with bile acids ( 500 m ) in presence of pres1 or a control peptide ( 400 nm ) for 48 hr . total rna from three biological replicates per condition from one experiment was then extracted using the reliaprep kit ( promega ) , and 200 ng was subjected to genome - wide transcriptome profiling using humanht-12 beadarray ( illumina ) , following the manufacturer s protocol . raw scanned data were normalized by cubic spline algorithm using illumina normalizer module of genepattern genomic analysis toolkit ( http://software.broadinstitute.org/cancer/software/genepattern ) , as previously described ( hoshida et al . , 2008 ) . modulated molecular pathways were determined using gsea ( subramanian et al . , 2005 ) . the number of independent experiments as well as the total number of biological replicates ( n ) are indicated in the figure legends . statistical analyses were performed using mann - whitney u test by comparing values from every biological replicate per study ( indicated by n in the figure legends ) ; p < 0.05 ( ) , p < 0.01 ( ) , and p < 0.001 ( ) were considered statistically significant . significant p values are indicated by asterisks in the individual figures . for microarray analyses , two - tailed unpaired student s t test was performed by comparing the values from three biological replicates ( p < 0.05 was considered statistically significant ) .

summarychronic hepatitis b , c , and d virus ( hbv , hcv , and hdv ) infections are the leading causes of liver disease and cancer worldwide . recently , the solute carrier and sodium taurocholate co - transporter ntcp has been identified as a receptor for hbv and hdv . here , we uncover ntcp as a host factor regulating hcv infection . using gain- and loss - of - function studies , we show that ntcp mediates hcv infection of hepatocytes and is relevant for cell - to - cell transmission . ntcp regulates hcv infection by augmenting the bile - acid - mediated repression of interferon - stimulated genes ( isgs ) , including ifitm3 . in conclusion , our results uncover ntcp as a mediator of innate antiviral immune responses in the liver , and they establish a role for ntcp in the infection process of multiple viruses via distinct mechanisms . collectively , our findings suggest a role for solute carriers in the regulation of innate antiviral responses , and they have potential implications for virus - host interactions and antiviral therapies .

Introduction Results Discussion Experimental Procedures Author Contributions

hepatitis b and c viruses ( hbv and hcv ) are the leading causes of chronic liver disease worldwide ( el - serag , 2012 ) . with 400 million individuals chronically infected with hbv or hcv and at risk for severe liver disease , these viruses are a major global health burden ( el - serag , 2012 , wedemeyer et al . although hbv and hcv differ in their genomic organization and life cycles , these viruses exclusively infect human hepatocytes ( baumert et al . , 2014 ) , suggesting that liver - specific factors are important for the life cycle of both viruses . hcv requires a number of host proteins , including cluster of differentiation 81 ( cd81 ) , scavenger receptor bi ( sr - bi ) , claudin-1 ( cldn1 ) , occludin ( ocln ) , and accessory factors , such as epidermal growth factor receptor ( egfr ) , to enter hepatocytes ( baumert et al . , 2013 ) some of these host factors interact directly with hcv glycoproteins , whereas others contribute to hcv internalization by promoting co - receptor associations and inducing intracellular signaling pathways ( kim et al . , the sodium taurocholate co - transporting polypeptide ( ntcp ) , a bile acid transporter expressed at the hepatocyte basolateral membrane ( claro da silva et al . , 2013 ) , was identified previously as a receptor for hbv and hepatitis d virus ( hdv ) ( ni et al . hdv can use hbv envelope proteins to assemble infectious particles and is widely used as a surrogate model to study hbv entry ( sureau , 2010 ) . , 2012 ) , although the mechanisms are not yet defined . furthermore , whether bile acid transporters such as ntcp play a role in infection of alternative hepatotropic viruses , such as hcv , is unclear . in this study , we tested the role of ntcp in hcv infection , and we identified the mechanisms by which a solute carrier affects infectivity of three major hepatotropic viruses . to investigate the effect of ntcp expression on hcv infection , we transduced the huh7.5.1 cell line ( zhong et al . to confirm that ntcp is functional as a viral host factor in these cells , huh7.5.1-ntcp cells supported hdv infection , as demonstrated by the presence of hdv rna and delta antigen ( hdag ) 7 days post - inoculation ( figure 1b ) . moreover , hdv infection was inhibited by the hbv pres1-derived peptide ( figure 1b ) , which binds to ntcp and prevents hbv / hdv entry ( schieck et al . to assess if ntcp expression affects hcv infection , we used cell culture - derived hcv ( hcvcc ) and lentiviral particles pseudotyped with hcv e1e2 glycoproteins ( hcvpp ) of different genotypes . expression of ntcp significantly enhanced hcvcc ( jc1 and jcr2a ) infection and hcvpp ( genotypes 1b , 2a , 3a , and 4 ) entry compared to the parental cells ( normalized as 100% ) ( figures 1c and 1d ) . furthermore , the effect of ntcp on hcv entry / infection was independent of the infectivity levels of different hcvpp and hcvcc strains ( figures s1c and s1d ) . since hcv can infect cells by direct cell - to - cell transmission ( meredith et al . , 2013 , 2008 ) , we evaluated the role of ntcp in this mode of viral dissemination . hcv - replicating huh7.5.1 cells were co - cultured with gfp - expressing huh7.5 cells ( lupberger et al . in this assay , the presence of an anti - envelope e2 antibody inhibits cell - free infection , allowing the specific assessment of cell - to - cell transmission . ntcp expression significantly increased the percentage of gfp - positive hcv - infected cells , suggesting that ntcp also plays a role in cell - to - cell spread ( figures 1f and 1 g ) . we confirmed the role of ntcp in hcv infection by silencing ntcp expression in huh7.5.1-ntcp cells using a small interfering rna ( sirna ) targeting ntcp , prior to infection with hcvcc . to rule out potential off - target effects , we tested multiple ntcp - targeting small hairpin rna ( shrna ) sequences in huh7.5.1-ntcp cells . although pres1 inhibited hdv infection of huh7.5.1-ntcp cells after 1 hr pre - treatment , it had no effect on hcvpp entry under these conditions ( figure 3d ) , indicating that the hbv pres1-binding domain of ntcp is not required to promote hcv entry . , 2014 ) , for 24 , 48 , and 72 hr prior to the addition of hdv , hcvpp , or vsvpp ( figures 3e3 g ) . hcvcc infection also was decreased by 72-hr pres1 treatment ( figure 3h ) , confirming that our observations are relevant for the infectious virus and the full hcv life cycle . taken together , our data suggest that long - term pres1 inhibition of ntcp - mediated bile acid transport perturbs cellular physiology to modulate hcv entry . since pres1 only affected hcv entry after long - term treatment ( figure 3 ) , we hypothesized that the bile acid transport activity of ntcp induces metabolic or transcriptional changes that regulate virus infection . , 2015 ) , were among the pres1-induced genes ( figure 4b ) . however , as endogenous ifitm2 expression in huh7.5.1 and huh7.5.1-ntcp cells was low and at the limit of detection , we focused on ifitm3 in further functional studies . notably , ifitm3 expression was decreased at the protein level by 60% in huh7.5.1-ntcp cells compared to parental cells ( figure 4e ) , suggesting that ntcp modulates ifitm3 expression . to confirm that the changes in gene expression were directly related to ntcp and not to off - target effects of pres1 , we selected ifitm3 and two other genes involved in ifn responses ( parp9 and cxcl10 ) which were induced by pres1 treatment ( figure 4b ) , and we compared their expression levels in huh7.5.1 and huh7.5.1-ntcp cells . as expected , huh7.5.1-ntcp cells had decreased mrna expression of ifitm3 , parp9 , and cxcl10 compared to parental cells ( figure 4f ) , confirming the specific role of ntcp in the suppression of these genes . the gene expression analyses implied that ntcp facilitates hcv entry by altering the expression of interferon - stimulated genes ( isgs ) . given that the physiological function of ntcp is to transport bile acids , and bile acids are known to affect isg expression in hepatocytes ( graf et al . , 1999 ) , we hypothesized that bile acid transport by ntcp regulates the expression of isgs and viral infection . since ifitm3 functions as an hcv restriction factor in our model system ( figure 4d ) , we selected ifitm3 as a representative isg for functional studies to probe the link between ntcp and the ifn response . to ensure that ifn responses are indeed functional in huh7.5.1-ntcp cells , we treated cells with poly(i : c ) and ifn2 , and we evaluated isg induction by the expression of ifitm3 . the addition of bile acid ( 100 m sodium taurocholate ) to huh7.5.1-ntcp cells decreased ifitm3 mrna expression even further , whereas blocking bile acid transport with the pres1 peptide restored ifitm3 mrna expression to the levels observed in huh7.5.1 cells ( figure 5a ) . these findings suggest that the effect of ntcp on isg expression and hcv infection is dependent on bile acid . indeed , the addition of supplementary bile acid in the cell culture medium dose - dependently increased hcvcc infection in huh7.5.1-ntcp cells , but not in parental cells ( figure 5b ) . we next evaluated the effect of pres1 treatment on ifitm3 protein expression and hcv infection in the presence of bile acids . reflecting our observations of the mrna level ( figure 5a ) , ifitm3 protein expression was decreased in huh7.5.1-ntcp cells compared to huh7.5.1 cells in the presence of bile acid ( figure 5c ) . we did not observe these effects in huh7.5.1 cells ( figures 5c and 5d ) , confirming that the bile acid - mediated effect is dependent on ntcp . interestingly , when we silenced ifitm3 expression ( figure 5e ) in the presence of bile acid , we observed an increase in hcv infection ( figure 5f ) . however , this effect was no longer observed in the presence of pres1 to block bile acid uptake into the cells or in the absence of bile acid ( figure 5f ) . these results suggest that bile acid - mediated suppression of other isgs ( which may otherwise compensate the absence of an individual isg ) is necessary to observe a functional effect of ifitm3 silencing on hcv infection . differences in bile acid levels in cell culture medium also may explain why ifitm3 was not identified as an hcv restriction factor in previous screens ( brass et al . , we investigated the role of ntcp during hcv entry into primary human hepatocytes ( phhs ) . following transfection of sintcp , the expression of ntcp protein was reduced by 50% ( figure 6a ) . , phhs in the absence of bile acid ) ( figure 6d ) . these findings are consistent with our microarray analyses in huh7.5.1-ntcp cells ( figure 4 ) , where expression of genes involved in ifn responses was regulated by treatment with pres1 . interestingly , ifitm1 , ifitm2 , and ifitm3 were among the genes suppressed by bile acid treatment of phhs ( figure 6c ) , and their expression could be rescued by the addition of pres1 ( figure 6d ) . moreover , the effects we observed in phhs were dependent on the presence of bile acid , as treatment with pres1 in the absence of bile acid did not have a major impact on the expression of these genes ( data not shown ) . treatment of phhs with bile acid increased hcvpp genotype 1b entry , and the addition of pres1 under these conditions restored the level of infection to that observed in the absence of bile acid ( figure 6e ) . interestingly , the concentration of bile acid required to see a robust effect for hcvpp entry into phhs was higher than for hcvcc infection of huh7.5.1-ntcp cells ( figure 5b ) , which may reflect different bile acid uptake efficiency in phhs or that the effect is more potent when the full hcv life cycle is measured and isgs targeting different steps of the viral infection cycle are involved . finally , to confirm that the activity of pres1 is linked to ifn responses , we tested the effect of pres1 in the presence of an antibody blocking the type i ifn receptor . in the presence of this antibody , pres1 no longer inhibited hcvpp entry into phhs ( figure 6f ) , suggesting that the inhibitory effect of pres1 against hcv entry is indeed mediated by the ifn signal transduction cascade and resulting ifn responses in phhs . exogenous expression of ntcp in huh7.5.1 hepatoma cells increased hcv infection , whereas silencing of ntcp expression reduced hcv entry ( figures 1 and 2 ) . using microarray analyses in cell lines ( figure 4 ) and phhs ( figure 6 ) , we discovered that ntcp - mediated bile acid transport regulates innate antiviral responses , thereby inhibiting hcv infection and uncovering a role for ntcp as a regulator of antiviral immunity . innate antiviral responses mediated by isgs have been shown to target multiple steps during viral infection , including entry ( liu et al . , 2014 ) , have been shown to exert broad antiviral activity against a range of viruses , including vsv , hiv , dengue virus , influenza virus , and zika virus ( savidis et al . given that bile acids have been shown to modulate cellular antiviral responses ( graf et al . , 1999 ) , we hypothesized that ntcp affects the induction of isg expression via its bile acid transport activity . indeed , the expression of isgs ( including ifitm1 , ifitm2 , and ifitm3 ) in phhs was suppressed by the addition of bile acid ( figure 6c ) . however , expression of these isgs in phhs was restored by addition of the pres1 peptide , which blocks ntcp - mediated bile acid uptake ( figure 6d ) . furthermore , modulation of the expression of ifitm3 by bile acid or pres1 treatment had a clear functional effect on hcv infection ( figures 5 and 6 ) . our findings are consistent with a previous report showing that bile acids affect hcv replication ( chhatwal et al . in this study , we selected ifitm3 as a representative isg for functional characterization , but the expression of other isgs is also affected by bile acids ( figure 6 ) , likely contributing further to the overall effect on hcv infection . our data suggest that ntcp facilitates hcv infection by modulating bile acid transport and isg expression . isgs also restrict hcv cell - to - cell transmission ( meredith et al . , 2014 ) , consistent with our finding that ntcp contributes to hcv cell - to - cell spread ( figures 1e1 g ) . huh7.5.1 cells are derived from huh7.5 cells , which differ from huh7 cells by a single point mutation in the retinoic acid - inducible gene - i ( bartenschlager and pietschmann , 2005 , zhong et al . moreover , differences in bile acid concentrations in cell culture medium may be responsible for differences in the effect of ntcp on cell entry of hcv ( figure 5c ) . however , our loss - of - function studies and microarray analyses in phhs ( figure 6 ) clearly demonstrate the impact of ntcp on hcv infection in primary cells with physiological innate immune responses . , bile acid uptake ) , and ntcp ligands can abrogate hbv / hdv infection ( oehler et al . we confirmed the inhibitory effect of the hbv pres1-derived peptide on hdv infection following 1-hr treatment ( figure 3d ) , but we did not see a corresponding effect on hcv infection under these conditions . furthermore , the effect of ntcp on hcv infection does not appear to involve hcv e2 or binding factors cd81 and sr - bi ( pileri et al . indeed , modulation of ntcp bile acid transport activity by pres1 affected ifn responses , but also bile acid metabolism and cholesterol homeostasis in huh7.5.1-ntcp cells as well as other pathways ( table 1 ) . in particular , modulation of bile acid metabolism and cholesterol homeostasis by pres1 would alter cellular cholesterol pools , which has been shown to affect the activation of type i ifn responses ( york et al . our results demonstrate that ntcp , acting by distinct mechanisms , is relevant for the three major viruses causing chronic hepatitis and liver disease . targeting viral cell entry with receptor antagonists , antibodies , peptides , and receptor kinase inhibitors has provided perspectives to prevent and treat chronic hepatitis b and c infections ( colpitts et al . 2013 ) , has been shown to protect against hbv infection ( petersen et al . , 2013 ) , and to decrease hdv viral load in patients in a phase ii clinical trial ( bogomolov et al . ntcp - targeting agents in clinical development also may inhibit hcv infection , which is of interest particularly for patients with hbv / hdv / hcv co - infections . ntcp is a member of the solute carrier ( slc ) family of proteins , a group of membrane proteins having crucial roles in many physiological functions ( csar - razquin et al . we uncover ntcp as a mediator of innate antiviral responses in hepatocytes and establish a role for ntcp in the entry process of multiple viruses . our data uncover an important role of slcs in virus - host interactions by linking their function to the regulation of innate immune responses . overall , we have identified ntcp as a regulator of innate immune responses in the human liver . these findings improve the understanding of virus - host interactions in the human liver , and they may open perspectives for the development of broad antiviral therapies targeting hepatotropic viruses that cause chronic liver disease and cancer . after 3 days , the cells were expanded and selected for ntcp expression with 1.8 g / ml puromycin . lentiviral pseudoparticles expressing hcv envelope glycoproteins ( hcvpp strains hcv - j [ 1b ] , jfh1 [ 2a ] , nih s52 [ 3a ] , and ukn4.21.16 ) , vsvpp , mlvpp , as well as cell culture - derived hcvcc ( luc - jc1 and luc - jcr2a ) were generated as described ( fofana et al . total rna from three biological replicates per condition from one experiment was then extracted using the reliaprep kit ( promega ) , and 200 ng was subjected to genome - wide transcriptome profiling using humanht-12 beadarray ( illumina ) , following the manufacturer s protocol . raw scanned data were normalized by cubic spline algorithm using illumina normalizer module of genepattern genomic analysis toolkit ( http://software.broadinstitute.org/cancer/software/genepattern ) , as previously described ( hoshida et al . statistical analyses were performed using mann - whitney u test by comparing values from every biological replicate per study ( indicated by n in the figure legends ) ; p < 0.05 ( ) , p < 0.01 ( ) , and p < 0.001 ( ) were considered statistically significant . significant p values are indicated by asterisks in the individual figures .

streptomyces clavuligerus is an important industrial microorganism , which produces the lactam antibiotic cephamycin c ( martn and liras , 1989 ) and the lactamase inhibitor clavulanic acid ( saudagar et al . , 2008 ) . clavulanic acid is produced worldwide on a large scale , and coformulated with amoxicillin in augmentin ( brogden et al . , 1981 ) . two biotechnological strain optimization approaches have been utilized to increase the production of clavulanic acid by the bacterium : rational metabolic engineering and iterative optimization through random mutagenesis and screening . in the rational approach , a specific gene is knocked out or overexpressed to divert metabolic fluxes towards the antibiotic biosynthetic pathways . arguably , the best example comes from the work of li and townsend ( 2006 ) , who reengineered the s. clavuligerus glycolytic pathway by constructing a deletion mutant of the glyceraldehyde 3phosphate dehydrogenase gene gap1 to increase the pool of the clavulanic acid precursor glycerol 3phosphate ( g3p ) . overexpression of the regulatory proteins ccar and clar also led to clavulanic acid overproduction ( hung et al . , 2007 ) , and the two strategies have recently been combined successfully in a single strain ( jnawali et al . , 2010 ) . yet , most or even all production strains that are used in industry have been obtained by classical strain improvement ( adrio and demain , 2006 ) , based on mutagenesis with mutagens such as nitrosoguanidine ( ntg ) . little is known about the exact genetic changes through which high production titers are achieved in these mutants . recently , we published the genome sequence of s. clavuligerus atcc 27064 ( medema et al . , 2010 ) . we now employed this information to perform a genomewide transcriptome study on an industrial production strain , which has been generated from the atcc 27064 type strain ( higgens and kastner , 1971 ) by several iterations of mutagenesis and screening , and produces clavulanic acid at levels approximately 100 that of the wildtype . the majority of observed changes consist of increased transcript levels of the antibiotic biosynthesis gene clusters . these observations are in agreement with fluxbalance analysis ( fba ) predictions using a constraintbased genomescale metabolic model constructed for s. clavuligerus . however , we also detected some potentially crucial transcript level changes in primary metabolism that could contribute to the increased production of clavulanic acid by redirection of fluxes , mimicking strategies utilized in rational approaches . comparing gene transcript levels of s. clavuligerus wildtype and ds48802 strains during stationary phase using microarrays , almost all genes ranking high in a differential transcriptome analysis belong to the complete clavulanic acid / cephamycin c supercluster , which is significantly overexpressed ( between twofold and eightfold ) in the ds48802 strain compared to the wildtype . they are located within the same supercluster and their products have been shown to regulate it positively ( alexander and jensen , 1998 ; paradkar et al . , 1998 ) . additionally , the clavams gene cluster ( cvm1245 and cas1 ) and the paralogous alanylclavam cluster ( orfabcd and ceas1/pah1/bls1/oat1 ) are significantly overexpressed ( fig . 1 ) , as is the twocomponent system involving cvm7p ( sclav_p1079p1080 ) that induces expression of the alanylclavam cluster ( tahlan et al . , 2007 ) ccar is an unlikely candidate for such a common regulatory factor , as it does not appear to control the paralogous cluster ( tahlan et al . , 2004 ) ; the pleiotropic regulator adpa ( sclav_1957 ) is a more likely candidate , as it is known to induce clavulanic acid expression ( lopezgarca et al . , 2010 ) and differential gene expression in s. clavuligerus ds48802 and atcc 27064 . sliding window plot ( size = 50 ) of the difference in gene expression between s. clavuligerus ds48802 and wildtype atcc 27064 . see table s2 for description of the ten gene clusters shown . for gene expression analysis , cultivations were performed in shake flasks directly inoculated with spore suspensions at 28c and 280 r.p.m . the semisynthetic growth medium used consisted of 30 g l glycerol , 5 g l wheat gluten , 3.5 g l asparagine monohydrate , 1.5 g lllysine , 0.7 g l kh2po4 , 0.3 g l mgso47h2o , 0.2 g l cacl22h2o , 0.2 g l feso47h2o , 10 g l mops , 0.1 ml l basilodon and 1 ml l trace elements solution at ph 7.0 . the trace element solution consisted of 20.4 g l h2so4 , 50 g l citric acid h2o , 16.75 g l znso47h2o , 1.6 g l cuso4 . 5 h2o , 1.5 g l mncl24h2o , 2 g l h3bo3 and 2 g l na2moo42h2o . after 70 h of cultivation , the cells were harvested by centrifugation , treated with rnaprotect ( qiagen ) and directly frozen with liquid nitrogen and stored at 80c . to isolate total rna , the frozen mycelium was ground in a mortar , resuspended in te buffer with 5 mg l lysozyme and incubated for 5 min at room temperature . rna isolation and purification were performed using phenol extraction ( trizol reagent , invitrogen ) and rneasy kit ( qiagen ) . the rna was quantified by measuring the absorbance at 260 nm . biotinylated cdna was prepared after fragmentation according to the standard affymetrix protocol using gc rich ( average 72% ) primers from 10 g total rna . for hybridization , 5 g and 7 g biotinylated cdna were used per affymetrix gene chip . microarray data have been deposited at gene expression omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number gse24033 . fluxbalance analysis was performed using a recently published genomescale metabolic model of s. clavuligerus ( medema et al . , 2010 ) . in this study , we slightly changed our objective function and included both clavulanic acid and cephamycin c biosynthesis pathways . we dynamically changed the antibiotic concentration in the biomass composition based on experimental observations of romero and colleagues ( 1984 ) and optimized the objective function for different concentrations of each antibiotic . among the 785 genes that the model contains , 497 genes showed nonzero flux for at least one antibiotic concentration . we calculated spearman correlation of fluxes of each reaction with increasing antibiotic concentrations . if an enzyme was involved in multiple reactions , we assigned the flux which had the highest r. in contrast to , for example , the intrachromosomal amplification of the kanamycin biosynthesis gene cluster in streptomyces kanamyceticus ( yanai et al . , 2006 ) , hybridization of s. clavuligerus ds48802 genomic dna to the microarrays revealed no amplifications of genes or gene clusters ( data not shown ) . the overproduction that we observe therefore appears to be caused by transcriptional ( and posttranscriptional ) changes only . while many changes have occurred during the generation of the ds48802 strain through mutagenesis , it is important to note that changes in transcription levels of many genes may be due to random mutations that have no impact on antibiotic biosynthesis . in order to assess which changes could be causatively linked to antibiotic overproduction , we computationally predicted the metabolic fluxes during antibiotic overproduction , using a constraintsbased genomescale metabolic network model of s. clavuligerus ( medema et al . , 2010 ) . we dynamically modelled the metabolic flux changes during increased production of clavulanic acid and cephamycin c with different rates of antibiotic production relative to the biomass , based on the experimental observations of romero and colleagues ( 1984 ) . interestingly , the computational predictions made through dynamic fba are largely in line with the observed expression changes . r > 0.6 ) and 129 genes were predicted to be downregulated ( negatively correlated ; r < 0.6 ) at increasing antibiotic production levels . forty per cent ( 15/37 ) of the genes that actually showed increased transcript levels ( fold change > 2 ) were also predicted to do so according to fba , and these include all genes encoding key biosynthetic enzymes known to be involved in clavulanic acid and cephamycin c biosynthesis . even though 40% does not appear to be a very large percentage , these predictions are statistically very significant according to a fisher 's exact test ( p = 0.0005 ; see table s1 ) . one should note that fba predicts the flux for every reaction , not for every gene product , as multiple gene products can be involved in a single reaction . therefore , in these cases the same flux was assigned to all gene products involved in that particular reaction , which is not necessarily the case in the actual gene expression , as only a single homologue or isoenzyme could be actively performing the reaction and thus would be differentially expressed . out of the 47 different enzymatic reactions predicted to be upregulated ( associated with 87 genes ) , 26 ( 55% ) have at least one gene linked to them , which showed increased transcript levels . as both our fba and gene transcript analysis pointed to an increased expression of the clavulanic acid and cephamycin core biosynthesis genes , we suggest that this is a crucial change required for antibiotic overproduction in this strain . moreover , as the fba indicated that the absolute fluxes required for high production of the secondary metabolites are minor compared to other fluxes involved in maintenance and cellular growth , a complete redirection of primary metabolism appears not to be necessary for overproduction . nonetheless , because clavulanic acid is synthesized from the precursors g3p and larginine , which play important roles in primary metabolism , specific changes in the primary metabolism of ds48802 could have occurred during the various random mutagenesis rounds , so that the intracellular pools of these intermediates are increased . indeed , glycerol uptake and metabolism ( sclav_06310632 and sclav_08770879 ) is clearly upregulated over twofold in ds48802 , indicating an improved utilization of glycerol as a carbon source as well as increased production of the clavulanic acid precursor g3p ( fig . 2 ) . moreover , the aconitase and citrate synthase from the citric acid cycle appear to be downregulated . a likely explanation for this is that the carbon flux from g3p in this direction is reduced and is partly redirected to clavulanic acid biosynthesis . this situation is remarkably similar to the result of the rationally constructed gap1 deletion that blocked g3p conversion into 1,3bisphosphoglycerate , thus improving clavulanic acid biosynthesis by increasing the intracellular g3p pool ( li and townsend , 2006 ) . however , an advantage of the situation in ds48802 , which seems to have an incomplete downregulation of the flux , could be that a considerable pool of acetylcoa is maintained , e.g. for the biosynthesis of ornithine from glutamate . ds48802 also seems to avoid the potential negative effects of a complete deletion of the aconitase and citrate synthase genes : a complete absence of these enzyme activities could lead to acidogenesis with negative consequences for secondary metabolite production as shown by viollier and colleagues ( 2001a , b ) . moreover , ds48802 still seems to be able to synthesize ketoglutarate ( a cosubstrate required for clavaminic acid biosynthesis ; salowe et al . , 1990 ) , while achieving the benefits of higher acetylcoa and/or g3p pools that have made these genes attractive targets for rational engineering to improve antibiotic production ( viollier et al . a potentially important observation that we can not explain yet from the current data are the differential transcript level changes of the two pyruvate kinase isoenzyme genes , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . changes in gene expression in s. clavuligerus ds48802 compared to the wildtype atcc 27064 projected onto a metabolic map . green arrows represent reactions catalyzed by genes expressed over twofold higher in ds48802 than in the wildtype . the orange arrow represents the reaction catalyzed by pyruvate kinase , for which two isoenzymes exist which have changed in expression differently , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . black arrows represent unchanged steps ; solid arrows represent single biosynthetic steps ; and dashed arrows represent multiple steps . we also observed a remarkable upregulation of glutamine synthetases i and ii ( sclav_1416 and sclav_1431 ) , glutamate synthetases ( sclav_1231 ) and glutamate importers ( sclav_46604663 ) . this conversion takes place through the urea cycle involving ornithine as an intermediate ( rodrguezgarca et al . , 2000 ) , addition of which to the medium has been shown to strongly enhance clavulanic acid biosynthesis ( cheng et al . , 2003 ) . probably to overcome nitrogen and phosphate limitations , genes encoding the transporters for ammonia ( sclav_4534 ) and phosphate ( sclav_31663169 ) are also observed to be more highly transcribed in ds48802 . this may be caused by the increased expression of the pathwayspecific activator genes phou ( sclav_3220 , ghorbel et al . , 2006 ) and glnb ( sclav_4535 , drepper et al . , 2003 ) , which both show over twofold increased transcription . comparing gene transcript levels of s. clavuligerus wildtype and ds48802 strains during stationary phase using microarrays , almost all genes ranking high in a differential transcriptome analysis belong to the complete clavulanic acid / cephamycin c supercluster , which is significantly overexpressed ( between twofold and eightfold ) in the ds48802 strain compared to the wildtype . they are located within the same supercluster and their products have been shown to regulate it positively ( alexander and jensen , 1998 ; paradkar et al . , 1998 ) . additionally , the clavams gene cluster ( cvm1245 and cas1 ) and the paralogous alanylclavam cluster ( orfabcd and ceas1/pah1/bls1/oat1 ) are significantly overexpressed ( fig . 1 ) , as is the twocomponent system involving cvm7p ( sclav_p1079p1080 ) that induces expression of the alanylclavam cluster ( tahlan et al . , 2007 ) ccar is an unlikely candidate for such a common regulatory factor , as it does not appear to control the paralogous cluster ( tahlan et al . , 2004 ) ; the pleiotropic regulator adpa ( sclav_1957 ) is a more likely candidate , as it is known to induce clavulanic acid expression ( lopezgarca et al . , 2010 ) and differential gene expression in s. clavuligerus ds48802 and atcc 27064 . sliding window plot ( size = 50 ) of the difference in gene expression between s. clavuligerus ds48802 and wildtype atcc 27064 . see table s2 for description of the ten gene clusters shown . for gene expression analysis , cultivations were performed in shake flasks directly inoculated with spore suspensions at 28c and 280 r.p.m . the semisynthetic growth medium used consisted of 30 g l glycerol , 5 g l wheat gluten , 3.5 g l asparagine monohydrate , 1.5 g lllysine , 0.7 g l kh2po4 , 0.3 g l mgso47h2o , 0.2 g l cacl22h2o , 0.2 g l feso47h2o , 10 g l mops , 0.1 ml l basilodon and 1 ml l trace elements solution at ph 7.0 . the trace element solution consisted of 20.4 g l h2so4 , 50 g l citric acid h2o , 16.75 g l znso47h2o , 1.6 g l cuso4 . 5 h2o , 1.5 g l mncl24h2o , 2 g l h3bo3 and 2 g l na2moo42h2o . after 70 h of cultivation , the cells were harvested by centrifugation , treated with rnaprotect ( qiagen ) and directly frozen with liquid nitrogen and stored at 80c . to isolate total rna , the frozen mycelium was ground in a mortar , resuspended in te buffer with 5 mg l lysozyme and incubated for 5 min at room temperature . rna isolation and purification were performed using phenol extraction ( trizol reagent , invitrogen ) and rneasy kit ( qiagen ) . the rna was quantified by measuring the absorbance at 260 nm . biotinylated cdna was prepared after fragmentation according to the standard affymetrix protocol using gc rich ( average 72% ) primers from 10 g total rna . for hybridization , 5 g and 7 g biotinylated cdna were used per affymetrix gene chip . microarray data have been deposited at gene expression omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number gse24033 . fluxbalance analysis was performed using a recently published genomescale metabolic model of s. clavuligerus ( medema et al . , 2010 ) . in this study , we slightly changed our objective function and included both clavulanic acid and cephamycin c biosynthesis pathways . we dynamically changed the antibiotic concentration in the biomass composition based on experimental observations of romero and colleagues ( 1984 ) and optimized the objective function for different concentrations of each antibiotic . among the 785 genes that the model contains , 497 genes showed nonzero flux for at least one antibiotic concentration . we calculated spearman correlation of fluxes of each reaction with increasing antibiotic concentrations . if an enzyme was involved in multiple reactions , we assigned the flux which had the highest r. in contrast to , for example , the intrachromosomal amplification of the kanamycin biosynthesis gene cluster in streptomyces kanamyceticus ( yanai et al . , 2006 ) , hybridization of s. clavuligerus ds48802 genomic dna to the microarrays revealed no amplifications of genes or gene clusters ( data not shown ) . the overproduction that we observe therefore appears to be caused by transcriptional ( and posttranscriptional ) changes only . while many changes have occurred during the generation of the ds48802 strain through mutagenesis , it is important to note that changes in transcription levels of many genes may be due to random mutations that have no impact on antibiotic biosynthesis . in order to assess which changes could be causatively linked to antibiotic overproduction , we computationally predicted the metabolic fluxes during antibiotic overproduction , using a constraintsbased genomescale metabolic network model of s. clavuligerus ( medema et al . , 2010 ) . we dynamically modelled the metabolic flux changes during increased production of clavulanic acid and cephamycin c with different rates of antibiotic production relative to the biomass , based on the experimental observations of romero and colleagues ( 1984 ) . interestingly , the computational predictions made through dynamic fba are largely in line with the observed expression changes . r > 0.6 ) and 129 genes were predicted to be downregulated ( negatively correlated ; r < 0.6 ) at increasing antibiotic production levels . forty per cent ( 15/37 ) of the genes that actually showed increased transcript levels ( fold change > 2 ) were also predicted to do so according to fba , and these include all genes encoding key biosynthetic enzymes known to be involved in clavulanic acid and cephamycin c biosynthesis . even though 40% does not appear to be a very large percentage , these predictions are statistically very significant according to a fisher 's exact test ( p = 0.0005 ; see table s1 ) . one should note that fba predicts the flux for every reaction , not for every gene product , as multiple gene products can be involved in a single reaction . therefore , in these cases the same flux was assigned to all gene products involved in that particular reaction , which is not necessarily the case in the actual gene expression , as only a single homologue or isoenzyme could be actively performing the reaction and thus would be differentially expressed . out of the 47 different enzymatic reactions predicted to be upregulated ( associated with 87 genes ) , 26 ( 55% ) have at least one gene linked to them , which showed increased transcript levels . as both our fba and gene transcript analysis pointed to an increased expression of the clavulanic acid and cephamycin core biosynthesis genes , we suggest that this is a crucial change required for antibiotic overproduction in this strain . moreover , as the fba indicated that the absolute fluxes required for high production of the secondary metabolites are minor compared to other fluxes involved in maintenance and cellular growth , a complete redirection of primary metabolism appears not to be necessary for overproduction . nonetheless , because clavulanic acid is synthesized from the precursors g3p and larginine , which play important roles in primary metabolism , specific changes in the primary metabolism of ds48802 could have occurred during the various random mutagenesis rounds , so that the intracellular pools of these intermediates are increased . indeed , glycerol uptake and metabolism ( sclav_06310632 and sclav_08770879 ) is clearly upregulated over twofold in ds48802 , indicating an improved utilization of glycerol as a carbon source as well as increased production of the clavulanic acid precursor g3p ( fig . 2 ) . moreover , the aconitase and citrate synthase from the citric acid cycle appear to be downregulated . a likely explanation for this is that the carbon flux from g3p in this direction is reduced and is partly redirected to clavulanic acid biosynthesis . this situation is remarkably similar to the result of the rationally constructed gap1 deletion that blocked g3p conversion into 1,3bisphosphoglycerate , thus improving clavulanic acid biosynthesis by increasing the intracellular g3p pool ( li and townsend , 2006 ) . however , an advantage of the situation in ds48802 , which seems to have an incomplete downregulation of the flux , could be that a considerable pool of acetylcoa is maintained , e.g. for the biosynthesis of ornithine from glutamate . ds48802 also seems to avoid the potential negative effects of a complete deletion of the aconitase and citrate synthase genes : a complete absence of these enzyme activities could lead to acidogenesis with negative consequences for secondary metabolite production as shown by viollier and colleagues ( 2001a , b ) . moreover , ds48802 still seems to be able to synthesize ketoglutarate ( a cosubstrate required for clavaminic acid biosynthesis ; salowe et al . , 1990 ) , while achieving the benefits of higher acetylcoa and/or g3p pools that have made these genes attractive targets for rational engineering to improve antibiotic production ( viollier et al . , 2001a ) . a potentially important observation that we can not explain yet from the current data are the differential transcript level changes of the two pyruvate kinase isoenzyme genes , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . changes in gene expression in s. clavuligerus ds48802 compared to the wildtype atcc 27064 projected onto a metabolic map . green arrows represent reactions catalyzed by genes expressed over twofold higher in ds48802 than in the wildtype . the orange arrow represents the reaction catalyzed by pyruvate kinase , for which two isoenzymes exist which have changed in expression differently , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . black arrows represent unchanged steps ; solid arrows represent single biosynthetic steps ; and dashed arrows represent multiple steps . we also observed a remarkable upregulation of glutamine synthetases i and ii ( sclav_1416 and sclav_1431 ) , glutamate synthetases ( sclav_1231 ) and glutamate importers ( sclav_46604663 ) . this conversion takes place through the urea cycle involving ornithine as an intermediate ( rodrguezgarca et al . , 2000 ) , addition of which to the medium has been shown to strongly enhance clavulanic acid biosynthesis ( cheng et al . , 2003 ) . probably to overcome nitrogen and phosphate limitations , genes encoding the transporters for ammonia ( sclav_4534 ) and phosphate ( sclav_31663169 ) are also observed to be more highly transcribed in ds48802 . this may be caused by the increased expression of the pathwayspecific activator genes phou ( sclav_3220 , ghorbel et al . , 2006 ) and glnb ( sclav_4535 , drepper et al . , 2003 ) , which both show over twofold increased transcription . our data show that a strain improvement program by random mutagenesis and screening has caused gene transcript changes in both primary and secondary metabolism . the overlap with results obtained by rational metabolic engineering through clar / ccar overexpression and gap1 deletion is intriguing . new leads from transcript changes observed in this study , such as the increased transcription of glutamine and glutamate synthetase genes , and of those encoding ammonium and phosphate transporters , can now be combined to rationally design novel highproducer strains . this approach might avoid the introduction of unwanted adverse effects from random mutagenesis , and provide strains suited for industrial application in a more efficient way . in this manner , functional genomics allows two key strategies applied in biotechnology random mutagenesis and rational engineering to become increasingly complementary . additional supporting information may be found in the online version of this article : table s1 . statistical significance and comparison of transcriptional changes with fba predictions using different cutoffs . . please note : wiley - blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors . any queries ( other than missing material ) should be directed to the corresponding author for the article .

summaryto increase production of the important pharmaceutical compound clavulanic acid , a lactamase inhibitor , both random mutagenesis approaches and rational engineering of streptomyces clavuligerus strains have been extensively applied . here , for the first time , we compared genomewide gene expression of an industrial s. clavuligerus strain , obtained through iterative mutagenesis , with that of the wildtype strain . intriguingly , we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters . a few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid . in general , the observed changes largely coincide with genes that have been targeted by rational engineering in recent years , yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology .

Introduction Results and discussion Increased transcription of secondary metabolite biosynthesis gene clusters in strain DS48802 Fluxbalance analysis of increased clavulanic acid production correlates well with transcriptomic data Gene expression changes in primary metabolism Conclusions Supporting Information

streptomyces clavuligerus is an important industrial microorganism , which produces the lactam antibiotic cephamycin c ( martn and liras , 1989 ) and the lactamase inhibitor clavulanic acid ( saudagar et al . clavulanic acid is produced worldwide on a large scale , and coformulated with amoxicillin in augmentin ( brogden et al . two biotechnological strain optimization approaches have been utilized to increase the production of clavulanic acid by the bacterium : rational metabolic engineering and iterative optimization through random mutagenesis and screening . in the rational approach , a specific gene is knocked out or overexpressed to divert metabolic fluxes towards the antibiotic biosynthetic pathways . arguably , the best example comes from the work of li and townsend ( 2006 ) , who reengineered the s. clavuligerus glycolytic pathway by constructing a deletion mutant of the glyceraldehyde 3phosphate dehydrogenase gene gap1 to increase the pool of the clavulanic acid precursor glycerol 3phosphate ( g3p ) . overexpression of the regulatory proteins ccar and clar also led to clavulanic acid overproduction ( hung et al . yet , most or even all production strains that are used in industry have been obtained by classical strain improvement ( adrio and demain , 2006 ) , based on mutagenesis with mutagens such as nitrosoguanidine ( ntg ) . recently , we published the genome sequence of s. clavuligerus atcc 27064 ( medema et al . we now employed this information to perform a genomewide transcriptome study on an industrial production strain , which has been generated from the atcc 27064 type strain ( higgens and kastner , 1971 ) by several iterations of mutagenesis and screening , and produces clavulanic acid at levels approximately 100 that of the wildtype . the majority of observed changes consist of increased transcript levels of the antibiotic biosynthesis gene clusters . these observations are in agreement with fluxbalance analysis ( fba ) predictions using a constraintbased genomescale metabolic model constructed for s. clavuligerus . however , we also detected some potentially crucial transcript level changes in primary metabolism that could contribute to the increased production of clavulanic acid by redirection of fluxes , mimicking strategies utilized in rational approaches . comparing gene transcript levels of s. clavuligerus wildtype and ds48802 strains during stationary phase using microarrays , almost all genes ranking high in a differential transcriptome analysis belong to the complete clavulanic acid / cephamycin c supercluster , which is significantly overexpressed ( between twofold and eightfold ) in the ds48802 strain compared to the wildtype . additionally , the clavams gene cluster ( cvm1245 and cas1 ) and the paralogous alanylclavam cluster ( orfabcd and ceas1/pah1/bls1/oat1 ) are significantly overexpressed ( fig . 1 ) , as is the twocomponent system involving cvm7p ( sclav_p1079p1080 ) that induces expression of the alanylclavam cluster ( tahlan et al . , 2004 ) ; the pleiotropic regulator adpa ( sclav_1957 ) is a more likely candidate , as it is known to induce clavulanic acid expression ( lopezgarca et al . , 2010 ) and differential gene expression in s. clavuligerus ds48802 and atcc 27064 . sliding window plot ( size = 50 ) of the difference in gene expression between s. clavuligerus ds48802 and wildtype atcc 27064 . see table s2 for description of the ten gene clusters shown . for gene expression analysis , cultivations were performed in shake flasks directly inoculated with spore suspensions at 28c and 280 r.p.m . the semisynthetic growth medium used consisted of 30 g l glycerol , 5 g l wheat gluten , 3.5 g l asparagine monohydrate , 1.5 g lllysine , 0.7 g l kh2po4 , 0.3 g l mgso47h2o , 0.2 g l cacl22h2o , 0.2 g l feso47h2o , 10 g l mops , 0.1 ml l basilodon and 1 ml l trace elements solution at ph 7.0 . the trace element solution consisted of 20.4 g l h2so4 , 50 g l citric acid h2o , 16.75 g l znso47h2o , 1.6 g l cuso4 . after 70 h of cultivation , the cells were harvested by centrifugation , treated with rnaprotect ( qiagen ) and directly frozen with liquid nitrogen and stored at 80c . microarray data have been deposited at gene expression omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number gse24033 . fluxbalance analysis was performed using a recently published genomescale metabolic model of s. clavuligerus ( medema et al . in this study , we slightly changed our objective function and included both clavulanic acid and cephamycin c biosynthesis pathways . among the 785 genes that the model contains , 497 genes showed nonzero flux for at least one antibiotic concentration . if an enzyme was involved in multiple reactions , we assigned the flux which had the highest r. in contrast to , for example , the intrachromosomal amplification of the kanamycin biosynthesis gene cluster in streptomyces kanamyceticus ( yanai et al . , 2006 ) , hybridization of s. clavuligerus ds48802 genomic dna to the microarrays revealed no amplifications of genes or gene clusters ( data not shown ) . while many changes have occurred during the generation of the ds48802 strain through mutagenesis , it is important to note that changes in transcription levels of many genes may be due to random mutations that have no impact on antibiotic biosynthesis . in order to assess which changes could be causatively linked to antibiotic overproduction , we computationally predicted the metabolic fluxes during antibiotic overproduction , using a constraintsbased genomescale metabolic network model of s. clavuligerus ( medema et al . we dynamically modelled the metabolic flux changes during increased production of clavulanic acid and cephamycin c with different rates of antibiotic production relative to the biomass , based on the experimental observations of romero and colleagues ( 1984 ) . interestingly , the computational predictions made through dynamic fba are largely in line with the observed expression changes . forty per cent ( 15/37 ) of the genes that actually showed increased transcript levels ( fold change > 2 ) were also predicted to do so according to fba , and these include all genes encoding key biosynthetic enzymes known to be involved in clavulanic acid and cephamycin c biosynthesis . even though 40% does not appear to be a very large percentage , these predictions are statistically very significant according to a fisher 's exact test ( p = 0.0005 ; see table s1 ) . therefore , in these cases the same flux was assigned to all gene products involved in that particular reaction , which is not necessarily the case in the actual gene expression , as only a single homologue or isoenzyme could be actively performing the reaction and thus would be differentially expressed . as both our fba and gene transcript analysis pointed to an increased expression of the clavulanic acid and cephamycin core biosynthesis genes , we suggest that this is a crucial change required for antibiotic overproduction in this strain . moreover , as the fba indicated that the absolute fluxes required for high production of the secondary metabolites are minor compared to other fluxes involved in maintenance and cellular growth , a complete redirection of primary metabolism appears not to be necessary for overproduction . nonetheless , because clavulanic acid is synthesized from the precursors g3p and larginine , which play important roles in primary metabolism , specific changes in the primary metabolism of ds48802 could have occurred during the various random mutagenesis rounds , so that the intracellular pools of these intermediates are increased . indeed , glycerol uptake and metabolism ( sclav_06310632 and sclav_08770879 ) is clearly upregulated over twofold in ds48802 , indicating an improved utilization of glycerol as a carbon source as well as increased production of the clavulanic acid precursor g3p ( fig . a likely explanation for this is that the carbon flux from g3p in this direction is reduced and is partly redirected to clavulanic acid biosynthesis . this situation is remarkably similar to the result of the rationally constructed gap1 deletion that blocked g3p conversion into 1,3bisphosphoglycerate , thus improving clavulanic acid biosynthesis by increasing the intracellular g3p pool ( li and townsend , 2006 ) . however , an advantage of the situation in ds48802 , which seems to have an incomplete downregulation of the flux , could be that a considerable pool of acetylcoa is maintained , e.g. for the biosynthesis of ornithine from glutamate . ds48802 also seems to avoid the potential negative effects of a complete deletion of the aconitase and citrate synthase genes : a complete absence of these enzyme activities could lead to acidogenesis with negative consequences for secondary metabolite production as shown by viollier and colleagues ( 2001a , b ) . , 1990 ) , while achieving the benefits of higher acetylcoa and/or g3p pools that have made these genes attractive targets for rational engineering to improve antibiotic production ( viollier et al . a potentially important observation that we can not explain yet from the current data are the differential transcript level changes of the two pyruvate kinase isoenzyme genes , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . changes in gene expression in s. clavuligerus ds48802 compared to the wildtype atcc 27064 projected onto a metabolic map . green arrows represent reactions catalyzed by genes expressed over twofold higher in ds48802 than in the wildtype . the orange arrow represents the reaction catalyzed by pyruvate kinase , for which two isoenzymes exist which have changed in expression differently , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . , 2000 ) , addition of which to the medium has been shown to strongly enhance clavulanic acid biosynthesis ( cheng et al . this may be caused by the increased expression of the pathwayspecific activator genes phou ( sclav_3220 , ghorbel et al . comparing gene transcript levels of s. clavuligerus wildtype and ds48802 strains during stationary phase using microarrays , almost all genes ranking high in a differential transcriptome analysis belong to the complete clavulanic acid / cephamycin c supercluster , which is significantly overexpressed ( between twofold and eightfold ) in the ds48802 strain compared to the wildtype . they are located within the same supercluster and their products have been shown to regulate it positively ( alexander and jensen , 1998 ; paradkar et al . additionally , the clavams gene cluster ( cvm1245 and cas1 ) and the paralogous alanylclavam cluster ( orfabcd and ceas1/pah1/bls1/oat1 ) are significantly overexpressed ( fig . 1 ) , as is the twocomponent system involving cvm7p ( sclav_p1079p1080 ) that induces expression of the alanylclavam cluster ( tahlan et al . , 2010 ) and differential gene expression in s. clavuligerus ds48802 and atcc 27064 . sliding window plot ( size = 50 ) of the difference in gene expression between s. clavuligerus ds48802 and wildtype atcc 27064 . see table s2 for description of the ten gene clusters shown . for gene expression analysis , cultivations were performed in shake flasks directly inoculated with spore suspensions at 28c and 280 r.p.m . the trace element solution consisted of 20.4 g l h2so4 , 50 g l citric acid h2o , 16.75 g l znso47h2o , 1.6 g l cuso4 . after 70 h of cultivation , the cells were harvested by centrifugation , treated with rnaprotect ( qiagen ) and directly frozen with liquid nitrogen and stored at 80c . microarray data have been deposited at gene expression omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number gse24033 . in this study , we slightly changed our objective function and included both clavulanic acid and cephamycin c biosynthesis pathways . among the 785 genes that the model contains , 497 genes showed nonzero flux for at least one antibiotic concentration . if an enzyme was involved in multiple reactions , we assigned the flux which had the highest r. in contrast to , for example , the intrachromosomal amplification of the kanamycin biosynthesis gene cluster in streptomyces kanamyceticus ( yanai et al . , 2006 ) , hybridization of s. clavuligerus ds48802 genomic dna to the microarrays revealed no amplifications of genes or gene clusters ( data not shown ) . while many changes have occurred during the generation of the ds48802 strain through mutagenesis , it is important to note that changes in transcription levels of many genes may be due to random mutations that have no impact on antibiotic biosynthesis . in order to assess which changes could be causatively linked to antibiotic overproduction , we computationally predicted the metabolic fluxes during antibiotic overproduction , using a constraintsbased genomescale metabolic network model of s. clavuligerus ( medema et al . we dynamically modelled the metabolic flux changes during increased production of clavulanic acid and cephamycin c with different rates of antibiotic production relative to the biomass , based on the experimental observations of romero and colleagues ( 1984 ) . interestingly , the computational predictions made through dynamic fba are largely in line with the observed expression changes . forty per cent ( 15/37 ) of the genes that actually showed increased transcript levels ( fold change > 2 ) were also predicted to do so according to fba , and these include all genes encoding key biosynthetic enzymes known to be involved in clavulanic acid and cephamycin c biosynthesis . even though 40% does not appear to be a very large percentage , these predictions are statistically very significant according to a fisher 's exact test ( p = 0.0005 ; see table s1 ) . as both our fba and gene transcript analysis pointed to an increased expression of the clavulanic acid and cephamycin core biosynthesis genes , we suggest that this is a crucial change required for antibiotic overproduction in this strain . moreover , as the fba indicated that the absolute fluxes required for high production of the secondary metabolites are minor compared to other fluxes involved in maintenance and cellular growth , a complete redirection of primary metabolism appears not to be necessary for overproduction . nonetheless , because clavulanic acid is synthesized from the precursors g3p and larginine , which play important roles in primary metabolism , specific changes in the primary metabolism of ds48802 could have occurred during the various random mutagenesis rounds , so that the intracellular pools of these intermediates are increased . indeed , glycerol uptake and metabolism ( sclav_06310632 and sclav_08770879 ) is clearly upregulated over twofold in ds48802 , indicating an improved utilization of glycerol as a carbon source as well as increased production of the clavulanic acid precursor g3p ( fig . a likely explanation for this is that the carbon flux from g3p in this direction is reduced and is partly redirected to clavulanic acid biosynthesis . this situation is remarkably similar to the result of the rationally constructed gap1 deletion that blocked g3p conversion into 1,3bisphosphoglycerate , thus improving clavulanic acid biosynthesis by increasing the intracellular g3p pool ( li and townsend , 2006 ) . for the biosynthesis of ornithine from glutamate . ds48802 also seems to avoid the potential negative effects of a complete deletion of the aconitase and citrate synthase genes : a complete absence of these enzyme activities could lead to acidogenesis with negative consequences for secondary metabolite production as shown by viollier and colleagues ( 2001a , b ) . , 1990 ) , while achieving the benefits of higher acetylcoa and/or g3p pools that have made these genes attractive targets for rational engineering to improve antibiotic production ( viollier et al . a potentially important observation that we can not explain yet from the current data are the differential transcript level changes of the two pyruvate kinase isoenzyme genes , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . changes in gene expression in s. clavuligerus ds48802 compared to the wildtype atcc 27064 projected onto a metabolic map . green arrows represent reactions catalyzed by genes expressed over twofold higher in ds48802 than in the wildtype . the orange arrow represents the reaction catalyzed by pyruvate kinase , for which two isoenzymes exist which have changed in expression differently , one being downregulated ( sclav_4329 ) and the other being upregulated ( sclav_1203 ) . , 2000 ) , addition of which to the medium has been shown to strongly enhance clavulanic acid biosynthesis ( cheng et al . this may be caused by the increased expression of the pathwayspecific activator genes phou ( sclav_3220 , ghorbel et al . our data show that a strain improvement program by random mutagenesis and screening has caused gene transcript changes in both primary and secondary metabolism . the overlap with results obtained by rational metabolic engineering through clar / ccar overexpression and gap1 deletion is intriguing . this approach might avoid the introduction of unwanted adverse effects from random mutagenesis , and provide strains suited for industrial application in a more efficient way . in this manner , functional genomics allows two key strategies applied in biotechnology random mutagenesis and rational engineering to become increasingly complementary . statistical significance and comparison of transcriptional changes with fba predictions using different cutoffs . any queries ( other than missing material ) should be directed to the corresponding author for the article .

depression is a major public health problem that is supposedly twice as common in women during the childbearing age as it is in men . . it accounts for the greatest burden of disease among all mental health problems , and it is expected to become the second most prevalent of all general health problems globally by 2020 [ 2 , 3 ] . according to describe depression not just as a syndrome but also as an affective state that might be experienced by anyone at some point in their daily lives . according to them , depression is a mood disorder where mood refers to the prolonged emotions that colour psychic life . affects , on the other hand , are the feeling tones or emotional states and their manifestations at a given moment . mood disorders affect anybody without distinction of class , ethnicity , gender , education , age , or religion . postnatal depression , a type of depression and , therefore , a mood disorder , is experienced by 1 in 10 women in the united kingdom ( uk ) . technically defined , postnatal depression is constructed as an affective mood disorder often occurring in women up to one year after childbirth . furthermore , it is often characterised by feelings of loss and sadness and , sometimes , the loss of self - esteem . the depressive scale of this disorder and its presentation ranges from mild depression requiring minimal intervention to puerperal psychosis which often requires multitherapy intervention , hospitalization , and long - term support . postnatal depression has profound effects on the quality of life , social functioning , and economic productivity of women and families . the health consequences could also lead to adverse effects on the long - term emotional and physical development of the infant [ 1012 ] . it is also predictive of child cognitive and behavioural disturbances at the age of 3 years [ 13 , 14 ] . moreover , failure by health professionals to identify postnatal depressed women often leads to safeguarding concerns for both mothers and infants [ 15 , 16 ] . in general , health professionals and , in particular , health visitors in the uk play a vital role in identifying and supporting women who experience postnatal depression in the community . their role includes supporting families during the period from the birth of the child to the age of five , thus enabling them to provide a prolonged period of contact and support for women affected by it . however , evidence suggests that most vulnerable women , including black minority ethnic ( bme ) groups in the uk , do not always access or demand this service . this is because the symptoms are either overlooked or endured in silence by the women themselves in some cases , or they are not picked up by the health professionals in others [ 1 , 5 , 1720 ] . in a study by and in the study reported here , some women were reluctant to expose frailty and stigma , thus making it difficult for health professionals to provide adequate diagnoses or treatment . there is a national recognition that over 6 million of the population of the united kingdom experience some form of mental illness ( depression ) ; however , about 2 million of these do not have access to psychological therapies . the fact that the bme population is growing in the uk and in south east london generates the need to explore and understand african women immigrants ' perception of postnatal depression in order to improve service development and outcomes for them . for example women who are affected by postnatal depression often find themselves isolated and unable to return to employment . this problem is further exacerbated by the overwhelming evidence of the link between depression and domestic violence , and the differing conceptualisations of postnatal depression among health professionals . this study presents the findings of an earlier exploration of the perception of postnatal depression in african women immigrants in south east london . the aims of this research were twofold : firstly , to establish cultural elements related to postnatal depression through women 's narratives regarding their daily life situations including the nuances and complexities present in postnatal depression , and secondly , to help health professionals understand and acknowledge postnatal depression signs in these immigrant women and some of the cultural ambiguities surrounding them . the literature on mental health illness addresses the fact that people from diverse cultural backgrounds might display different constructions of mental health illness and , therefore , various ways of handling and coping with it . for example , some studies on depression among south asian women , in particular punjabis , have identified a cultural idiom called sinking heart which they experience as a result of excessive heat , exhaustion , worry , or a feeling of social failure . similarly , among women of african descent in america who had experienced postpartum depression ( ppd ) in the past , a study reports that they described and managed their depression in culturally specific ways , such as relying on their religious beliefs and the counsel of family members as well as keeping the depression a secret in the family . the women also believed that only white women experience postnatal depression , as postnatal depression is considered a sign of weakness that does not represent a legitimate illness . some authors argued that the individual is bound by the rules of their culture which in turn shape and influence their behaviour [ 28 , 29 ] . similarly , cultural aspects of one 's social system have a major impact on one 's emotional life . major cultural differences influencing depression are family structure and dynamics , social organization , socially - sanctioned defence mechanisms , rituals , and social stresses . other cultural factors that may be important for a general understanding of depression include a distinctive language related to depression , the transmission of information among people about depression , and beliefs about healthcare and the healthcare system . studies in developed countries report prevalence rates of 10% or more for postpartum depression . in developing societies postnatal depression is thought to occur three times more in the developing societies than in developed ones , for example , in khayelitsha , cape town , in south africa , the prevalence rate of major depression was reported to be 34.7% at two months postpartum . other african studies that have looked at postpartum women have dealt with the prevalence of psychological distress in general rather than focusing on major depression or postnatal depression [ 3335 ] , which looked at prevalence of major depression at six weeks postpartum in uganda , found a figure of 6.1% . similarly , the literature on mental health illness addresses the fact that aspects such as perceptions and attitudes towards depression in different cultures may affect help - seeking behaviour and access to treatment [ 10 , 11 ] . most studies in the literature regarding women 's health - seeking behaviour coincide , for example , in pointing out that bme women tend to rely on family and religion as their main coping strategies . in the same way , for some women , postnatal depression is not perceived as an illness , yet they recognise the need to seek spiritual intervention . equally , additional findings suggest that some black caribbean women face difficulties describing or talking about perinatal depression due to their tendency to underreport their psychological feelings . thus , barriers to health - seeking behaviour relate very much to the reluctance of some bme women to discuss problems as well as the way in which problems are dealt with [ 21 , 27 , 36 , 37 ] . another significant and controversial aspect of the literature , particularly in regard to the uk , relates to the diagnosing of postnatal depression among bme women through the edinburgh postnatal depression scale ( epds ) . it comprises a ten - item self - rating questionnaire which is administered by health visitors approximately at eight weeks and at twelve months after childbirth . the epds is used by health visitors in the community . following its validation for use in the uk it was implemented across the country by health visitors as a universal method of identifying mothers who were at risk of postnatal depression argued that epds was originally designed as a screening test and was not intended as a diagnostic tool . however , many gp practices have continued to utilise the questionnaire as a single psychometric diagnostic tool . thus , the controversy in the literature regarding the use of edps displays two positions : the one that considers this tool as culturally insensitive for bme women [ 40 , 41 ] and the one that argues that it is effective [ 42 , 43 ] . the literature that considers the epds to be less culturally sensitive to the needs of women from black and ethnic minority backgrounds states that it does not translate into other languages , let alone cultures [ 44 , 45 ] . these authors also cautioned against direct translations of the tool , pointing out that some cultures do not have a word for depression , and suggesting that other screening methods should be considered depending on ethnicity . it is argued that the use of standardised western methods and diagnostic classification systems , even by local - but - westernised investigators , may be culturally insensitive and could increase the risk of practitioners missing symptoms or signs prevalent in non - western cultures [ 46 , 47 ] . using epds as the assessment tool for these women might result in them additional arguments related to this position in the literature claim that most research has been conducted in the western developed countries [ 31 , 48 ] and has not taken into account the range of different psychosocial experiences likely to be involved in childbirth , for example differences in rates of lone motherhood , the nature of marriage , family kinship , and variations in the support new mothers receive in different countries and cultures . for those who consider epds an effective tool for diagnosing postnatal depression the main evidence comes via some empirical studies that have screened women to check prevalence and associated factors in two groups : nigerian and black caribbean women reporting a significant level of diagnosis [ 42 , 43 ] . on the part of the health professionals , the literature addresses various factors that could contribute to the lack of awareness , late diagnoses , undetected cases or , worse , excessive medicalisation of symptoms . for example , in a study investigating the influences of cultural factors in relation to postpartum depression , found that mothers from different cultural backgrounds may display culturally explicit behaviours and actions when suffering from depression . another author argued that the way a person perceives and understands their health is related to the subjective cultural experience in her or his society . posits the idea that all cultures are unstable and subject to daily variations , innovations and change . similarly , clearly demonstrates this in a study on how women understood and responded to depression according to their cultural understanding of the disorder . according to , culture can be understood as shared beliefs , learned values , and attitudes which shape and influence perception and behaviour . in other words , african women immigrants in the uk could be seen as a group of people who share history , religion , language , thoughts and , overall , the experience of being immigrants . thus , how the cultural background of women is understood and constructed by the providers of health services and how these providers and the women communicate is a matter of great interest for researchers focusing on intercultural communication in the context of health services in various multicultural societies . ultimately , although postnatal depression affects all women regardless of ethnicity or social class , additional contributory risk factors include social exclusion , deprivation , and relationship complexities . thus , despite all the attempts in the literature to explain the causes of this illness , no single factor has been successfully identified as its cause . on the contrary , as discussed above , several explanations have been put forward by the literature . this qualitative study presented here expects to add to this ample range of explanations in the literature on postnatal depression particularly among african women immigrants in south east london . the qualitative study reported here used focus groups as a means of collecting data from participants . the focus group is an in - depth , open - ended group discussion that explores a specific set of issues on a predefined and limited topic . focus groups within feminist work have been devised to elicit and validate collective testimonies and group resistance narratives . these testimonies and narratives have been used by women and could be used by any subjugated group to unveil specific and little - researched aspects of women 's daily lives , their feelings , attitudes , hopes , and dreams [ 54 , page 835 ] . they can also facilitate the identification of cultural values and are said to be valuable when researching ethnic minority groups . the group targeted for the study was all african women immigrants registered on the general lists of the health visitors of four health centres in south east london . participants were contacted personally , through leaflets , and a phone call by the researchers but the gate keepers for the research were the health visitors . twenty - six immigrant women of african background between sixteen and forty - five years of age were purposively identified and selected . however after the selection of twenty six women , only seventeen were able to confirm and attend the focus groups ( see table 1 below ) . these were divided into two groups as seventeen people would had been a too large number for only one focus group . the women were then given the choice of the focus group they preferred to attend . the numbers of women for each group were within the standard methodological recommendation for a number of people in a focus group which can be from six to nine people in accordance with . furthermore , the purposive selection followed suggestion that a deliberate nonrandom sampling should include a group of people with particular characteristics , in this case : immigrant women of african background . during the two focus groups a psychologist was in attendance and acted as a research cofacilitator , taking field notes . they help to organise play activities for the toddlers who came with their mothers and with the signing of the consent forms . further , each focus group lasted for about two hours with fifteen - minute breaks in between . the two groups were held in a children 's centre , a familiar environment for the participants . having a cofacilitator meant that the two other researchers were able to lead the discussion while the cofacilitator was free to take notes and assist with the subsequent transcription of the data collected . all activities took place in the same large room and no major disruptions took place . the main inclusion criteria which applied to the women participating in the research were ( i ) women in the postnatal period who had delivered a baby up to a year earlier , ( ii ) immigrant women who identified themselves as being of african descent between the ages of 16 and 45 years ( the age range within which women 's fertility and reproductive capacity is at its peak ) , ( iii ) women who spoke and understood english , ( iv ) women whose bab(ies ) were in a good health , and ( v ) women who lived in the south east ( women with little children are always busy and will hesitate to attend any group if the distance is more than two miles ) and ( vi ) . the inclusion criteria were kept flexible . however , strict exclusion criteria were : ( i ) women who had children subject to the child protection procedures , ( ii ) women aged over 45 years , and ( iii ) women who were under 16 years of age ( parental consent issues ) . ethical approval for the research was obtained from the south london research and ethics committee . the national health service trust also gave the required research and development ( r&d ) approval . ethical research committees in the uk endorse the declaration of helsinki which seeks to minimize harm towards research participants . thus , in this case voluntary informed consent , confidentiality and anonymity were explained to the women at the beginning of each focus group . they also were informed about their right to withdraw at any point should any discomfort arise . they were also given a gift voucher as a token of appreciation for attending the focus groups . in order to collect women 's narratives , the logic of qualitative inquiry was used . in the discussion on research into sensitive issues such as postnatal depression , and the differences between qualitative and quantitative studies , an important phenomenon to recall is the ergodic hypothesis as postulated by george devereux in the seventies : the analysis of a great number of relatively superficial facts ampleness provides exactly the same insights as the in - depth analysis of only one phenomenon . ampleness is depth , rotating 90 degrees in horizontal position ; the depth is ampleness if the 90-degree turn is on a vertical position . the equivalence of the two phenomena takes exact root in the ergodica hypothesis in fact , under this premise , it is postulated that the same results are obtained whether throwing x number of coins simultaneously ( for example , 10 coins at the same time ) or only one coin x number of times ( 1 coin 10 times ) . in the context of social research , this yields an equivalence for a survey with one thousand answers of yes or no type questions , and three in - depth interviews of , say , 4 hours the analysis of a great number of relatively superficial facts ampleness provides exactly the same insights as the in - depth analysis of only one phenomenon . ampleness is depth , rotating 90 degrees in horizontal position ; the depth is ampleness if the 90-degree turn is on a vertical position . the equivalence of the two phenomena takes exact root in the ergodica hypothesis in fact , under this premise , it is postulated that the same results are obtained whether throwing x number of coins simultaneously ( for example , 10 coins at the same time ) or only one coin x number of times ( 1 coin 10 times ) . in the context of social research , this yields an equivalence for a survey with one thousand answers of yes or no type questions , and three in - depth interviews of , say , 4 hours the researcher asked the women at the very beginning of the two focus groups about their marital status and the kind of support network they had at home . studies have shown that women 's marital status and the kind of support network they have are significant risk factors that may predispose a woman to postnatal depression . similarly , their educational background or employment status may also affect their perception and the way they describe postnatal depression . thus , a basic task here was to map the sociodemographic characteristics of the participants as illustrated in table 1 . first , according to , in a group setting group norms may silence dissent ; indeed , in one of the focus groups a social hierarchy was observed . one of the women tended to dominate the group , silencing any mention of family problems and thus intimidating the less confident women within the group . this was dealt with by inviting the less vocal among the women to contribute their experiences . an attempt was also made to contact these individuals soon after the group to ensure they actually had a voice in the data collection process . secondly , an observation that became pretty obvious during the two focus groups was that those participants who were more educated , up to degree level , were more vocal and their perception of the symptoms of postnatal depression were freely expressed whereas those who had achieved gcse level were less expressive . in fact the most vocal and educated women in the first focus group contributed to within data saturation , since they articulated many themes setting the trend , that reemerged in the second focus group and , later , during the analysis of the data . following each focus group , theme analysis comprises the process of examining a piece of data as many times as possible until patterns or themes emerge . a theme , broadly speaking , is nothing more than a cluster of similar units of meaning that had been stated by the different participants in the focus group . in the particular case of the study reported here initial coding of the transcripts was performed with the goal of remaining open to all possible interpretations . codes either stored information about patient demographics or were far more analytical , representing links between the data and an idea . thus , once the data were sorted by units of meaning , the themes were identified . the themes were grouped and examined in all cases to make sure that all the descriptions of each theme had been captured and compared . table 2 shows some of the most significant stages in the thematic analysis of the data ( narratives ) . the first column on the left shows some significant excerpts from the statements provided by the women . however , this paper moves from just description of the themes to examine how the themes are interconnected according to pope et al . . the results discussed here present the data through the main themes and literal quotations ( narratives ) as stated by the women regarding events , episodes , points of view , settings , and comments related to the main focus of this research : postnatal depression among a group of african women immigrants in south east london . the main themes accordingly were : responses to their pregnancy , feelings before and after giving birth , social support or the lack of it , feeling alone , lack of information about health services , poverty , signs of postnatal depression , and not coping with their situation . this was to allow them to consider what their reaction had been at the time and to describe their emotional feelings . ranging from the ones who experienced difficulties in seeing the good side of being pregnant to the ones who were happily surprised , the main trend in this theme was that being pregnant is always a good thing for all women but particularly for african women immigrants who are married or cohabiting . it was considered that if a woman is not married , nobody cares whether she is pregnant or not ; once married , the expectation is that she is ready for the responsibility and that includes having babies . for example , describing her experience , a first - time mother educated up to secondary school level said : when i first became pregnant , i was scared , during the pregnancy it was quite difficult for me because i felt ill , sick all the time , lost a lot of weight and this happened throughout the pregnancy . when i first became pregnant , i was scared , during the pregnancy it was quite difficult for me because i felt ill , sick all the time , lost a lot of weight and this happened throughout the pregnancy . participants also reported their experiences and feelings during the antenatal period that made it extremely difficult for them to see the good side of being pregnant . i 'm very healthy but not so happy as there are all other stuff going on in my mind , such as not being with the father or married to him , only empty promises , happy but sad . so i realise it was the pregnancy , it was mixed feelings i 'm very healthy but not so happy as there are all other stuff going on in my mind , such as not being with the father or married to him , only empty promises , happy but sad . the women reported emotional feelings associated with being pregnant and almost all participants reported these . for example , a woman reported the emotional stress she incurred by getting pregnant while still at school and not finishing her secondary school education . the participants described feelings of being sad , angry , annoyed , and irritable , not with anyone else but with themselves ; they blamed themselves for being pregnant or evaluated the tasks before them and assessed their ability to cope with the tasks . as illustrated by another participant:this was my first baby , i was afraid and also i do n't have family here and was crying all the time and very lonely . this was my first baby , i was afraid and also i do n't have family here and was crying all the time and very lonely . the cultural ideal for describing the postnatal period was strongly endorsed by and pervasive amongst participants . this was accompanied by tacit acknowledgement that the actual experience of many women would fall short , leading to disappointment at unfulfilled expectations and potential risk of postnatal depression . rest was considered a necessity following the demands of pregnancy and childbirth but the participants were totally disappointed as the shock of having a crying baby hit them . they received no help from husbands or family members and had to cope with the demands that came with this new , vulnerable child , who needed help and care constantly.so i suppose i was happy to become a mum , emotionally i was sad , and i but i just kept crying and she was quite shocked and she spoke to me and said to me oh it 's ok to cry because if you do n't cry you can become very depressed . so i suppose i was happy to become a mum , emotionally i was sad , and i but i just kept crying and she was quite shocked and she spoke to me and said to me oh it 's ok to cry because if you do n't cry you can become very depressed . these feelings were attributed to practical problems : for example , having little opportunity to rest , and lack of support at home . these are serious issues in women 's lives so , for them , it was a case of finding a way to cope with feelings nobody else understood or which they could not explain to others for fear of being labelled or misunderstood . as various participants stated:so the first week was very difficult for me to cope with changing him , feeding him , it was n't easy , it was my first time , so i would cry sometimes ; and not until when my mum came , things were a little bit easier for me . so the first week was very difficult for me to cope with changing him , feeding him , it was n't easy , it was my first time , so i would cry sometimes ; and not until when my mum came , things were a little bit easier for me . sometimes like i said , it was mixed feelings . here symptoms are very much associated with a state of unhappiness following delivery , although by no means all recognised it as an illness postnatal depression or a requirement for treatment by healthcare professionals . the participants , being relatively recent immigrants to the uk , spoke of the difficulties they faced in this country , which promotes equality between men and women . on average as narrated by various women , they did all the household chores regardless of the number of days that had elapsed since the birth . they received no help from their husbands nor even from their mothers - in - law ; in fact , some women reported that the in - laws made matters worse . i mean having a baby is difficult but i did all the cooking , my in - laws were around , i was frustrated , nobody helped me do anything for six months , i changed the baby 's diapers all the time , nobody helped me once . i was cooking , i was going to the market , and there was no help for me . i cried a lot , felt rejected by my husband , so by the time i got pregnant the second time , i was n't looking forward to having the baby , i was . [ the in laws ] would come around just to see the baby , ask me how i 'm doing , and leave when they should actually be helping me , but they did n't do any of that . so that was one of the things that really got to me , like i 've got help here but . i mean having a baby is difficult but i did all the cooking , my in - laws were around , i was frustrated , nobody helped me do anything for six months , i changed the baby 's diapers all the time , nobody helped me once . i was cooking , i was going to the market , and there was no help for me . i cried a lot , felt rejected by my husband , so by the time i got pregnant the second time , i was n't looking forward to having the baby , i was . [ the in laws ] would come around just to see the baby , ask me how i 'm doing , and leave when they should actually be helping me , but they did n't do any of that . so that was one of the things that really got to me , like i 've got help here but . and although most of the participants had husbands they emphasised that they did not help at all . they described it as an african thing and called for awareness training for some african men in order to educate them on issues of the postnatal period . we do it this way . why do you want to do it that way ? so then whatever you want to say has been short out of you or stays in you . if you want to with mum , i 'll deal with the person who 's trying to tell you what you have to do . i mean for african perspective , this is how we do it . we do it this way . why do you want to do it that way ? so then whatever you want to say has been short out of you or stays in you . if you want to with mum , i 'll deal with the person who 's trying to tell you what you have to do . for some participants , their postnatal status meant that they were unable to escape and thus had to stay and endure the hardship . participants indicated that a major source of distress was being unable to share their emotions with their immediate family for fear of being seen as a failure . some participants spoke about talking to their husbands about their emotions while others did not hold any hope that it would improve the situation as the men do not see their role as providing help even though they live in the uk . participants felt there was no generally accepted person to talk to ; one participant pointed out that , even when the health visitors come , the mother - in - law is sometimes present , thus making her ( the participant ) uncomfortable about displaying her emotions in front of the mother - in - law . the quotations below are illustrative of this theme:it 's something nobody else can help you with . like someone can help you with the baby and help you with other things , but the way that you 're feeling , you do n't get help for that.that i 'm going mad , mentally and sometimes i 'd be crying the baby and i will be crying , and sometimes i feel like throwing the baby out , but i can't.i do n't know i ca n't imagine myself going to my mum , or my mother - in - law probably i can say to my mum , but i still did n't , i just could n't imagine saying to somebody , it 's just not it 's not something that you do , you just everybody expects you to get on with it and you get on with it . it 's something nobody else can help you with . like someone can help you with the baby and help you with other things , but the way that you 're feeling , you do n't get help for that . the baby and i will be crying , and sometimes i feel like throwing the baby out , but i ca n't . i ca n't imagine myself going to my mum , or my mother - in - law probably i can say to my mum , but i still did n't , i just could n't imagine saying to somebody , it 's just not it 's not something that you do , you just everybody expects you to get on with it and you get on with it . participants pointed out that there is a general lack of awareness among uk health visitors and healthcare professionals in identifying when african women immigrants are in distress or are crying out for help . a woman remarked that she was given a questionnaire ( referring to the epds ) , which to her made no sense:but they did n't really give me some of the other information that as a new mother i would have found really useful , without me having to look on the internet or buy a book . to me , the information given by the health visitor did not help me at all ; i had to search the internet myself for answers to some of my questions . but they did n't really give me some of the other information that as a new mother i would have found really useful , without me having to look on the internet or buy a book . to me , the information given by the health visitor did not help me at all ; i had to search the internet myself for answers to some of my questions . the same woman pointed out threats to her mental health arising from her postnatal experience . her inherent vulnerability and the potential consequences of her distressed mental state were not adequately dealt with , probably because of the professionals ' failure to capture the struggle and nuances , in this case , of what this woman was going through . this participant also felt she needed more information from the care providers that would have helped her cope better after the birth of her baby . another theme repeatedly raised by the participants is the issue of social class and working - class women . there is the issue of the father / partner , mother or both working long hours and not earning enough . there were also issues about providing for the family , because social welfare provision is not a practice in their country of origin or they do not have recourse to public funds due to their status as recent immigrants . as a result as another participant stated:with this baby i felt really depressed because i did n't want to have another child because i raised my other daughter on my own . i did n't want to do it all over again , so all the time i was up and down , some days i feel ok some days i feel really depressed . with this baby i felt really depressed because i did n't want to have another child because i raised my other daughter on my own . i did n't want to do it all over again , so all the time i was up and down , some days i feel ok some days i feel really depressed . participants described some of the signs of postnatal depression ; however , the main theme here is that they would not dare admit to their families or relatives that that was how they were feeling at that moment in their lives . the fears of being seen as a failure or being stigmatised or labelled as mad were some of the reasons women kept their feelings to themselves . i mean i was screaming at the midwife so i was just screaming , i cried , postnatal depression , so i said no , i want to go home . i mean i was screaming at the midwife so i was just screaming , i cried , postnatal depression , so i said no , i want to go home . participants also stated that there are instances when they feel out of control not coping and even sometimes the feeling of losing it completely . furthermore , almost all of the seventeen participants agreed that there is a problem with the present system of provision of healthcare services for african women immigrants in capturing these emotional distresses . they also recognised that they do not speak to professionals about their true emotional feelings for fear of being labelled depressed , which is a taboo or a stigma in both their country of origin and to a lesser extent in the uk . as another participant stated:so the urge to want to speak up and say hey , i actually need help you know , i 'm not coping here , superwoman , you know , i can do this , but you 're not . inside you 're not . so the urge to want to speak up and say hey , i actually need help you know , i 'm not coping here , superwoman , you know , i can do this , but you 're not . inside you 're not . the main findings were related to : response to participants ' pregnancies , feelings before and after giving birth , social support or the lack of it , feeling alone , lack of information about health services , poverty , signs of postnatal depression , and not coping with their situation . and as mentioned before , these were women 's statements that reached a good level of within - data saturation , since they emerged rapidly from the first focus group and kept being repeated in the subsequent focus group . in order to synthetize them , the statements are discussed in terms of the direct experience of the women ( feelings before and after giving birth , signs of postnatal depression , a feeling that they are not coping with the situation , feeling alone , and expectations of and responses to their pregnancy ) and , also in terms of perceived support from families ( husband and mother - in - law ) , community and health services ( lack of information and health visitors ) . the majority of the statements articulated by some of participants and related to their direct experiences indicated symptoms associated with a state of unhappiness following delivery , although by no means all recognised it as an illness postnatal depression or felt they required treatment by healthcare professionals as addressed too by the literature [ 10 , 11 , 21 , 27 , 36 , 37 ] . as one of the participants stated powerfully , rendering it invisible , it is something that you have to get on with . however , their narratives showed features such as distressed states manifested by sadness , irritability , anger and unhappiness , all of which characterise the medical construct of postnatal depression . so , certainly , the narrative of those states was comparable to the criteria for a diagnosis of postnatal depressive disorder but , as seen , these become invisible because they are not identified as postnatal depression as such . further descriptions of the women 's experiences related to their emotional feelings when facing lack of social support . cooking for the family , worrying about my finances and looking after the house . so , as the literature also shows , the lack of all types of social support can not be ignored here as a major risk factor for postnatal depression particular in immigrant women , whose close relatives are absent . in their narratives women expressed emotional distress as something different from physical illness , and that one may cause or influence the other . their descriptions fitted in many ways the psychosocial model of health as this conceptualises health as a bio - psychosocial issue [ 58 , 63 ] , rather than an exclusively biomedical issue . they acknowledged that emotional distress was a product of their social conditions combined with their own individual issues . according to the literature two major stressors for mothers of newborns going through the postnatal period are : ( i ) recovering from the immediate physiological changes caused by delivery ; ( ii ) returning to functional status ( which rarely returns to normal prior to six weeks ) during the days and weeks after delivery . thus , discussions with new mothers during this period must include those two stressors . in the case of women in the two focus groups , most of the stressors expressed by the women are included among the themes as , for example , mental stress such as loneliness , unmet expectations , birth plan disappointment and abandonment , and external stressors such as crying baby , sibling care , lack of support , and financial concerns . of course , while women in the study reported here did not experience all the stressors at once , most experienced some simultaneously . experiencing multiple stressors during the postnatal period can lead to sleeplessness , fatigue , and irritability , which were also part of the narratives . studies have shown that these multiple stressors are risk factors for postnatal depression [ 31 , 65 ] . from the women 's narratives it was apparent that their african cultural background has a bearing on their help - seeking behaviour . although some of them felt sad , unhappy , and stressed , they kept their feelings to themselves because , culturally , to admit having problems coping with the after - effects of childbirth is probably a sign of failure or weakness in front of the extended family husbands , mothers - in - law , and others . thus , as argues , the culturally appropriate terminology for depression seems to be an issue for further research here . also , as expressed in their narratives , the fact that women may choose to make their emotional problems invisible to health professionals could find explanation in their cultural backgrounds and their status as newly arrived immigrants . as the literature addresses , among various factors , cultural background is one that could lead to some women being undiagnosed , particularly african immigrant women in the uk . women from black ethnic minority backgrounds may not be diagnosed with postnatal depression because of their fear of being stigmatised and their cultural perception and understanding of postnatal depression [ 5 , 17 ] . the cultural background of immigrants as a factor in the concealment of postnatal symptoms is important because it is vital to recall that cultural practices are not frozen activities that determine unequivocally the behaviour of an individual . culture is reenacted by individuals daily and is responsible for the embedded ambiguity in the way they react . immigrants are neither in the old familiar place nor fully tailored to the new place including , among other things , their access to or demand for health services that are mainly biomedically oriented [ 28 , 29 , 67 ] . so the common conceptualization of culture by health services as a frozen element that determines people 's behaviour , attitudes , or understanding of the lifeworld should be modified to that of daily enactment and ambiguity which needs to be understood by the health professionals in each specific case . here , there is a need for skilful health professionals to work with women who experience this cultural ambiguity regarding postnatal depression . as some of the participants narrated , counting on their own mothers for support seems to have helped some participants cope with the distress they faced during this postnatal period although not in all cases , as a few never had the opportunity for their own mothers to be present due to their immigrant status . similarly , as described in the literature , turning to prayer in order to cope with their stress levels was seen by other participants as a coping mechanism , particularly for those with strong religious beliefs . interestingly , the two focus groups welcomed the idea of a group such as the focus group which allowed them to communicate their internal emotional struggles and feelings with women who were going through the same experience . another important and recurrent finding as presented in the narratives of the participants in the two focus groups was the mention of the mother - in - law as one of the sources of unhappiness in the dynamics of the household once the baby has arrived . as described by participants in some african groups , once a woman gets married , it is expected that , in the next few months , she will become pregnant . the absence of babies causes unfulfilled expectations in both families the woman is seen as an alien and the longer she remains without a child the more sadness she experiences . she receives unfriendly comments from her mother - in - law and other members of her husband 's family , and not even the husband will intervene in this matter . in the study reported here , this fact was revalidated by the comments of two of the participants who were married for about a year before conceiving . this happiness , however , was cut short by the arrival of their mothers - in - law , who did not help them at all but rather caused them distress . after the mother - in - law , the second most culpable figure seen as failing to provide any social support was the husband . and , although most of the participants had husbands , they emphasised that they did not help at all . they described it as an african thing and called for awareness training for some african men in order to educate them on issues of the postnatal period . ultimately , without denying the biomedical mechanisms present in postnatal depression , the interesting issue to discuss here in closing this discussion is the fact that the women through their narratives volunteer the sources of solutions based on the psychosocial model of health . social support from family , practical and emotional support from partners and having someone to talk to were unanimously expressed as the remedies for what they were experiencing : isolation and self - inflicted suffering endured in silence because of the fear of being labelled and stigmatised as emotionally unstable by the immediate family ( partners and in - laws ) . this overwhelming result found by the study reported here was also very well addressed by the literature on postnatal depression [ 21 , 27 , 3337 ] . contextualising the most important findings discussed above ( acknowledgement of postnatal depression , social support , and emotional distress , cultural identification and coping strategies ) in some of the recent discussions in social sciences , three issues are addressed below , particularly for health professionals to consider when offering health services for people of different cultural backgrounds , in this case african immigrant women . the dialogue between social sciences and nursing science is imperative in the context of framing health as a bio - psychosocial , cultural and political issue rather than a biomedical one . the first issue is that of the acknowledgement of suffering , which has been addressed by medical anthropology . in particular , have addressed the issue of the role of language in the incommunicability of pain and suffering . as the authors brilliantly state:to be in pain is to be certain about this knowledge . to be asked to react to another person 's pain is to be in doubt about its existence . from the perspective of theories of social suffering , such a preoccupation with individual certainty and doubt simply seems a less interesting , less important question to ask than that of how such suffering is produced in societies and how acknowledgement of pain , as a cultural process , is given or withheld . after all , to be ignorant or incapable of imagining another person 's pain does signal blindness in moral sensibility in the same way in which the incapacity to acknowledge that pain does to be in pain is to be certain about this knowledge . to be asked to react to another person 's pain is to be in doubt about its existence . from the perspective of theories of social suffering , such a preoccupation with individual certainty and doubt simply seems a less interesting , less important question to ask than that of how such suffering is produced in societies and how acknowledgement of pain , as a cultural process , is given or withheld . after all , to be ignorant or incapable of imagining another person 's pain does signal blindness in moral sensibility in the same way in which the incapacity to acknowledge that pain does ( page.xiii ) . unquestionably , failure to acknowledge that african immigrant women to the uk are struggling in an ambiguous way with their emotions in their postnatal period calls into question the understanding of suffering and pain by the health services and health managers trapped in frameworks of accountability and quantitative indicators . can we imagine the health services , managers and professionals acknowledging the statement it is something that you have to get on with with something like : no , it is not something that you have to get on with ! ? the second issue has to do with the contemporary conceptualisation of power . according to this notion , power is a multiple force cross - cutting human relations and therefore spaces such as the domestic arena and even the clinical space . power , as described by the narratives of the women , plays a role in the dynamics of their households through the family politics and certainly underpins most of the features the themes are pointing out such as domineering husbands and lack of support for the women , powerful role of the mother - in - law , isolation , and self - inflicted suffering by the women themselves who remain silent about their stress . so , an understanding of the family politics that is , how all the members of the extended family display and wield power around the pregnancy issues as described by these african women immigrants is something that needs to be incorporated in the bio - psychosocial model of health services when working with them . these compelling features should make it clear to the health professionals that they are not working with the assumed normality that is the reconstructed british nuclear family . of course , this is not say that such politics are not also played out among the reconstructed british nuclear family . the third issue to be discussed here has to do with intercultural communication in the health setting between these immigrant women and health professionals . as demonstrated by the narratives , on the one hand the women 's concealment of the emotional stressors from themselves , complemented by the fear of failure , generates some self - inflicted suffering . on the other hand , the family members ' disregard and stigmatisation of the stressors , along with the professionals ' failure to acknowledge the cultural ambiguities , adds to the women 's suffering . the fact that professionals often report women as being well because they have hidden their internal turmoil is significant evidence of the lack of awareness of intercultural communications among health professionals in observing culture and power at play . and this could be the result of the lack of education among health professionals in the discussion of contemporary social sciences as well as the new discussion on compassionate care . combining the features described above along with more specific tips for health professionals , it is evident that the main one comes via the approach used by health service managers and professionals , at least in the in the uk , to conceptualising health . if health is a bio - psychosocial issue , the acknowledgement of the suffering of these immigrant women , the household politics they face and the intercultural communication between them and the health professionals are issues that can definitely be accommodated . but this ethical and academic decision lies in the hands of the health services . feminist research has demonstrated that the best outcomes in improving women 's lives come from the work done with both men and women and not just women . health professionals probably need to understand that postnatal depression in african immigrant women is not the exclusive issue for these women , as the research study reported here has tried to demonstrate . this has implications for health professionals working with postnatal women since their experiences should extend to and include the understanding of the entire family and not just the postnatal women . again , as points out when outlining the social suffering theory , suffering and pain as in the case presented here of postnatal depression , inasmuch as they are health and social problems , needs to take into account not only the individual but also his or her networks . there were four limitations in this study which eventually could relate to the literature concerning qualitative research with bme groups or other vulnerable groups [ 7174 ] . first , when carrying out research on health issues , the researcher has to abide by the ethnic self - identification of the person that the research is about rather than engage in labelling . in this case the women who participated in the study reported here self - identified themselves as african immigrants to the uk in the last decade . second , regarding the issue that research with people of different cultural backgrounds should ideally look for same cultural matching between researchers and participants has been challenged . in particular , argues that more than ethnic matching , researchers and research participants should look for a mutual or shared understanding regardless of their cultural background . in the case of this research thus while the ethnicity of the three main researchers involved in the data collection were , respectively , african , english , and south american , the gender mix involved two females and one male . so any possible limitations in the research reported here could be related to the these cultural backgrounds . third , the literature on qualitative research becomes controversial where data saturation regarding focus groups is reached . while some authors mention that this is obtained between three and six focus groups , other authors view it as a reemerge of a theme or a topic even if it is in one focus group . in the particular case of this research , data saturation took place from the first focus group due to the influence of the most vocal and educated women . however , as was also indicated , this trend was countered by taking into account the statements of the less vocal women . fourth , trustworthiness and rigour in qualitative research as suggested by should come through the dialogue or agreement between , on one hand , the reader of the research , and on the other , the detailed description of how the research process was conducted by the researcher . in the case of this research the materials and methods have been described as carefully as possible so that readers can follow the process by which this research with african immigrant women was conducted . equally transferability as part of trustworthiness and rigour in qualitative research comes through the description of the specific context in which the research has taken place . thus a possible limitation of this research could be that some of the situations discussed ( acknowledgement of postnatal depression , social support and emotional distress , cultural identification , and coping strategies ) in african immigrant women relate only to them while the same or other similar aspects discussed relate to other women from other cultural backgrounds . the study , using the logic of qualitative inquiry , the ergodic hypothesis as postulated by devereux , showed that african immigrant women in south east london received little practical and emotional support before , during and after delivery of their babies . as the narratives of the study illustrated , these women suffer and cope with their emotional distress alone and in silence , magnifying their suffering . they see emotional distress as something different from physical illness and very much framed in the bio - psychosocial model of health . their narratives also allow us to infer that support from the immediate extended family ( mainly in - laws and husband ) is inadequate and these family members barely understand what the mothers are going through . the same lack of acknowledgement and understanding was observed by the healthcare services . as demonstrated by the study , on the one hand , the women 's tendency to keep all the emotional stressors to themselves complemented by the fear of failing generates some self - inflicted suffering . on the other hand , when the family members ignore the stressors and stigmatise the mothers , and the professionals fail to pick up any of these cultural clues , the women 's suffering is compounded . the fact that professionals often report women as being well because they have hidden their inner turmoil is significant evidence of the lack of acknowledgement of the suffering the women are going through as well as the lack of awareness of intercultural communication between health professionals and the women , in this particular case african immigrants in london . there is a need for health professionals to embed cultural ambiguities in their daily work routine , as culture is not a frozen equivocal determinant of peoples ' lived world . again , in this particular case involving immigrant women , can we imagine health services , managers and professionals acknowledging the women 's statement it is something that you have to get on with with something like : no , it is not something that you have to get on with ! ? the discussion of contemporary social sciences could help here immensely as well as the new discussion on compassionate care . simultaneous with the acknowledgement of the women 's suffering , it was seen as important that the health professionals understand the family politics of any household . the inclusion of family politics in the bio - psychosocial model of health services is imperative . as the narratives showed , health professionals need to understand that , in a multicultural society , there is more than one assumed normality besides the reconstructed british nuclear family . but , as has was already been stated , this and other ethical and academic decisions lie in the hands of the health services .

postnatal depression has profound effects on the quality of life , social functioning , and economic productivity of women and families . this paper presents the findings of an earlier exploration of the perception of postnatal depression in african women immigrants in south east london . the aims of this research were twofold : firstly , to establish cultural elements related to postnatal depression through women 's narratives regarding their daily life situations , including the nuances and complexities present in postnatal depression , and secondly , to help health professionals understand and acknowledge postnatal depression signs in these immigrant women and some of the cultural ambiguities surrounding them . the study used a qualitative approach mainly through the implementation of two focus groups . thematic analysis of the women 's narratives suggested that almost half of the participants in the study struggle with some signs of postnatal depression . the women did not perceive the signs as related to illness but as something else in their daily lives , that is , the notion that you have to get on with it . the study also highlights the fact that the signs were not identified by health visitors , despite prolonged contact with the women , due to the lack of acknowledgement of women 's silence regarding their emotional struggle , household and family politics , and intercultural communication in health services .

1. Introduction 2. The Multicultural Literature Concerning Postnatal Depression 3. Materials and Method 4. Results 5. Discussion 6. Limitations of This Study 7. Conclusion

postnatal depression has profound effects on the quality of life , social functioning , and economic productivity of women and families . in general , health professionals and , in particular , health visitors in the uk play a vital role in identifying and supporting women who experience postnatal depression in the community . the fact that the bme population is growing in the uk and in south east london generates the need to explore and understand african women immigrants ' perception of postnatal depression in order to improve service development and outcomes for them . this problem is further exacerbated by the overwhelming evidence of the link between depression and domestic violence , and the differing conceptualisations of postnatal depression among health professionals . this study presents the findings of an earlier exploration of the perception of postnatal depression in african women immigrants in south east london . the aims of this research were twofold : firstly , to establish cultural elements related to postnatal depression through women 's narratives regarding their daily life situations including the nuances and complexities present in postnatal depression , and secondly , to help health professionals understand and acknowledge postnatal depression signs in these immigrant women and some of the cultural ambiguities surrounding them . other cultural factors that may be important for a general understanding of depression include a distinctive language related to depression , the transmission of information among people about depression , and beliefs about healthcare and the healthcare system . in developing societies postnatal depression is thought to occur three times more in the developing societies than in developed ones , for example , in khayelitsha , cape town , in south africa , the prevalence rate of major depression was reported to be 34.7% at two months postpartum . similarly , the literature on mental health illness addresses the fact that aspects such as perceptions and attitudes towards depression in different cultures may affect help - seeking behaviour and access to treatment [ 10 , 11 ] . another significant and controversial aspect of the literature , particularly in regard to the uk , relates to the diagnosing of postnatal depression among bme women through the edinburgh postnatal depression scale ( epds ) . following its validation for use in the uk it was implemented across the country by health visitors as a universal method of identifying mothers who were at risk of postnatal depression argued that epds was originally designed as a screening test and was not intended as a diagnostic tool . using epds as the assessment tool for these women might result in them additional arguments related to this position in the literature claim that most research has been conducted in the western developed countries [ 31 , 48 ] and has not taken into account the range of different psychosocial experiences likely to be involved in childbirth , for example differences in rates of lone motherhood , the nature of marriage , family kinship , and variations in the support new mothers receive in different countries and cultures . on the part of the health professionals , the literature addresses various factors that could contribute to the lack of awareness , late diagnoses , undetected cases or , worse , excessive medicalisation of symptoms . another author argued that the way a person perceives and understands their health is related to the subjective cultural experience in her or his society . in other words , african women immigrants in the uk could be seen as a group of people who share history , religion , language , thoughts and , overall , the experience of being immigrants . thus , how the cultural background of women is understood and constructed by the providers of health services and how these providers and the women communicate is a matter of great interest for researchers focusing on intercultural communication in the context of health services in various multicultural societies . this qualitative study presented here expects to add to this ample range of explanations in the literature on postnatal depression particularly among african women immigrants in south east london . these testimonies and narratives have been used by women and could be used by any subjugated group to unveil specific and little - researched aspects of women 's daily lives , their feelings , attitudes , hopes , and dreams [ 54 , page 835 ] . the group targeted for the study was all african women immigrants registered on the general lists of the health visitors of four health centres in south east london . participants were contacted personally , through leaflets , and a phone call by the researchers but the gate keepers for the research were the health visitors . the main inclusion criteria which applied to the women participating in the research were ( i ) women in the postnatal period who had delivered a baby up to a year earlier , ( ii ) immigrant women who identified themselves as being of african descent between the ages of 16 and 45 years ( the age range within which women 's fertility and reproductive capacity is at its peak ) , ( iii ) women who spoke and understood english , ( iv ) women whose bab(ies ) were in a good health , and ( v ) women who lived in the south east ( women with little children are always busy and will hesitate to attend any group if the distance is more than two miles ) and ( vi ) . in the discussion on research into sensitive issues such as postnatal depression , and the differences between qualitative and quantitative studies , an important phenomenon to recall is the ergodic hypothesis as postulated by george devereux in the seventies : the analysis of a great number of relatively superficial facts ampleness provides exactly the same insights as the in - depth analysis of only one phenomenon . in the context of social research , this yields an equivalence for a survey with one thousand answers of yes or no type questions , and three in - depth interviews of , say , 4 hours the researcher asked the women at the very beginning of the two focus groups about their marital status and the kind of support network they had at home . secondly , an observation that became pretty obvious during the two focus groups was that those participants who were more educated , up to degree level , were more vocal and their perception of the symptoms of postnatal depression were freely expressed whereas those who had achieved gcse level were less expressive . in fact the most vocal and educated women in the first focus group contributed to within data saturation , since they articulated many themes setting the trend , that reemerged in the second focus group and , later , during the analysis of the data . in the particular case of the study reported here initial coding of the transcripts was performed with the goal of remaining open to all possible interpretations . table 2 shows some of the most significant stages in the thematic analysis of the data ( narratives ) . the results discussed here present the data through the main themes and literal quotations ( narratives ) as stated by the women regarding events , episodes , points of view , settings , and comments related to the main focus of this research : postnatal depression among a group of african women immigrants in south east london . the main themes accordingly were : responses to their pregnancy , feelings before and after giving birth , social support or the lack of it , feeling alone , lack of information about health services , poverty , signs of postnatal depression , and not coping with their situation . ranging from the ones who experienced difficulties in seeing the good side of being pregnant to the ones who were happily surprised , the main trend in this theme was that being pregnant is always a good thing for all women but particularly for african women immigrants who are married or cohabiting . the participants , being relatively recent immigrants to the uk , spoke of the difficulties they faced in this country , which promotes equality between men and women . if you want to with mum , i 'll deal with the person who 's trying to tell you what you have to do . like someone can help you with the baby and help you with other things , but the way that you 're feeling , you do n't get help for that.that i 'm going mad , mentally and sometimes i 'd be crying the baby and i will be crying , and sometimes i feel like throwing the baby out , but i can't.i do n't know i ca n't imagine myself going to my mum , or my mother - in - law probably i can say to my mum , but i still did n't , i just could n't imagine saying to somebody , it 's just not it 's not something that you do , you just everybody expects you to get on with it and you get on with it . i ca n't imagine myself going to my mum , or my mother - in - law probably i can say to my mum , but i still did n't , i just could n't imagine saying to somebody , it 's just not it 's not something that you do , you just everybody expects you to get on with it and you get on with it . participants pointed out that there is a general lack of awareness among uk health visitors and healthcare professionals in identifying when african women immigrants are in distress or are crying out for help . a woman remarked that she was given a questionnaire ( referring to the epds ) , which to her made no sense:but they did n't really give me some of the other information that as a new mother i would have found really useful , without me having to look on the internet or buy a book . participants described some of the signs of postnatal depression ; however , the main theme here is that they would not dare admit to their families or relatives that that was how they were feeling at that moment in their lives . furthermore , almost all of the seventeen participants agreed that there is a problem with the present system of provision of healthcare services for african women immigrants in capturing these emotional distresses . the main findings were related to : response to participants ' pregnancies , feelings before and after giving birth , social support or the lack of it , feeling alone , lack of information about health services , poverty , signs of postnatal depression , and not coping with their situation . in order to synthetize them , the statements are discussed in terms of the direct experience of the women ( feelings before and after giving birth , signs of postnatal depression , a feeling that they are not coping with the situation , feeling alone , and expectations of and responses to their pregnancy ) and , also in terms of perceived support from families ( husband and mother - in - law ) , community and health services ( lack of information and health visitors ) . the majority of the statements articulated by some of participants and related to their direct experiences indicated symptoms associated with a state of unhappiness following delivery , although by no means all recognised it as an illness postnatal depression or felt they required treatment by healthcare professionals as addressed too by the literature [ 10 , 11 , 21 , 27 , 36 , 37 ] . as one of the participants stated powerfully , rendering it invisible , it is something that you have to get on with . so , certainly , the narrative of those states was comparable to the criteria for a diagnosis of postnatal depressive disorder but , as seen , these become invisible because they are not identified as postnatal depression as such . further descriptions of the women 's experiences related to their emotional feelings when facing lack of social support . so , as the literature also shows , the lack of all types of social support can not be ignored here as a major risk factor for postnatal depression particular in immigrant women , whose close relatives are absent . in the case of women in the two focus groups , most of the stressors expressed by the women are included among the themes as , for example , mental stress such as loneliness , unmet expectations , birth plan disappointment and abandonment , and external stressors such as crying baby , sibling care , lack of support , and financial concerns . of course , while women in the study reported here did not experience all the stressors at once , most experienced some simultaneously . although some of them felt sad , unhappy , and stressed , they kept their feelings to themselves because , culturally , to admit having problems coping with the after - effects of childbirth is probably a sign of failure or weakness in front of the extended family husbands , mothers - in - law , and others . also , as expressed in their narratives , the fact that women may choose to make their emotional problems invisible to health professionals could find explanation in their cultural backgrounds and their status as newly arrived immigrants . the cultural background of immigrants as a factor in the concealment of postnatal symptoms is important because it is vital to recall that cultural practices are not frozen activities that determine unequivocally the behaviour of an individual . so the common conceptualization of culture by health services as a frozen element that determines people 's behaviour , attitudes , or understanding of the lifeworld should be modified to that of daily enactment and ambiguity which needs to be understood by the health professionals in each specific case . as some of the participants narrated , counting on their own mothers for support seems to have helped some participants cope with the distress they faced during this postnatal period although not in all cases , as a few never had the opportunity for their own mothers to be present due to their immigrant status . interestingly , the two focus groups welcomed the idea of a group such as the focus group which allowed them to communicate their internal emotional struggles and feelings with women who were going through the same experience . another important and recurrent finding as presented in the narratives of the participants in the two focus groups was the mention of the mother - in - law as one of the sources of unhappiness in the dynamics of the household once the baby has arrived . in the study reported here , this fact was revalidated by the comments of two of the participants who were married for about a year before conceiving . ultimately , without denying the biomedical mechanisms present in postnatal depression , the interesting issue to discuss here in closing this discussion is the fact that the women through their narratives volunteer the sources of solutions based on the psychosocial model of health . contextualising the most important findings discussed above ( acknowledgement of postnatal depression , social support , and emotional distress , cultural identification and coping strategies ) in some of the recent discussions in social sciences , three issues are addressed below , particularly for health professionals to consider when offering health services for people of different cultural backgrounds , in this case african immigrant women . unquestionably , failure to acknowledge that african immigrant women to the uk are struggling in an ambiguous way with their emotions in their postnatal period calls into question the understanding of suffering and pain by the health services and health managers trapped in frameworks of accountability and quantitative indicators . can we imagine the health services , managers and professionals acknowledging the statement it is something that you have to get on with with something like : no , it is not something that you have to get on with ! power , as described by the narratives of the women , plays a role in the dynamics of their households through the family politics and certainly underpins most of the features the themes are pointing out such as domineering husbands and lack of support for the women , powerful role of the mother - in - law , isolation , and self - inflicted suffering by the women themselves who remain silent about their stress . so , an understanding of the family politics that is , how all the members of the extended family display and wield power around the pregnancy issues as described by these african women immigrants is something that needs to be incorporated in the bio - psychosocial model of health services when working with them . these compelling features should make it clear to the health professionals that they are not working with the assumed normality that is the reconstructed british nuclear family . the third issue to be discussed here has to do with intercultural communication in the health setting between these immigrant women and health professionals . as demonstrated by the narratives , on the one hand the women 's concealment of the emotional stressors from themselves , complemented by the fear of failure , generates some self - inflicted suffering . on the other hand , the family members ' disregard and stigmatisation of the stressors , along with the professionals ' failure to acknowledge the cultural ambiguities , adds to the women 's suffering . the fact that professionals often report women as being well because they have hidden their internal turmoil is significant evidence of the lack of awareness of intercultural communications among health professionals in observing culture and power at play . and this could be the result of the lack of education among health professionals in the discussion of contemporary social sciences as well as the new discussion on compassionate care . combining the features described above along with more specific tips for health professionals , it is evident that the main one comes via the approach used by health service managers and professionals , at least in the in the uk , to conceptualising health . if health is a bio - psychosocial issue , the acknowledgement of the suffering of these immigrant women , the household politics they face and the intercultural communication between them and the health professionals are issues that can definitely be accommodated . health professionals probably need to understand that postnatal depression in african immigrant women is not the exclusive issue for these women , as the research study reported here has tried to demonstrate . again , as points out when outlining the social suffering theory , suffering and pain as in the case presented here of postnatal depression , inasmuch as they are health and social problems , needs to take into account not only the individual but also his or her networks . in this case the women who participated in the study reported here self - identified themselves as african immigrants to the uk in the last decade . in the case of this research thus while the ethnicity of the three main researchers involved in the data collection were , respectively , african , english , and south american , the gender mix involved two females and one male . so any possible limitations in the research reported here could be related to the these cultural backgrounds . in the particular case of this research , data saturation took place from the first focus group due to the influence of the most vocal and educated women . fourth , trustworthiness and rigour in qualitative research as suggested by should come through the dialogue or agreement between , on one hand , the reader of the research , and on the other , the detailed description of how the research process was conducted by the researcher . in the case of this research the materials and methods have been described as carefully as possible so that readers can follow the process by which this research with african immigrant women was conducted . thus a possible limitation of this research could be that some of the situations discussed ( acknowledgement of postnatal depression , social support and emotional distress , cultural identification , and coping strategies ) in african immigrant women relate only to them while the same or other similar aspects discussed relate to other women from other cultural backgrounds . the study , using the logic of qualitative inquiry , the ergodic hypothesis as postulated by devereux , showed that african immigrant women in south east london received little practical and emotional support before , during and after delivery of their babies . as demonstrated by the study , on the one hand , the women 's tendency to keep all the emotional stressors to themselves complemented by the fear of failing generates some self - inflicted suffering . on the other hand , when the family members ignore the stressors and stigmatise the mothers , and the professionals fail to pick up any of these cultural clues , the women 's suffering is compounded . the fact that professionals often report women as being well because they have hidden their inner turmoil is significant evidence of the lack of acknowledgement of the suffering the women are going through as well as the lack of awareness of intercultural communication between health professionals and the women , in this particular case african immigrants in london . there is a need for health professionals to embed cultural ambiguities in their daily work routine , as culture is not a frozen equivocal determinant of peoples ' lived world . again , in this particular case involving immigrant women , can we imagine health services , managers and professionals acknowledging the women 's statement it is something that you have to get on with with something like : no , it is not something that you have to get on with ! simultaneous with the acknowledgement of the women 's suffering , it was seen as important that the health professionals understand the family politics of any household .

poverty and poor health infrastructure continue to be an impediment to effective health care service delivery . on a global scale , life - threatening infections caused by these pathogenic prokaryotic microorganisms have become an important cause of morbidity and mortality in immune - compromised patients in developing countries . despite the availability of a wide range of antibiotics , bacteria are constantly developing resistance to these agents , which makes it difficult for the concerted effort of combating infectious diseases . from the advent of antimicrobial application in treatment of bacterial diseases , bacteria responded by manifesting varied forms of mechanisms of resistance . with passage of time the level and complexity of the resistance mechanisms by bacterial pathogens increase . drug resistant bacteria , particularly staphylococci species , klebsiella pneumoniae , and pseudomonas species , are becoming commonplace in healthcare institutions and are possibly contributing to the occurrence of treatment failures . virulence factors aid bacteria in invading tissues , evade the defenses mechanisms , and cause disease in the host . it is fundamental to understand how pathogenic bacteria interact with their hosts to produce disease as these mechanisms may be targets in drug development . the ability to form biofilms confers a selected advantage for bacteria to strive under harsh environmental conditions and provide resistance to antimicrobial agents . pseudomonas aeruginosa , mycobacterium tuberculosis , streptococcus pneumoniae , and staphylococcus aureus are examples of bacteria that form biofilms . staphylococcus aureus and pseudomonas aeruginosa are typical gram - positive and gram - negative pathogens , respectively , that have been significantly characterized in community - acquired and hospital - acquired infections . staphylococcus aureus is a facultative anaerobe that exists normally as part of the skin and nasal flora and estimates are that 20% percent of the human population are long term carriers of the bacteria . this bacterium also occurs in animals , sewage , and food and on household and environmental surfaces . the presence of s. aureus in humans as part of normal flora means that the bacteria can infect human tissues such as the skin dermis or mucosal linings when defensive barriers have been breached . this leads to topical skin infections like furuncles , carbuncles , acne , boils , and severe staphylococcal - scalded skin syndrome . life - threatening systemic infections such as pneumonia , meningitis , and sepsis may also result especially in immunocompromised patients . p. aeruginosa is a ubiquitous microbe as it can catabolise a wide range of organic chemicals like benzoate ; as such , it has been found in environments such as soil , water , and hospitals and in humans , plants , and sewage . an outer membrane in the cell structure of p. aeruginosa confers pronounced resistance to xenobiotics including antibacterials . p. aeruginosa rarely causes diseases in health individuals with a competent immune set - up ; it , however , is an opportunistic pathogen that infects immunocompromised patients , especially those with aids and cystic fibrosis and chemotherapy patients . p. aeruginosa gains entry to burns , breached skin , or mucosal linings using its flagellum and pili and replicates to create an infectious critical mass . more seriously , the exotoxins and endotoxins released by p. aeruginosa continue to cause inflammation and harm even after the bacteremia has been treated with antibiotic , which makes infections by p. aeruginosa life - threatening . one of the mechanisms of manifesting resistance to antibacterial agents is the acquisition of efflux pumps that extrude the antibacterial agent from the cell before it can reach its target site of action . the efflux pumps prevent accumulation of drugs within the bacterium from achieving bactericidal or bacteriostatic concentrations at the target sites . the efflux pumps often work in synergism with limited permeability of the p. aeruginosa outer membrane to produce resistance to -lactams , fluoroquinolones , tetracycline , chloramphenicol , macrolides , and aminoglycosides . analysis of s. aureus isolates that were resistant to antibacterial agents and were causing nosocomial infections revealed that these strains contained plasmids coding for transporters . these resistance mechanisms have been attributed to the occurrence of strains like methicillin resistant staphylococcus aureus ( mrsa ) which is resistant to methicillin , tetracycline , chloramphenicol , and aminoglycosides . mrsa is a major source of hospital - acquired infections and old antibiotics like vancomycin are now being used for treatment of mrsa infections despite their unfavorable side effects . the worldwide escalation in both community- and hospital - acquired antimicrobial bacteria is threatening the effective treatment of patients , emphasizing the need for continued surveillance , prudent infection control , and new treatment alternatives . antibiotic resistant bacteria are increasingly prevalent and new antimicrobials are needed to control these pathogens yet development of resistance is unavoidable as it is a pivotal aspect of microbial evolution . thus , there is an urgent need for development of novel antibacterial products that act on molecular targets that act against bacterial resistance mechanisms . herbal medicine use employed either in traditional medicine practice or complementary and alternative medicine ( cam ) is popular for 80% percent of the world in asia , latin america , and africa and is reported to have minimal side effects . furthermore , with concerns of rising costs of drug development , plants have turned out to be some of the most cost - effective and most affordable alternative sources of drugs . vernonia adoensis is an herbaceous plant of the asteraceae family , which is indigenous and commonly distributed throughout zimbabwe , where it is locally known in vernacular shona language as musikavakadzi . its habitat is commonly miombo woodlands and wooded grassland especially near streams [ 20 , 21 ] . this plant has been traditionally used in african ethnomedicine for the treatment of fever and upper respiratory tract infections and currently for tb in traditional medicine practice . callistemon citrinus , commonly known as bottlebrush , is a woody shrub widely distributed in the temperate regions , notably australia particularly on the east and southwest coasts , south america , and tropical asia , and , thus , is exotic to zimbabwe . the different parts of this herb have been used in folk medicine as a common remedy for treatment of diarrhea , dysentery , rheumatism , and antibronchitis . herbal preparations are now readily available as over - the - counter products with numerous medical claims , some of whose scientific proof remains lacking . it is , therefore , essential that , as herbal medicine use is increasingly becoming popular , scientific research is fundamental to back up such uses and medical claims and allay fears of potential herb - drug interactions with conventional medicines . promising results from work on crude extracts have shown the potential for antibacterial activity from these two plants and , hence , the need to evaluate the alkaloid extracts with a view to understanding the mechanisms of action needed to thwart the effect of both hospital- and community - acquired multidrug resistant organisms . the main objective was to evaluate alkaloid extracts isolated from c. citrinus and v. adoensis for activity against s. aureus and p. aeruginosa . plants were collected from mashonaland central for vernonia adoensis ( centenary : 16.8000 s , 31.1167 e ) , and harare for callistemon citrinus ( university of zimbabwe : 17.7840 s , 31.0530 e ) on the basis of indigenous knowledge uses . the leaf samples c1 e7 ( vernonia adoensis ) and uz2 e7 ( callistemon citrinus ) were authenticated by a taxonomist , mr . leaves of callistemon citrinus and vernonia adoensis were separately predried in a labcon orbital incubator ( labotec co. , cape town , s. a. ) at 50c . the leaves were then separately ground in a two - speed blender ( bl2 , abb , moulinex , france ) . approximately 5 g of callistemon citrinus and vernonia adoensis comminuted leaf samples were mixed with 15 ml and 20 ml of 10% v / v ammonia solution , respectively . extraction was done with 30 ml and 50 ml ethanol for callistemon citrinus and vernonia adoensis samples , respectively , in a 40c water bath for 10 minutes . a whatman filter paper number 1 was used for filtration into separately labeled 50 ml falcon tubes . ethanol was left to evaporate in an incubator at 50c for 48 hours . a constant dry weight of each plant alkaloidal extract was obtained . a 1 l broth medium was prepared by dissolving 15 g tryptone , 3 g yeast , 6 g sodium , and 1 g glucose in 1 l of hot distilled water and poured in a 1 l glass jar . the broth medium was sterilised in an autoclave machine ( accu steriliser , vwr scientific products co. , usa ) . staphylococcus aureus ( atcc 9144 ) and pseudomonas aeruginosa ( atcc 27853 ) were obtained from the division of microbiology , department of biological sciences at the university of botswana . the strains were maintained as stock strains in 50% glycerol in eppendorf microtubes and kept at 33c until resuscitation . approximately 20 ml of the broth was inoculated with 25 l of staphylococcus aureus and pseudomonas aeruginosa each in its own falcon tube . the inoculated bacteria were incubated for 24 hours at 37c in a lab - companion incubator ( si300 incubated shaker , jeiotech , korea ) . standardization of the bacterial cultures was done and bacterial broth cultures of 1 10 cfu / ml for both p. aeruginosa and s. aureus were made . the alkaloid extracts of callistemon citrinus and vernonia adoensis made up to a concentration of 25 mg / ml were separately applied to 96-well plates in 20 l volumes so as to expose the test cell cultures with 500 g ( 1.67 mg / ml ) of extracts in each well . staphylococcus aureus and pseudomonas aeruginosa at a concentration of 1 10 cfu / ml and broth media were diluted into the 96-well microplate wells . a positive control containing ampicillin was also set up in which 500 g was applied in each well containing media and bacterial broth culture . relevant negative controls containing the media only or the media with extract were also set up . preincubation absorption measurements were determined using a microplate reader ( spectramax plus384 , molecular devices co. usa ) at 600 nm . postincubation cell density measurements were also determined after incubation at 37c in a lab - companion incubator for 24 h ( si300 incubated shaker , jeiotech co. , korea ) . the alkaloid extracts were then separately serially diluted twofold from 1.67 mg / ml to 0.0032 mg / ml in a 96-well polystyrene microplate to obtain 10 dilution concentrations . in a separate 96-well microplate , 20 l of each dilution was sequentially added in triplicate into wells . broth cultures of s. aureus and p. aeruginosa ( 1 10 cfu / ml ) were then separately added to the wells in 100 l volumes , each species in its own microplate after which 180 l broth media were added to the well to obtain a total volume of 300 l in each well . rows of wells with broth media containing 100 l of cell cultures were used as positive control ; also controls of media containing extract only and extract - free broth media were also used as negative controls in each well . preincubation absorbance values were read from a microplate reader ( spectramax plus384 absorbance microplate reader , molecular devices co. , usa ) . the microplates were then incubated at 37c in a lab - companion incubator ( si300 incubated shaker , jeiotech co. , korea ) for 24 h ; thereafter , absorbance values were read and recorded . mtt was used to identify the mic values in the well where the purple colouration was the least visible in each test row . s. aureus and p. aeruginosa were grown in two separate culture flasks overnight at 37c with shaking at 120 rpm in a an incubator . the bacterial cultures were then poured into 50 ml centrifuge tubes and centrifuged using a centrifuge machine ( mse minor 35 , mse ltd . , england ) at 3 000 rpm for ten minutes and the supernatant was discarded . the pellet was washed twice with phosphate buffered saline ( pbs ) and resuspended in the buffer . the resuspended cells were poured in preweighed centrifuge tubes and spun again at 4 000 rpm for 5 minutes . the supernatant was decanted and the cells were washed again in pbs , before being centrifuged again at 4 000 rpm for 5 minutes . the pellets , both of s. aureus and p. aeruginosa , were suspended at 40 mg / ml in pbs containing 10 mm sodium azide ( nan3 ) . rhodamine 6 g was immediately added to a final concentration of 10 m and incubated the cells with 90 rpm agitation for 1 h in a lab - companion incubator ( si300 incubated shaker , jeiotech co. , korea ) . the tubes were then divided into two centrifuge tubes in the ratio 1 : 3 , to give tube a and tube b of each of the two species . the tubes were then centrifuged at 4 000 rpm for 5 minutes centronic sp selecta centrifuge ( barcelona , spain ) . the supernatant was discarded and cells from tube a of each of the two bacterial species were resuspended in pbs alone at 40 mg / ml . cells from tube b of each of the two species were resuspended in pbs containing 1 m glucose . the cells of tube b of both bacteria were then divided into 5 ml portions in 8 different falcon tubes ; of them , two tubes separately for glucose only , glucose + reserpine , glucose + c. citrinus alkaloid extracts , glucose + v. adoensis alkaloid extracts , and cells from tube a were divided into two 5 ml portions . this was done for each of the pellets from s. aureus and p. aeruginosa . to tubes marked glucose + reserpine , reserpine was added to a final concentration of 61 g / ml and the alkaloids added to their respective tubes to a final concentration of 61 g / ml . the centrifuge tubes were mixed by shaking on a vortex mixer ( thermolyne maxi mix ii , iowa , usa ) and placed in a shaking incubator ( si300 lab - companion co. , korea ) at 37c for 30 minutes at 90 rpm . after 30 minutes , the tubes were centrifuged at 4 000 rpm for 10 minutes in a rotofix 32 centrifuge ( hettich , zentrifugen , germany ) and the supernatant was collected to quantitate the amount r6 g pumped out from the cell . the pellet in each tube was lysed by resuspending in 5 ml 3 m glycine ph 3 . the tubes were mixed by shaking on a vortex mixer and then incubated with shaking for 24 h at 37c . the mixture was centrifuged at 4 000 rpm for 10 minutes and the supernatant was collected and quantified for the amount of r6 g that was accumulated in the cells . r6 g was determined from the samples using a standard r6 g calibration curve after determining the absorbance in 96-well microplates , at 527 nm , using a microplate reader ( spectramax plus384 , molecular devices co. , usa ) . data analyses were performed using graphpad instat software ( graphpad prism inc . , san diego , ca , usa ) . levels of significance were determined using anova using dunnett 's posttest where all columns of treatments were compared to the control . the effects of c. citrinus and v. adoensis extracts on bacterial species were determined by measuring preincubation and postincubation absorbance readings at 600 nm . at an initial screening concentration of 1.67 mg / ml , both alkaloids from c. citrinus and v. adoensis inhibited bacterial growth significantly ( p < 0.001 , figure 1 ) . c. citrinus alkaloids were the most potent in inhibiting growth of p. aeruginosa with the mean absorbance for the cells exposed to this extract being 0.023 au whilst that of v. adoensis alkaloids had 0.125 au . ampicillin as was expected almost killed all the cells in the positive controls as the recorded mean absorbance was 0.0062 au . generally , p. aeruginosa was less susceptible to the antibacterial effect of the alkaloids and ampicillin as compared to s. aureus . the mic was identified for the well in which there was the least visible mtt color . the extracts from c. citrinus were the most potent with the lowest mic of 0.025 mg / ml against s. aureus and 0.21 mg / ml against p. aeruginosa . the least potent extracts were those from v. adoensis , the mic being 0.21 mg / ml against s. aureus . c. citrinus extracts only had a bactericidal action against s. aureus only with an mbc of 0.835 mg / ml ( table 1 ) whereas the mbc for ampicillin was 0.008 mg / ml . the effects of the extracts on drug accumulation were determined using rhodamine 6 g , a fluorescent dye that is actively pumped out by the atp - dependent efflux pumps of both species of bacteria in this study . the highest accumulation of rhodamine 6 g was by c. citrinus alkaloids against p. aeruginosa which was 121% increase from the glucose only control . s. aureus was shown to be less susceptible to efflux pump inhibition as an increase in accumulation of 114% was obtained . for v. adoensis , thus , c. citrinus alkaloids inhibited efflux pumps to a greater extent as compared to v. adoensis alkaloids . interestingly , p. aeruginosa proved to be more susceptible to efflux pump inhibition than s. aureus as the standard inhibitor reserpine 's activity was more notable against p. aeruginosa than against s. aureus ( figure 2 and table 2 ) . the search for antimicrobial compounds for the benefit of humanity is necessitated by the inherent ability of pathogens to develop and adopt mechanisms of resistance against antibiotics . potentially harmful side effects associated with use of new chemical entities synthesized artificially and the unsustainably high costs of drug development are slowly shifting the focus to plant derived phytochemicals of medicinal significance . coformulation of naturally sourced antimicrobial adjuvants like efflux pump inhibitors with old and new generation antibiotics remains a novel mechanism of countering antimicrobial resistance . the effects of c. citrinus and v. adoensis extracts were determined by carrying out antibacterial susceptibility tests , minimum inhibitory concentration ( mic ) , and minimum bactericidal concentration ( mbc ) determinations . results from this study showed s. aureus to be more susceptible to both alkaloid extracts than p. aeruginosa which was expected as previous studies confirm that gram - positive strains are more sensitive to malicious xenobiotics than gram - negative bacteria . similar margins of inhibition were obtained by aliyu et al . when chloroform fractions of vernonia species were tested against methicillin resistant s. aureus ( mrsa ) and gram - negative bacteria as well . chloroform and ethanol extracts of c. citrinus were found to be active against both s. aureus and p. aeruginosa and other bacteria like e. coli and s. typhi via disc diffusion methods [ 28 , 29 ] . although the extracts used were crude , alkaloids have been found to be present in the chloroform and ethanol solvents of extraction . in this regard , the alkaloid extract of c. citrinus and v. adoensis might have broad spectrum activity since they exhibited activity against both gram - positive and gram - negative bacteria . the mic and mbc assay confirmed that alkaloids form c. citrinus were more potent extracts against s. aureus which had an mic value of 0.0025 mg / ml and an mbc of 0.835 mg / ml . the mbc of ampicillin was 0.008 mg / ml ; hence , the activity of the alkaloids is far outweighed by the standard antibiotic . v. adoensis alkaloids were least potent with mic of 0.42 mg / ml and 0.21 mg / ml against p. aeruginosa and s. aureus , respectively . bactericidal effects were shown by c. citrinus alkaloids against s. aureus only ( table 1 ) . a bacteriostatic effect against p. aeruginosa by all plant alkaloid extracts could be due to the pathogen 's thick outer membrane that is highly hydrophobic and possibly provided a permeability barrier to the extract . this bacterium is generally less sensitive to antimicrobials . in a previous study by krishna et al . , chloroform extracts of alkaloids from c. citrinus were found to be inhibitors of growth in bacteria . mic values obtained for the gram - positive ( b. subtilis and b. pumilis ) and gram - negative bacteria ( e. coli ) were less than that of streptomycin , the standard antibiotic used in that study . atp - dependent efflux pumps are one of the main mechanisms of preventing accumulation of effective concentrations of antibiotics at molecular target sites inside the bacterial cell . the major efflux pump systems studied in gram - negative strains like p. aeruginosa are mexxy - oprm or mexcd - oprj which have been associated with acquired multidrug resistance [ 15 , 16 ] . in gram - positive bacteria such as s. aureus transporters of the major facilitator superfamily ( mfs ) , qaca , qacb , nora , and norb were found in strains causing hospital - acquired infections and conferring resistance to fluoroquinolones and puromycin . inhibition of efflux pumps is a plausible mechanism that can be employed to effectively combat the consequences of resistance . inhibition of efflux pumps was also found to decrease the mics for both antibiotic susceptible and resistant bacteria and reversed acquired resistance among selected p. aeruginosa strains . the alkaloids extract from both plants was able to cause accumulation of rhodamine 6 g in the cells of s. aureus and p. aeruginosa . however , c. citrinus alkaloids were potent inhibitors compared to alkaloids from v. adoensis and caused 141% and 121% increases in accumulation of r6 g in s. aureus and p. aeruginosa , respectively . v. adoensis alkaloid extracts only managed to make the cells accumulate only a 14% increase in the two bacterial species . previous work on efflux pump activity revealed v. adoensis crude extracts to be more active in inhibiting s. aureus than c. citrinus ; in light of the results of this present study , such activity of v. adoensis might have been due to other phytoconstituents present in the crude extracts . the greatest accumulation of r6 g was caused by exposure of cells to c. citrinus alkaloids against p. aeruginosa . these results are in agreement with the work of chitemerere and mukanganyama in which c. citrinus crude extracts caused the most significant accumulation in p. aeruginosa as well . therefore , p. aeruginosa was more sensitive to efflux pump inhibition by phytoconstituents than gram - positive microbes , although this bacterium was less sensitive to growth inhibition . this is an unexpected finding which was also observed with crude extracts that exhibited significant inhibition of 64100% of drug accumulation of rhodamine 6 g in p. aeruginosa cell . it is important to identify the agents that block efflux of drugs from within the bacteria as they are potential sources of adjuvants in coformulations with conventional antibiotics . a clinical example is the successful coformulation of clavulanic acid , a penicillinase inhibitor , with amoxicillin , an old conventional beta - lactam antibiotic in the coamoxiclav generic . in vitro studies of synergistic effects of combinations of an mdr pump inhibitor and other antimicrobial phytoconstituents as well as conventional antibiotics previous studies have already proved the effectiveness of c. citrinus extract for in vitro antibacterial activity [ 28 , 29 , 32 ] . in conclusion , the alkaloid extracts from c. citrinus and v. adoensis have shown some antibacterial activity on s. aureus and p. aeruginosa . the c. citrinus alkaloid extract showed more potent growth inhibitory activity than that from v. adoensis with some bactericidal effects . p. aeruginosa was most susceptible to efflux pump inhibition by alkaloids extracted from c. citrinus which also caused notable accumulation in s. aureus . the demonstration of p. aeruginosa as being more susceptible to efflux pump inhibition means that formulations of medicinal products with epi activity may have potential acceptable efficacy against this bacterial species . further work needs to be done to isolate the specific chemicals that have antibacterial activity .

the development of new antibiotics from new chemical entities is becoming more and more expensive , time - consuming , and compounded by emerging strains that are drug resistant . alkaloids are plant secondary metabolites which have been shown to have potent pharmacological activities . the effect of alkaloids from callistemon citrinus and vernonia adoensis leaves on bacterial growth and efflux pump activity was evaluated on staphylococcus aureus and pseudomonas aeruginosa . at a concentration of 1.67 mg / ml , the alkaloids inhibited bacterial growth with comparable effects to ampicillin , a standard antibiotic . the alkaloids from c. citrinus were the most potent against s. aureus with an mic of 0.0025 mg / ml and mbc of 0.835 mg / ml . it was shown that effects on p. aeruginosa by both plant alkaloids were bacteriostatic . p. aeruginosa was most susceptible to drug efflux pump inhibition by c. citrinus alkaloids which caused an accumulation of rhodamine 6 g of 121% compared to the control . thus , c. citrinus alkaloids showed antibacterial activity as well as inhibiting atp - dependent transport of compounds across the cell membrane . these alkaloids may serve as potential courses of compounds that can act as lead compounds for the development of plant - based antibacterials and/or their adjunct compounds .

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

despite the availability of a wide range of antibiotics , bacteria are constantly developing resistance to these agents , which makes it difficult for the concerted effort of combating infectious diseases . drug resistant bacteria , particularly staphylococci species , klebsiella pneumoniae , and pseudomonas species , are becoming commonplace in healthcare institutions and are possibly contributing to the occurrence of treatment failures . virulence factors aid bacteria in invading tissues , evade the defenses mechanisms , and cause disease in the host . pseudomonas aeruginosa , mycobacterium tuberculosis , streptococcus pneumoniae , and staphylococcus aureus are examples of bacteria that form biofilms . staphylococcus aureus and pseudomonas aeruginosa are typical gram - positive and gram - negative pathogens , respectively , that have been significantly characterized in community - acquired and hospital - acquired infections . this bacterium also occurs in animals , sewage , and food and on household and environmental surfaces . the presence of s. aureus in humans as part of normal flora means that the bacteria can infect human tissues such as the skin dermis or mucosal linings when defensive barriers have been breached . this leads to topical skin infections like furuncles , carbuncles , acne , boils , and severe staphylococcal - scalded skin syndrome . p. aeruginosa is a ubiquitous microbe as it can catabolise a wide range of organic chemicals like benzoate ; as such , it has been found in environments such as soil , water , and hospitals and in humans , plants , and sewage . an outer membrane in the cell structure of p. aeruginosa confers pronounced resistance to xenobiotics including antibacterials . p. aeruginosa rarely causes diseases in health individuals with a competent immune set - up ; it , however , is an opportunistic pathogen that infects immunocompromised patients , especially those with aids and cystic fibrosis and chemotherapy patients . p. aeruginosa gains entry to burns , breached skin , or mucosal linings using its flagellum and pili and replicates to create an infectious critical mass . more seriously , the exotoxins and endotoxins released by p. aeruginosa continue to cause inflammation and harm even after the bacteremia has been treated with antibiotic , which makes infections by p. aeruginosa life - threatening . one of the mechanisms of manifesting resistance to antibacterial agents is the acquisition of efflux pumps that extrude the antibacterial agent from the cell before it can reach its target site of action . the efflux pumps prevent accumulation of drugs within the bacterium from achieving bactericidal or bacteriostatic concentrations at the target sites . the efflux pumps often work in synergism with limited permeability of the p. aeruginosa outer membrane to produce resistance to -lactams , fluoroquinolones , tetracycline , chloramphenicol , macrolides , and aminoglycosides . analysis of s. aureus isolates that were resistant to antibacterial agents and were causing nosocomial infections revealed that these strains contained plasmids coding for transporters . these resistance mechanisms have been attributed to the occurrence of strains like methicillin resistant staphylococcus aureus ( mrsa ) which is resistant to methicillin , tetracycline , chloramphenicol , and aminoglycosides . the worldwide escalation in both community- and hospital - acquired antimicrobial bacteria is threatening the effective treatment of patients , emphasizing the need for continued surveillance , prudent infection control , and new treatment alternatives . antibiotic resistant bacteria are increasingly prevalent and new antimicrobials are needed to control these pathogens yet development of resistance is unavoidable as it is a pivotal aspect of microbial evolution . thus , there is an urgent need for development of novel antibacterial products that act on molecular targets that act against bacterial resistance mechanisms . herbal medicine use employed either in traditional medicine practice or complementary and alternative medicine ( cam ) is popular for 80% percent of the world in asia , latin america , and africa and is reported to have minimal side effects . furthermore , with concerns of rising costs of drug development , plants have turned out to be some of the most cost - effective and most affordable alternative sources of drugs . callistemon citrinus , commonly known as bottlebrush , is a woody shrub widely distributed in the temperate regions , notably australia particularly on the east and southwest coasts , south america , and tropical asia , and , thus , is exotic to zimbabwe . the different parts of this herb have been used in folk medicine as a common remedy for treatment of diarrhea , dysentery , rheumatism , and antibronchitis . promising results from work on crude extracts have shown the potential for antibacterial activity from these two plants and , hence , the need to evaluate the alkaloid extracts with a view to understanding the mechanisms of action needed to thwart the effect of both hospital- and community - acquired multidrug resistant organisms . the main objective was to evaluate alkaloid extracts isolated from c. citrinus and v. adoensis for activity against s. aureus and p. aeruginosa . plants were collected from mashonaland central for vernonia adoensis ( centenary : 16.8000 s , 31.1167 e ) , and harare for callistemon citrinus ( university of zimbabwe : 17.7840 s , 31.0530 e ) on the basis of indigenous knowledge uses . the leaf samples c1 e7 ( vernonia adoensis ) and uz2 e7 ( callistemon citrinus ) were authenticated by a taxonomist , mr . leaves of callistemon citrinus and vernonia adoensis were separately predried in a labcon orbital incubator ( labotec co. , cape town , s. a. ) approximately 5 g of callistemon citrinus and vernonia adoensis comminuted leaf samples were mixed with 15 ml and 20 ml of 10% v / v ammonia solution , respectively . extraction was done with 30 ml and 50 ml ethanol for callistemon citrinus and vernonia adoensis samples , respectively , in a 40c water bath for 10 minutes . a 1 l broth medium was prepared by dissolving 15 g tryptone , 3 g yeast , 6 g sodium , and 1 g glucose in 1 l of hot distilled water and poured in a 1 l glass jar . staphylococcus aureus ( atcc 9144 ) and pseudomonas aeruginosa ( atcc 27853 ) were obtained from the division of microbiology , department of biological sciences at the university of botswana . approximately 20 ml of the broth was inoculated with 25 l of staphylococcus aureus and pseudomonas aeruginosa each in its own falcon tube . standardization of the bacterial cultures was done and bacterial broth cultures of 1 10 cfu / ml for both p. aeruginosa and s. aureus were made . the alkaloid extracts of callistemon citrinus and vernonia adoensis made up to a concentration of 25 mg / ml were separately applied to 96-well plates in 20 l volumes so as to expose the test cell cultures with 500 g ( 1.67 mg / ml ) of extracts in each well . staphylococcus aureus and pseudomonas aeruginosa at a concentration of 1 10 cfu / ml and broth media were diluted into the 96-well microplate wells . the alkaloid extracts were then separately serially diluted twofold from 1.67 mg / ml to 0.0032 mg / ml in a 96-well polystyrene microplate to obtain 10 dilution concentrations . broth cultures of s. aureus and p. aeruginosa ( 1 10 cfu / ml ) were then separately added to the wells in 100 l volumes , each species in its own microplate after which 180 l broth media were added to the well to obtain a total volume of 300 l in each well . s. aureus and p. aeruginosa were grown in two separate culture flasks overnight at 37c with shaking at 120 rpm in a an incubator . the pellets , both of s. aureus and p. aeruginosa , were suspended at 40 mg / ml in pbs containing 10 mm sodium azide ( nan3 ) . rhodamine 6 g was immediately added to a final concentration of 10 m and incubated the cells with 90 rpm agitation for 1 h in a lab - companion incubator ( si300 incubated shaker , jeiotech co. , korea ) . the supernatant was discarded and cells from tube a of each of the two bacterial species were resuspended in pbs alone at 40 mg / ml . the cells of tube b of both bacteria were then divided into 5 ml portions in 8 different falcon tubes ; of them , two tubes separately for glucose only , glucose + reserpine , glucose + c. citrinus alkaloid extracts , glucose + v. adoensis alkaloid extracts , and cells from tube a were divided into two 5 ml portions . this was done for each of the pellets from s. aureus and p. aeruginosa . to tubes marked glucose + reserpine , reserpine was added to a final concentration of 61 g / ml and the alkaloids added to their respective tubes to a final concentration of 61 g / ml . after 30 minutes , the tubes were centrifuged at 4 000 rpm for 10 minutes in a rotofix 32 centrifuge ( hettich , zentrifugen , germany ) and the supernatant was collected to quantitate the amount r6 g pumped out from the cell . the mixture was centrifuged at 4 000 rpm for 10 minutes and the supernatant was collected and quantified for the amount of r6 g that was accumulated in the cells . r6 g was determined from the samples using a standard r6 g calibration curve after determining the absorbance in 96-well microplates , at 527 nm , using a microplate reader ( spectramax plus384 , molecular devices co. , usa ) . levels of significance were determined using anova using dunnett 's posttest where all columns of treatments were compared to the control . the effects of c. citrinus and v. adoensis extracts on bacterial species were determined by measuring preincubation and postincubation absorbance readings at 600 nm . at an initial screening concentration of 1.67 mg / ml , both alkaloids from c. citrinus and v. adoensis inhibited bacterial growth significantly ( p < 0.001 , figure 1 ) . c. citrinus alkaloids were the most potent in inhibiting growth of p. aeruginosa with the mean absorbance for the cells exposed to this extract being 0.023 au whilst that of v. adoensis alkaloids had 0.125 au . generally , p. aeruginosa was less susceptible to the antibacterial effect of the alkaloids and ampicillin as compared to s. aureus . the mic was identified for the well in which there was the least visible mtt color . the extracts from c. citrinus were the most potent with the lowest mic of 0.025 mg / ml against s. aureus and 0.21 mg / ml against p. aeruginosa . the least potent extracts were those from v. adoensis , the mic being 0.21 mg / ml against s. aureus . c. citrinus extracts only had a bactericidal action against s. aureus only with an mbc of 0.835 mg / ml ( table 1 ) whereas the mbc for ampicillin was 0.008 mg / ml . the effects of the extracts on drug accumulation were determined using rhodamine 6 g , a fluorescent dye that is actively pumped out by the atp - dependent efflux pumps of both species of bacteria in this study . the highest accumulation of rhodamine 6 g was by c. citrinus alkaloids against p. aeruginosa which was 121% increase from the glucose only control . s. aureus was shown to be less susceptible to efflux pump inhibition as an increase in accumulation of 114% was obtained . for v. adoensis , thus , c. citrinus alkaloids inhibited efflux pumps to a greater extent as compared to v. adoensis alkaloids . interestingly , p. aeruginosa proved to be more susceptible to efflux pump inhibition than s. aureus as the standard inhibitor reserpine 's activity was more notable against p. aeruginosa than against s. aureus ( figure 2 and table 2 ) . the search for antimicrobial compounds for the benefit of humanity is necessitated by the inherent ability of pathogens to develop and adopt mechanisms of resistance against antibiotics . potentially harmful side effects associated with use of new chemical entities synthesized artificially and the unsustainably high costs of drug development are slowly shifting the focus to plant derived phytochemicals of medicinal significance . coformulation of naturally sourced antimicrobial adjuvants like efflux pump inhibitors with old and new generation antibiotics remains a novel mechanism of countering antimicrobial resistance . the effects of c. citrinus and v. adoensis extracts were determined by carrying out antibacterial susceptibility tests , minimum inhibitory concentration ( mic ) , and minimum bactericidal concentration ( mbc ) determinations . results from this study showed s. aureus to be more susceptible to both alkaloid extracts than p. aeruginosa which was expected as previous studies confirm that gram - positive strains are more sensitive to malicious xenobiotics than gram - negative bacteria . when chloroform fractions of vernonia species were tested against methicillin resistant s. aureus ( mrsa ) and gram - negative bacteria as well . chloroform and ethanol extracts of c. citrinus were found to be active against both s. aureus and p. aeruginosa and other bacteria like e. coli and s. typhi via disc diffusion methods [ 28 , 29 ] . although the extracts used were crude , alkaloids have been found to be present in the chloroform and ethanol solvents of extraction . in this regard , the alkaloid extract of c. citrinus and v. adoensis might have broad spectrum activity since they exhibited activity against both gram - positive and gram - negative bacteria . the mic and mbc assay confirmed that alkaloids form c. citrinus were more potent extracts against s. aureus which had an mic value of 0.0025 mg / ml and an mbc of 0.835 mg / ml . the mbc of ampicillin was 0.008 mg / ml ; hence , the activity of the alkaloids is far outweighed by the standard antibiotic . v. adoensis alkaloids were least potent with mic of 0.42 mg / ml and 0.21 mg / ml against p. aeruginosa and s. aureus , respectively . bactericidal effects were shown by c. citrinus alkaloids against s. aureus only ( table 1 ) . a bacteriostatic effect against p. aeruginosa by all plant alkaloid extracts could be due to the pathogen 's thick outer membrane that is highly hydrophobic and possibly provided a permeability barrier to the extract . , chloroform extracts of alkaloids from c. citrinus were found to be inhibitors of growth in bacteria . mic values obtained for the gram - positive ( b. subtilis and b. pumilis ) and gram - negative bacteria ( e. coli ) were less than that of streptomycin , the standard antibiotic used in that study . atp - dependent efflux pumps are one of the main mechanisms of preventing accumulation of effective concentrations of antibiotics at molecular target sites inside the bacterial cell . the major efflux pump systems studied in gram - negative strains like p. aeruginosa are mexxy - oprm or mexcd - oprj which have been associated with acquired multidrug resistance [ 15 , 16 ] . in gram - positive bacteria such as s. aureus transporters of the major facilitator superfamily ( mfs ) , qaca , qacb , nora , and norb were found in strains causing hospital - acquired infections and conferring resistance to fluoroquinolones and puromycin . inhibition of efflux pumps was also found to decrease the mics for both antibiotic susceptible and resistant bacteria and reversed acquired resistance among selected p. aeruginosa strains . the alkaloids extract from both plants was able to cause accumulation of rhodamine 6 g in the cells of s. aureus and p. aeruginosa . however , c. citrinus alkaloids were potent inhibitors compared to alkaloids from v. adoensis and caused 141% and 121% increases in accumulation of r6 g in s. aureus and p. aeruginosa , respectively . previous work on efflux pump activity revealed v. adoensis crude extracts to be more active in inhibiting s. aureus than c. citrinus ; in light of the results of this present study , such activity of v. adoensis might have been due to other phytoconstituents present in the crude extracts . the greatest accumulation of r6 g was caused by exposure of cells to c. citrinus alkaloids against p. aeruginosa . these results are in agreement with the work of chitemerere and mukanganyama in which c. citrinus crude extracts caused the most significant accumulation in p. aeruginosa as well . therefore , p. aeruginosa was more sensitive to efflux pump inhibition by phytoconstituents than gram - positive microbes , although this bacterium was less sensitive to growth inhibition . this is an unexpected finding which was also observed with crude extracts that exhibited significant inhibition of 64100% of drug accumulation of rhodamine 6 g in p. aeruginosa cell . in vitro studies of synergistic effects of combinations of an mdr pump inhibitor and other antimicrobial phytoconstituents as well as conventional antibiotics previous studies have already proved the effectiveness of c. citrinus extract for in vitro antibacterial activity [ 28 , 29 , 32 ] . in conclusion , the alkaloid extracts from c. citrinus and v. adoensis have shown some antibacterial activity on s. aureus and p. aeruginosa . the c. citrinus alkaloid extract showed more potent growth inhibitory activity than that from v. adoensis with some bactericidal effects . p. aeruginosa was most susceptible to efflux pump inhibition by alkaloids extracted from c. citrinus which also caused notable accumulation in s. aureus . the demonstration of p. aeruginosa as being more susceptible to efflux pump inhibition means that formulations of medicinal products with epi activity may have potential acceptable efficacy against this bacterial species . further work needs to be done to isolate the specific chemicals that have antibacterial activity .

osteoporosis is a chronic disease of the skeleton characterized by loss of bone mass and disruption of bone microarchitecture , increasing the risk of fracture11,18 ) . nowadays , the incidence of osteoporosis is increasing rapidly in the elderly population , especially in postmenopausal women16,17,20 ) . bisphosphonates ( bps ) are potent antiresorptive agents widely used as the mainstay of treatment of osteoporosis . because bps accumulate in bone and are released for months or years after treatment is stopped , physicians need to be aware of the potential adverse effects of long - term use of bps in osteoporosis3,4,7,8,21 ) . 461due to the possible undesirable effects associated with pharmacological treatments , natural alternatives for the prevention and treatment of osteoporosis are highly desirable1,3,6,23 ) . curcumin is found in the rhizomes of the popular indian spice turmeric plant ( curcumin longa l. ) , a member of the ginger family . it is well known that curcumin has diverse biologic effects , including anti - inflammatory , antioxidant , antiviral and anti - infectious5,13,19 ) . in addition , some studies investigated the effects of curcumin on the regulation of bone remodeling . it has known as a therapeutic oriental plant that have shown to improve bone quality9 ) . in a recent study by french et al.6 ) , the long - term effects of curcumin administration in ovariectomized rats were examined , and it was concluded that curcumin produced beneficial changes in bone turnover and an increase in bone strength in the ovariectomized mature rat model of postmenopausal osteoporosis . curcumin has been consumed as a dietary spice at doses of up to approximately 1.5 mg / kg / day19 ) . however , according to the recent study of folwarczna et al.5 ) , curcumin at a dietary achievable dose may not be useful for the prevention or treatment of osteoporosis . although curcumin has a protective effect on bone remodeling2,5,6,9,13,19,22 ) , appropriate therapeutic concentrations of curcumin are not well known as therapeutic drugs for osteoporosis . thus the aim of the present study was to compare the bone sparing effect of treatment of low - dose and high - dose curcumin after ovariectomy in rats . we studied normal female rats and rats with estrogen deficiency ( bilaterally ovariectomized ) as a model of postmenopausal osteoporosis . all experiments involving animals were performed in accordance with the animal care guidelines issued by the national institutes of health , and were approved by the institutional animal care committee at our institute . forty female sprague - dawley rats ( 11 weeks old ) were purchased from samtako bio inc . ( osan , korea ) and acclimated to conditions for one week before the experiment . animals were housed in an air - conditioned room ( relative humidity 45 - 65% ) under a 12-h light / dark cycle at 222 and given free access to food and tap water . animals were fed phytoestrogen reduced food ( harlan teklad , madison , wi , usa ) and were housed in tall cages in order to provide a level of physical activity and mechanical stress considered to be osteogenic . the following week , 30 animals underwent bilateral ovariectomy ( ovx ) using the double dorso - lateral approach technique described in detail by park et al.12 ) . the remaining 10 animals underwent sham surgery and were designated as group 1 ( sham group ) . the 30 ovariectomized animals were randomly distributed among three groups ; untreated ovx group , low - dose curcumin ( 10 mg / kg ) administered group and high - dose curcumin ( 50 mg / kg ) administered group . before and four weeks after the ovx or sham operations , eight weeks after ovx or sham operations , all animals were sacrificed and blood samples were collected by cardiac puncture for serum isolation . curcumin was administered by a stomach tube ( po ) daily for eight weeks , and dietary intakes were limited to 20 g d in order to avoid excessive body weight gains following ovariectomy . serum was separated by centrifugation ( at 1500g ) and then stored at -80 until required for bone metabolic marker assays . serum estrogen ( estradiol , e2 ) was determined using an estradiol elisa kit ( elisa , drg instruments gmbh , marburg , germany ) . in addition , the 4th lumbar vertebrae were removed , fixed in a 3.7% formaldehyde in phosphate - buffered saline solution ( ph 7.4 ) for 16 h and then stored ( 4 ) in 80% ethanol for bone mass measurements . serum osteocalcin levels were determined using osteocalcin eia kits ( nordic bioscience diagnostics , herlev , denmark ) and alkaline phosphatase ( alp ) activities used quantichrome alp assay kits ( dalp-250 , bioassay systems , ca , usa ) . serum levels of c - terminal telopeptide fragment of type i collagen c - terminus ( ctx-1 ) , which is generated by the osteoclast and is a marker of bone resorption , were determined using ratlaps elisa kits ( nordic bioscience diagnostics ) . bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system ( explore locus sp , ge healthcare , london , ontario , canada ) with x - ray energy settings of 80 kv and 80 a . for bone analysis , . bone mineral densities ( bmd ) , trabecular bone volume fractions ( bv / tv , % ) , and cortical bone mineral densities ( crbmd ) were used for the quantitative analysis , which was performed using 2.0 + aba microview software provided with the micro - ct system . each bone was positioned on the two lower supports of the anvil of a universal testing machine ( instron 4202 ; instron , canton , ma , usa ) . load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests . the load versus displacement data were recorded automatically by the instron software ( instron series ix automated materials tester , version 8.04.00 , canton , ma , usa ) , which calculates the mechanical parameters from the load - displacement curves . repeated measure anova was used to compare body weights in the ovx and sham groups . one - way anova was used to identify significant differences between the groups , and p values of 0.05 were considered significant . all experiments involving animals were performed in accordance with the animal care guidelines issued by the national institutes of health , and were approved by the institutional animal care committee at our institute . forty female sprague - dawley rats ( 11 weeks old ) were purchased from samtako bio inc . ( osan , korea ) and acclimated to conditions for one week before the experiment . animals were housed in an air - conditioned room ( relative humidity 45 - 65% ) under a 12-h light / dark cycle at 222 and given free access to food and tap water . animals were fed phytoestrogen reduced food ( harlan teklad , madison , wi , usa ) and were housed in tall cages in order to provide a level of physical activity and mechanical stress considered to be osteogenic . the following week , 30 animals underwent bilateral ovariectomy ( ovx ) using the double dorso - lateral approach technique described in detail by park et al.12 ) . the remaining 10 animals underwent sham surgery and were designated as group 1 ( sham group ) . the 30 ovariectomized animals were randomly distributed among three groups ; untreated ovx group , low - dose curcumin ( 10 mg / kg ) administered group and high - dose curcumin ( 50 mg / kg ) administered group . before and four weeks after the ovx or sham operations , eight weeks after ovx or sham operations , all animals were sacrificed and blood samples were collected by cardiac puncture for serum isolation . curcumin was administered by a stomach tube ( po ) daily for eight weeks , and dietary intakes were limited to 20 g d in order to avoid excessive body weight gains following ovariectomy . serum was separated by centrifugation ( at 1500g ) and then stored at -80 until required for bone metabolic marker assays . serum estrogen ( estradiol , e2 ) was determined using an estradiol elisa kit ( elisa , drg instruments gmbh , marburg , germany ) . in addition , the 4th lumbar vertebrae were removed , fixed in a 3.7% formaldehyde in phosphate - buffered saline solution ( ph 7.4 ) for 16 h and then stored ( 4 ) in 80% ethanol for bone mass measurements . serum osteocalcin levels were determined using osteocalcin eia kits ( nordic bioscience diagnostics , herlev , denmark ) and alkaline phosphatase ( alp ) activities used quantichrome alp assay kits ( dalp-250 , bioassay systems , ca , usa ) . serum levels of c - terminal telopeptide fragment of type i collagen c - terminus ( ctx-1 ) , which is generated by the osteoclast and is a marker of bone resorption , were determined using ratlaps elisa kits ( nordic bioscience diagnostics ) . bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system ( explore locus sp , ge healthcare , london , ontario , canada ) with x - ray energy settings of 80 kv and 80 a . for bone analysis , . bone mineral densities ( bmd ) , trabecular bone volume fractions ( bv / tv , % ) , and cortical bone mineral densities ( crbmd ) were used for the quantitative analysis , which was performed using 2.0 + aba microview software provided with the micro - ct system . each bone was positioned on the two lower supports of the anvil of a universal testing machine ( instron 4202 ; instron , canton , ma , usa ) . load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests . the load versus displacement data were recorded automatically by the instron software ( instron series ix automated materials tester , version 8.04.00 , canton , ma , usa ) , which calculates the mechanical parameters from the load - displacement curves . repeated measure anova was used to compare body weights in the ovx and sham groups . one - way anova was used to identify significant differences between the groups , and p values of 0.05 were considered significant . table 1 shows mean body weights of the 40 rats euthanized 8 weeks after ovx or sham operations . at the start of the experiment , however , at 4 weeks after surgery , the mean body weight in the untreated ovx group and treated groups ( low - dose and high - dose curcumin group ) was significantly greater than in the sham group , and this significant difference was maintained throughout the experimental period ( p<0.05 at 4 and 8 weeks ) . the mean body weight in the untreated ovx group and treated groups ( low - dose and hig - dose curcumin group ) were similar throughout the experiment period . serum estrogen ( estradiol , e2 ) levels were expressed as percentage of the serum levels of the sham groups ( fig . as expected , serum estrogen levels in the untreated ovx group were significantly decreased after ovariectomy ( p<0.05 at 4 and 8 weeks ) . serum estrogen levels in low - dose curcumin treated group were slightly decreased compared than that of the sham group , but , which were not statistically significant ( p>0.05 ) . also , serum estrogen levels in the high - dose curcumin treated group were slightly higher compared to that of the sham group , which were not statistically significant ( p>0.05 ) . 2a , b show the serum levels of the bone formation biochemical markers , osteocalcin and alp , and fig . 2c shows the serum levels of ctx-1 , which is a sensitive marker of bone resorption . results have been expressed as percentage of the serum levels of the biochemical markers of the sham group . as expected , in the untreated ovx group , serum levels of osteocalcin , alp , and ctx-1 were significantly increased compared than that of the sham group . at 4 and 8 weeks after surgery , the concentrations of the bone formation markers ( osteocalcin and alp ) in the curcumin administered groups were significantly lower than in the untreated ovx group . compared with the low - dose curcumin group , the high - dose curcumin group had lower osteocalcin and alp levels at 8 weeks , statistically significant differences . 2c ) , the low - dose curcumin group had similar serum levels of ctx-1 compared than that of the sham group . in contrast , the high - dose curcumin group had significantly lower ctx-1 concentration at 4 and 8 weeks compared with the sham group as well as with the low - dose curcumin group . as shown in the representative micro - ct images of the 4th lumbar spine ( fig . 3 ) , the untreated ovx rats had fewer trabecular bone structures than that of the sham controls at 8 weeks after ovariectomy . the high - dose curcumin group had more trabecular bone structures compared with the low - dose curcumin group as well as the untreated ovx group . 4 shows the changes of bone histomorphometric parameters of the 4th lumbar vertebra 8 weeks after ovariectomy . all values represent 100% minus the percentage of the value of the sham group / the value of the experimental groups . in the analyses of micro - ct scans of 4th lumbar vertebrae , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a sustained increase in spine bmd and crbmd compared with the untreated ovx group . the high - dose curcumin treated group had a significant increase in bmd ( p=0.028 ) and crbmd ( p=0.036 ) compared with the low - dose curcumin treated group . considering bv / tv , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a significant increase compared with the untreated ovx group ( p=0.035 and p=0.001 ) . the high - dose curcumin group showed a sustained increase compared with the low - dose curcumin treated group , however , which was not statistically significant ( p=0.077 ) . although the three point bending test showed that the 4th lumbar vertebrae of the low - dose and high - dose curcumin treated groups had a greater maximal load value compared to the untreated ovx group , the only high - dose curcumin treated group differed significantly from the untreated ovx group ( p=0.015 ) ( fig . table 1 shows mean body weights of the 40 rats euthanized 8 weeks after ovx or sham operations . at the start of the experiment , however , at 4 weeks after surgery , the mean body weight in the untreated ovx group and treated groups ( low - dose and high - dose curcumin group ) was significantly greater than in the sham group , and this significant difference was maintained throughout the experimental period ( p<0.05 at 4 and 8 weeks ) . the mean body weight in the untreated ovx group and treated groups ( low - dose and hig - dose curcumin group ) were similar throughout the experiment period . serum estrogen ( estradiol , e2 ) levels were expressed as percentage of the serum levels of the sham groups ( fig . as expected , serum estrogen levels in the untreated ovx group were significantly decreased after ovariectomy ( p<0.05 at 4 and 8 weeks ) . serum estrogen levels in low - dose curcumin treated group were slightly decreased compared than that of the sham group , but , which were not statistically significant ( p>0.05 ) . also , serum estrogen levels in the high - dose curcumin treated group were slightly higher compared to that of the sham group , which were not statistically significant ( p>0.05 ) . 2a , b show the serum levels of the bone formation biochemical markers , osteocalcin and alp , and fig . 2c shows the serum levels of ctx-1 , which is a sensitive marker of bone resorption . results have been expressed as percentage of the serum levels of the biochemical markers of the sham group . as expected , in the untreated ovx group , serum levels of osteocalcin , alp , and ctx-1 were significantly increased compared than that of the sham group . at 4 and 8 weeks after surgery , the concentrations of the bone formation markers ( osteocalcin and alp ) in the curcumin administered groups were significantly lower than in the untreated ovx group . compared with the low - dose curcumin group , the high - dose curcumin group had lower osteocalcin and alp levels at 8 weeks , statistically significant differences . 2c ) , the low - dose curcumin group had similar serum levels of ctx-1 compared than that of the sham group . in contrast , the high - dose curcumin group had significantly lower ctx-1 concentration at 4 and 8 weeks compared with the sham group as well as with the low - dose curcumin group . as shown in the representative micro - ct images of the 4th lumbar spine ( fig . 3 ) , the untreated ovx rats had fewer trabecular bone structures than that of the sham controls at 8 weeks after ovariectomy . the high - dose curcumin group had more trabecular bone structures compared with the low - dose curcumin group as well as the untreated ovx group . 4 shows the changes of bone histomorphometric parameters of the 4th lumbar vertebra 8 weeks after ovariectomy . all values represent 100% minus the percentage of the value of the sham group / the value of the experimental groups . in the analyses of micro - ct scans of 4th lumbar vertebrae , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a sustained increase in spine bmd and crbmd compared with the untreated ovx group . the high - dose curcumin treated group had a significant increase in bmd ( p=0.028 ) and crbmd ( p=0.036 ) compared with the low - dose curcumin treated group . considering bv / tv , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a significant increase compared with the untreated ovx group ( p=0.035 and p=0.001 ) . the high - dose curcumin group showed a sustained increase compared with the low - dose curcumin treated group , however , which was not statistically significant ( p=0.077 ) . although the three point bending test showed that the 4th lumbar vertebrae of the low - dose and high - dose curcumin treated groups had a greater maximal load value compared to the untreated ovx group , the only high - dose curcumin treated group differed significantly from the untreated ovx group ( p=0.015 ) ( fig . although bisphosphonates , potent antiresorptive agents , have been the most popular first - line drugs for the treatment of osteoporosis for some time , many side effects have become evident in recent years4,8,18,21 ) . some reports suggested a link between bps use and the development of atypical insufficiency fractures or osteonecrosis of the jaw14,15 ) . these are thought to be due to long term oversuppression of bone turnover leading to impaired bone remodeling , accumulation of microdamage in bone and increased skeletal fragility . therefore , there is increasing interest in the discovery of natural substances that could favorably affect the skeletal system , which could be used in place of pharmacological treatment for osteoporosis . curcumin is a nonsteroidal , naturally occurring compound in the rhizomes of the popular indian spice turmeric plant ( curcumin longa l. ) , which is commonly used as a dietary pigment . in addition to a variety of pharmacologic effects , including anti - inflammatory , anti - infectious and antioxidant activities , which are traditionally known5,13,19 ) , recent studies investigated the protective effects of curcumin on the regulation of bone remodeling . it is well known that curcumin has action similar to bisphosphonate , the inhibition of osteoclastogenesis4 - 7,9,23 ) . ozaki et al.10 ) showed that curcumin is a potent stimulator of osteoclast apoptosis and also an inhibitor of bone resorption caused by rabbit osteoclast . it has also been shown in murine cells that curcumin inhibits osteoclastogenesis induced by receptor activation of nf - kb ligand2 ) . in the study by folwarczna et al.5 ) , 10 mg / kg curcumin was administered po daily for 4 weeks to normal and bilaterally ovariectomized rats . in their study , although curcumin slightly improved some bone histomorphometric parameters impaired by estrogen deficiency , the effects on the skeletal system were ambiguous . they concluded that curcumin at a dietary achievable dose may not be useful for the prevention or treatment of osteoporosis . curcumin has been consumed as a dietary spice at doses of up to 100 mg / day19 ) , i.e. , approximately 1.5 mg / kg / day , assuming that human body mass is 65 - 70 kg . according to phase i clinical trials , humans can tolerate curcumin even at a dose of 8 g / day2 ) . because it is known that the oral bioavailability of curcumin in rats is about 1% and the half - life of curcumin is rather short22 ) , we hypothesized that a relatively high concentration of curcumin would have a protective effect of bone remodeling . thus in the present study , ovariectomized rats were treated either with low - dose ( 10 mg / kg ) or high - dose ( 50 mg / kg ) curcumin . we then compared the therapeutic effects on bone remodeling with different dose of curcumin treatment using bone turnover markers , histomorphometric parameters and bone strength . french et al.6 ) conducted a well - designed experiment about the bone sparing effect of curcumin or bisphosphonate ( etidronate ) in the ovariectomized rat . they used three different doses of curcumin ; 1.5 mg / kg , 3 mg / kg , 15 mg / kg , which are a relatively low concentration of curcumin compared with the doses of our study . in their study , there was no difference in lumbar spine bmd at two months after ovariectomy . at four months post - ovariectomy , all three curcumin groups demonstrated a sustained increase in spine bmd over ovariectomized animals , which was not statistically significant . there was a 50% increase in mechanical strength for all groups of animals that received curcumin . we think that this lack of a significant increase in spine bmd in the curcumin group in the study by french et al.6 ) may be due to administration of a dose of curcumin that was too low to produce a significant increase in spine bmd . in our study using a relatively high dose of curcumin , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a sustained increase in spine bmd and crbmd compared with the untreated ovx group . in the comparison between the different doses of curcumin , the high - dose curcumin treated group had a significant increase in bmd and crbmd compared with the low - dose curcumin treated group . considering mechanical strength , the low - dose and high - dose curcumin treated groups had a greater maximal load value compared to the untreated ovx group , but only high - dose curcumin treated group had a significant difference from the untreated ovx group ( p=0.015 ) . we strongly agree with their opinion , that curcumin at a dietary achievable dose may not be useful for the prevention or treatment of osteoporosis . we think high - dose curcumin treatment could lead to more advantages in the bone sparing effect . as mentioned above , curcumin has an action similar to bisphosphonate , the inhibition of bone resorption by osteoclast . biochemical markers of bone turnover have been widely used as measures of the status of bone remodeling . the extent of bone resorption could be checked by ctx-1 level ( a sensitive marker of bone resorption ) . french et al.6 ) noted that the curcumin treated group showed ctx concentrations very similar to those of ovariectomized animals given etidronate . in our previous study about a synergistic bone sparing effect of curcumin ( 50 mg / kg , daily ) and alendronate in ovariectomized rats , we achieved results comparable to french et al.6 ) ctx-1 concentration in curcumin administered group was similar to those of the alendronate administered group . in addition , the combination therapy ( 50 mg / kg curcumin and alendronate ) group had lower ctx-1 concentrations , which were statistically significant to the curcumin only and the alendronate only group3 ) . in the present study , the high - dose curcumin group had significantly lower ctx-1 concentration compared with the sham group and the low - dose curcumin group . this is further evidence of our hypothesis that high - dose curcumin treatment could lead to better bone sparing effect . the present study demonstrated that a high - dose curcumin has therapeutic advantages over a low - dose curcumin as to antiresorptive effect on bone remodeling , and improving bone mechanical strength .

objectivealthough curcumin has a protective effect on bone remodeling , appropriate therapeutic concentrations of curcumin are not well known as therapeutic drugs for osteoporosis . the purpose of this study was to compare the bone sparing effect of treatment of low - dose and high - dose curcumin after ovariectomy in rats.methodsforty female sprague - dawley rats underwent either a sham operation ( the sham group ) or bilateral ovariectomy ( ovx ) . the ovariectomized animals were randomly distributed among three groups ; untreated ovx group , low - dose ( 10 mg / kg ) curcumin administered group , and high - dose ( 50 mg / kg ) curcumin group . at 4 and 8 weeks after surgery , serum biochemical markers of bone turnover were analyzed . bone histomorphometric parameters of the 4th lumbar vertebrae were determined by micro - computed tomography ( ct ) . in addition , mechanical strength was determined by a three - point bending test.resultshigh-dose curcumin group showed significantly lower osteocalcin , alkaline phosphatase , and the telopeptide fragment of type i collagen c - terminus concentration at 4 and 8 weeks compared with the untreated ovx group as well as low - dose curcumin group . in the analyses of micro - ct scans of 4th lumbar vertebrae , the high - dose curcumin treated group showed a significant increase in bone mineral densities ( p=0.028 ) and cortical bone mineral densities ( p=0.036 ) compared with the low - dose curcumin treated group . only high - dose curcumin treated group had a significant increase of mechanical strength compared with the untreated ovx group ( p=0.015).conclusionthe present study results demonstrat that a high - dose curcumin has therapeutic advantages over a low - dose curcumin of an antiresorptive effect on bone remodeling and improving bone mechanical strength .

INTRODUCTION MATERIALS AND METHODS Animal model and drug administration Serum biochemical markers of bone metabolism Histomorphometric analysis using microcomputed tomography Mechanical testing Statistical analysis RESULTS Body weights and estrogen levels Serum biochemical markers of bone metabolism Bone histomorphometric analysis using micro-CT Mechanical strength DISCUSSION CONCLUSION

osteoporosis is a chronic disease of the skeleton characterized by loss of bone mass and disruption of bone microarchitecture , increasing the risk of fracture11,18 ) . bisphosphonates ( bps ) are potent antiresorptive agents widely used as the mainstay of treatment of osteoporosis . because bps accumulate in bone and are released for months or years after treatment is stopped , physicians need to be aware of the potential adverse effects of long - term use of bps in osteoporosis3,4,7,8,21 ) . in addition , some studies investigated the effects of curcumin on the regulation of bone remodeling . in a recent study by french et al.6 ) , the long - term effects of curcumin administration in ovariectomized rats were examined , and it was concluded that curcumin produced beneficial changes in bone turnover and an increase in bone strength in the ovariectomized mature rat model of postmenopausal osteoporosis . curcumin has been consumed as a dietary spice at doses of up to approximately 1.5 mg / kg / day19 ) . although curcumin has a protective effect on bone remodeling2,5,6,9,13,19,22 ) , appropriate therapeutic concentrations of curcumin are not well known as therapeutic drugs for osteoporosis . thus the aim of the present study was to compare the bone sparing effect of treatment of low - dose and high - dose curcumin after ovariectomy in rats . all experiments involving animals were performed in accordance with the animal care guidelines issued by the national institutes of health , and were approved by the institutional animal care committee at our institute . forty female sprague - dawley rats ( 11 weeks old ) were purchased from samtako bio inc . the following week , 30 animals underwent bilateral ovariectomy ( ovx ) using the double dorso - lateral approach technique described in detail by park et al.12 ) . the remaining 10 animals underwent sham surgery and were designated as group 1 ( sham group ) . the 30 ovariectomized animals were randomly distributed among three groups ; untreated ovx group , low - dose curcumin ( 10 mg / kg ) administered group and high - dose curcumin ( 50 mg / kg ) administered group . in addition , the 4th lumbar vertebrae were removed , fixed in a 3.7% formaldehyde in phosphate - buffered saline solution ( ph 7.4 ) for 16 h and then stored ( 4 ) in 80% ethanol for bone mass measurements . serum osteocalcin levels were determined using osteocalcin eia kits ( nordic bioscience diagnostics , herlev , denmark ) and alkaline phosphatase ( alp ) activities used quantichrome alp assay kits ( dalp-250 , bioassay systems , ca , usa ) . serum levels of c - terminal telopeptide fragment of type i collagen c - terminus ( ctx-1 ) , which is generated by the osteoclast and is a marker of bone resorption , were determined using ratlaps elisa kits ( nordic bioscience diagnostics ) . bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system ( explore locus sp , ge healthcare , london , ontario , canada ) with x - ray energy settings of 80 kv and 80 a . bone mineral densities ( bmd ) , trabecular bone volume fractions ( bv / tv , % ) , and cortical bone mineral densities ( crbmd ) were used for the quantitative analysis , which was performed using 2.0 + aba microview software provided with the micro - ct system . load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests . repeated measure anova was used to compare body weights in the ovx and sham groups . all experiments involving animals were performed in accordance with the animal care guidelines issued by the national institutes of health , and were approved by the institutional animal care committee at our institute . forty female sprague - dawley rats ( 11 weeks old ) were purchased from samtako bio inc . animals were fed phytoestrogen reduced food ( harlan teklad , madison , wi , usa ) and were housed in tall cages in order to provide a level of physical activity and mechanical stress considered to be osteogenic . the following week , 30 animals underwent bilateral ovariectomy ( ovx ) using the double dorso - lateral approach technique described in detail by park et al.12 ) . the 30 ovariectomized animals were randomly distributed among three groups ; untreated ovx group , low - dose curcumin ( 10 mg / kg ) administered group and high - dose curcumin ( 50 mg / kg ) administered group . in addition , the 4th lumbar vertebrae were removed , fixed in a 3.7% formaldehyde in phosphate - buffered saline solution ( ph 7.4 ) for 16 h and then stored ( 4 ) in 80% ethanol for bone mass measurements . serum osteocalcin levels were determined using osteocalcin eia kits ( nordic bioscience diagnostics , herlev , denmark ) and alkaline phosphatase ( alp ) activities used quantichrome alp assay kits ( dalp-250 , bioassay systems , ca , usa ) . serum levels of c - terminal telopeptide fragment of type i collagen c - terminus ( ctx-1 ) , which is generated by the osteoclast and is a marker of bone resorption , were determined using ratlaps elisa kits ( nordic bioscience diagnostics ) . bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system ( explore locus sp , ge healthcare , london , ontario , canada ) with x - ray energy settings of 80 kv and 80 a . bone mineral densities ( bmd ) , trabecular bone volume fractions ( bv / tv , % ) , and cortical bone mineral densities ( crbmd ) were used for the quantitative analysis , which was performed using 2.0 + aba microview software provided with the micro - ct system . load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests . table 1 shows mean body weights of the 40 rats euthanized 8 weeks after ovx or sham operations . at the start of the experiment , however , at 4 weeks after surgery , the mean body weight in the untreated ovx group and treated groups ( low - dose and high - dose curcumin group ) was significantly greater than in the sham group , and this significant difference was maintained throughout the experimental period ( p<0.05 at 4 and 8 weeks ) . the mean body weight in the untreated ovx group and treated groups ( low - dose and hig - dose curcumin group ) were similar throughout the experiment period . as expected , serum estrogen levels in the untreated ovx group were significantly decreased after ovariectomy ( p<0.05 at 4 and 8 weeks ) . serum estrogen levels in low - dose curcumin treated group were slightly decreased compared than that of the sham group , but , which were not statistically significant ( p>0.05 ) . also , serum estrogen levels in the high - dose curcumin treated group were slightly higher compared to that of the sham group , which were not statistically significant ( p>0.05 ) . 2a , b show the serum levels of the bone formation biochemical markers , osteocalcin and alp , and fig . results have been expressed as percentage of the serum levels of the biochemical markers of the sham group . as expected , in the untreated ovx group , serum levels of osteocalcin , alp , and ctx-1 were significantly increased compared than that of the sham group . at 4 and 8 weeks after surgery , the concentrations of the bone formation markers ( osteocalcin and alp ) in the curcumin administered groups were significantly lower than in the untreated ovx group . compared with the low - dose curcumin group , the high - dose curcumin group had lower osteocalcin and alp levels at 8 weeks , statistically significant differences . 2c ) , the low - dose curcumin group had similar serum levels of ctx-1 compared than that of the sham group . in contrast , the high - dose curcumin group had significantly lower ctx-1 concentration at 4 and 8 weeks compared with the sham group as well as with the low - dose curcumin group . as shown in the representative micro - ct images of the 4th lumbar spine ( fig . 3 ) , the untreated ovx rats had fewer trabecular bone structures than that of the sham controls at 8 weeks after ovariectomy . the high - dose curcumin group had more trabecular bone structures compared with the low - dose curcumin group as well as the untreated ovx group . 4 shows the changes of bone histomorphometric parameters of the 4th lumbar vertebra 8 weeks after ovariectomy . all values represent 100% minus the percentage of the value of the sham group / the value of the experimental groups . in the analyses of micro - ct scans of 4th lumbar vertebrae , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a sustained increase in spine bmd and crbmd compared with the untreated ovx group . the high - dose curcumin treated group had a significant increase in bmd ( p=0.028 ) and crbmd ( p=0.036 ) compared with the low - dose curcumin treated group . considering bv / tv , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a significant increase compared with the untreated ovx group ( p=0.035 and p=0.001 ) . the high - dose curcumin group showed a sustained increase compared with the low - dose curcumin treated group , however , which was not statistically significant ( p=0.077 ) . although the three point bending test showed that the 4th lumbar vertebrae of the low - dose and high - dose curcumin treated groups had a greater maximal load value compared to the untreated ovx group , the only high - dose curcumin treated group differed significantly from the untreated ovx group ( p=0.015 ) ( fig . table 1 shows mean body weights of the 40 rats euthanized 8 weeks after ovx or sham operations . at the start of the experiment , however , at 4 weeks after surgery , the mean body weight in the untreated ovx group and treated groups ( low - dose and high - dose curcumin group ) was significantly greater than in the sham group , and this significant difference was maintained throughout the experimental period ( p<0.05 at 4 and 8 weeks ) . the mean body weight in the untreated ovx group and treated groups ( low - dose and hig - dose curcumin group ) were similar throughout the experiment period . as expected , serum estrogen levels in the untreated ovx group were significantly decreased after ovariectomy ( p<0.05 at 4 and 8 weeks ) . serum estrogen levels in low - dose curcumin treated group were slightly decreased compared than that of the sham group , but , which were not statistically significant ( p>0.05 ) . also , serum estrogen levels in the high - dose curcumin treated group were slightly higher compared to that of the sham group , which were not statistically significant ( p>0.05 ) . 2a , b show the serum levels of the bone formation biochemical markers , osteocalcin and alp , and fig . results have been expressed as percentage of the serum levels of the biochemical markers of the sham group . as expected , in the untreated ovx group , serum levels of osteocalcin , alp , and ctx-1 were significantly increased compared than that of the sham group . at 4 and 8 weeks after surgery , the concentrations of the bone formation markers ( osteocalcin and alp ) in the curcumin administered groups were significantly lower than in the untreated ovx group . compared with the low - dose curcumin group , the high - dose curcumin group had lower osteocalcin and alp levels at 8 weeks , statistically significant differences . 2c ) , the low - dose curcumin group had similar serum levels of ctx-1 compared than that of the sham group . in contrast , the high - dose curcumin group had significantly lower ctx-1 concentration at 4 and 8 weeks compared with the sham group as well as with the low - dose curcumin group . as shown in the representative micro - ct images of the 4th lumbar spine ( fig . 3 ) , the untreated ovx rats had fewer trabecular bone structures than that of the sham controls at 8 weeks after ovariectomy . the high - dose curcumin group had more trabecular bone structures compared with the low - dose curcumin group as well as the untreated ovx group . 4 shows the changes of bone histomorphometric parameters of the 4th lumbar vertebra 8 weeks after ovariectomy . all values represent 100% minus the percentage of the value of the sham group / the value of the experimental groups . in the analyses of micro - ct scans of 4th lumbar vertebrae , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a sustained increase in spine bmd and crbmd compared with the untreated ovx group . the high - dose curcumin treated group had a significant increase in bmd ( p=0.028 ) and crbmd ( p=0.036 ) compared with the low - dose curcumin treated group . considering bv / tv , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a significant increase compared with the untreated ovx group ( p=0.035 and p=0.001 ) . the high - dose curcumin group showed a sustained increase compared with the low - dose curcumin treated group , however , which was not statistically significant ( p=0.077 ) . although the three point bending test showed that the 4th lumbar vertebrae of the low - dose and high - dose curcumin treated groups had a greater maximal load value compared to the untreated ovx group , the only high - dose curcumin treated group differed significantly from the untreated ovx group ( p=0.015 ) ( fig . some reports suggested a link between bps use and the development of atypical insufficiency fractures or osteonecrosis of the jaw14,15 ) . these are thought to be due to long term oversuppression of bone turnover leading to impaired bone remodeling , accumulation of microdamage in bone and increased skeletal fragility . in addition to a variety of pharmacologic effects , including anti - inflammatory , anti - infectious and antioxidant activities , which are traditionally known5,13,19 ) , recent studies investigated the protective effects of curcumin on the regulation of bone remodeling . it is well known that curcumin has action similar to bisphosphonate , the inhibition of osteoclastogenesis4 - 7,9,23 ) . in the study by folwarczna et al.5 ) , 10 mg / kg curcumin was administered po daily for 4 weeks to normal and bilaterally ovariectomized rats . in their study , although curcumin slightly improved some bone histomorphometric parameters impaired by estrogen deficiency , the effects on the skeletal system were ambiguous . , approximately 1.5 mg / kg / day , assuming that human body mass is 65 - 70 kg . because it is known that the oral bioavailability of curcumin in rats is about 1% and the half - life of curcumin is rather short22 ) , we hypothesized that a relatively high concentration of curcumin would have a protective effect of bone remodeling . thus in the present study , ovariectomized rats were treated either with low - dose ( 10 mg / kg ) or high - dose ( 50 mg / kg ) curcumin . we then compared the therapeutic effects on bone remodeling with different dose of curcumin treatment using bone turnover markers , histomorphometric parameters and bone strength . french et al.6 ) conducted a well - designed experiment about the bone sparing effect of curcumin or bisphosphonate ( etidronate ) in the ovariectomized rat . they used three different doses of curcumin ; 1.5 mg / kg , 3 mg / kg , 15 mg / kg , which are a relatively low concentration of curcumin compared with the doses of our study . we think that this lack of a significant increase in spine bmd in the curcumin group in the study by french et al.6 ) may be due to administration of a dose of curcumin that was too low to produce a significant increase in spine bmd . in our study using a relatively high dose of curcumin , the curcumin treated groups ( low - dose and high - dose curcumin groups ) showed a sustained increase in spine bmd and crbmd compared with the untreated ovx group . in the comparison between the different doses of curcumin , the high - dose curcumin treated group had a significant increase in bmd and crbmd compared with the low - dose curcumin treated group . considering mechanical strength , the low - dose and high - dose curcumin treated groups had a greater maximal load value compared to the untreated ovx group , but only high - dose curcumin treated group had a significant difference from the untreated ovx group ( p=0.015 ) . we think high - dose curcumin treatment could lead to more advantages in the bone sparing effect . as mentioned above , curcumin has an action similar to bisphosphonate , the inhibition of bone resorption by osteoclast . biochemical markers of bone turnover have been widely used as measures of the status of bone remodeling . french et al.6 ) noted that the curcumin treated group showed ctx concentrations very similar to those of ovariectomized animals given etidronate . in our previous study about a synergistic bone sparing effect of curcumin ( 50 mg / kg , daily ) and alendronate in ovariectomized rats , we achieved results comparable to french et al.6 ) ctx-1 concentration in curcumin administered group was similar to those of the alendronate administered group . in addition , the combination therapy ( 50 mg / kg curcumin and alendronate ) group had lower ctx-1 concentrations , which were statistically significant to the curcumin only and the alendronate only group3 ) . in the present study , the high - dose curcumin group had significantly lower ctx-1 concentration compared with the sham group and the low - dose curcumin group . this is further evidence of our hypothesis that high - dose curcumin treatment could lead to better bone sparing effect . the present study demonstrated that a high - dose curcumin has therapeutic advantages over a low - dose curcumin as to antiresorptive effect on bone remodeling , and improving bone mechanical strength .

two characteristics define embryonic stem cells ( escs ) , self - renewal ability and pluripotency . recently , ectopic expression of combinations of transcription factors ( oct4 , nanog , sox2 , c - myc , esrrb , and klf4 ) has been shown to reprogram mouse and human fibroblasts into a pluripotent state ( kaji et al . , 2009 ; okita et al . , 2007 ; takahashi et al . , 2007 ; takahashi and yamanaka , 2006 ; woltjen et al . , 2009 ; yu et al . , 2007 ) . the induced pluripotent stem cells ( ipscs ) are very similar to escs and retain the ability to self - renew and differentiate into the three germ layers and thus promise great therapeutic potential in regenerative medicine ( amabile and meissner , 2009 ; maherali et al . , 2007 ; wernig et al . , 2007 ) . despite the recent flurry of studies , our understanding of the molecular mechanisms and players that drive esc self - renewal and differentiation is still limited . the pou transcription factor oct4 , also termed pou5f1 , is a central player in esc self - renewal and differentiation into specific lineages . a decrease in oct4 levels by 50% induces differentiation toward the trophectoderm lineage , whereas a 50% increase causes differentiation into mesoderm and endoderm ( niwa et al . , 2000 ; shimozaki et al . , oct4 plays an essential role in early development given that loss of oct4 in the mouse embryo causes the failure of the inner cell mass to develop ( nichols et al . , 1998 ) . oct4 regulates transcriptional programs to maintain esc pluripotency primarily in collaboration with transcription factors sox2 and nanog ( boyer et al . , 2005 ; chew et al . , 2005 ; pan et al . , several genome - wide analyses of regulatory targets of key pluripotency factors has led to the identification of sets of jointly regulated or bound targets , highlighting a complex transcriptional circuitry responsible for esc maintenance ( babaie et al . , 2007 ; boyer et al . , 2005 ; ivanova et al . , 2006 ; kim et al . , 2008 ; loh et al . , 2006 ; matoba et al . , also recently , various other factors have been functionally linked to oct4 and nanog , after identification of their binding partners by affinity purification and mass spectrometry ( wang et al . these studies have revealed a compact regulatory module responsible for esc pluripotency ( orkin et al . , 2008 ) . to further elucidate the esc transcriptional network , we have carried out an unbiased and extensive study of oct4-associated proteins , using an affinity purification and mass spectrometry approach . in contrast with a previous similar study ( wang et al . , 2006 ) , epitope - tagged oct4 was expressed under the control of oct4 's endogenous promoter to keep the natural transcriptional regulation . the epitope tagging strategy circumvents the need for specific antibodies and facilitates a generic purification procedure that results in cleaner and higher yield samples than traditional immunoprecipitation experiments . our data significantly expands the current repertoire of oct4-associated proteins , thereby shedding light on the complex regulatory circuitries of escs . the oct4 interactome provides a useful resource to investigate the mechanisms of oct4 function and regulation and to explore the basic principles underlying stem cell biology . to investigate the molecular network around oct4/pou5f1 , we used an epitope - tagging affinity purification strategy . we modified the spa tag ( zeghouf et al . , 2004 ) containing the 3 flag epitope and a calmodulin binding peptide ( cbp ) separated by a tev cleavage site , by adding an extra tev site to improve cleavage efficiency ( figure s1a available online ) . the ftap was fused at the c terminus of the oct4 coding region by recombineering into a bac clone containing full - length oct4 . this was then integrated into the hprt locus of escs by recombinase - mediated cassette exchange ( rmce ) ( prosser et al . , 2008 ) . expression levels of the oct4-ftap fusion protein were 30% that of endogenous oct4 expressed from two alleles ( figure s1c ) , close to what should be expected given that it is expressed from an extra copy of the gene and avoiding interference with the esc phenotype , as shown by the expression of esc markers by the transgenic clone ( figure s1c ) . we first performed three independent one - step purifications on whole - cell extracts from both oct4-ftap - expressing and control unmodified cells ( figure 1a ) . eluates were separated by gel electrophoresis , and whole lanes were excised into several regions , digested , and analyzed by nano - liquid chromatrography / tandem mass spectrometry ( lc - ms / ms ) . ms results files from each lane the data is available in the pride database ( martens et al . , 2005 ) the data was converted with the pride converter ( barsnes et al . , 2009 ) mass spectrometry analysis resulted in the identification of 92 proteins ( excluding oct4 itself ) that were present in all oct4-ftap purifications , but not in controls ( table 1 ) . the identification of some of the interacting proteins was confirmed by western blotting ( figure 1b ) . these data considerably expand the list of published oct4 binding partners and represent a major extension of the sets reported in two similar studies ( liang et al . we detected 13 previously identified oct4 interacting proteins in our study ( table s6 ) . these included sall4 , arid3b , zfp219 , and sp1 ( wang et al . parp1 ( gao et al . , 2009 ) , and nurd complex members hdac1 , mta1/2 , and gatad2a / b ( liang et al . , 2008 ; wang et al . , 2006 ) . furthermore , we also identified sox2 and nanog , two of the best characterized oct4 binding partners ( ambrosetti et al . , 1997 ; chew et al . , 2005 ; liang et al . , 2008 ; wang et al . , 2006 ) , and zfp281 , requiem / dpf2 , yy1 , rybp , dax1 , esrrb , and arid3a , recently shown to physically interact with oct4 ( donohoe et al . , 2009 ; sun et al . , 2009 ; van den berg et al . , 2008 ; wang et al . , 2006 ; wang et al . , 2008 ) in one or two ( arid3a and esrrb ) purifications , but because of our strict criteria of result reproducibility , we did not include them in the final data set . we also identified proteins reported to be linked to oct4 through association with some of its interactors , namely sall1 and smarcc1 ( wang et al . eight previously identified oct4-interacting proteins were either not detected , namely ews , nf45 , cdk1 ( wang et al . , 2006 ) , and zfp206 ( yu et al . , 2009 ) , or found also in controls , such as beta - catenin ( takao et al . , 2007 ) , hdac2 ( liang et al . , 2008 ) , ctcf ( donohoe et al . , 2009 ) , and wwp2 ( xu et al . , 2009 ; we next performed tandem affinity purification , although yields were not high because of the low levels of tagged oct4 and purification efficiency . we identified seven proteins of the 92 , mainly members of nurd , sall proteins , and transcription factors e3 and eb ( table s6 ) . we believe these constitute the highest - affinity interactors , given that they can endure a more stringent purification . although yielding small numbers of interactors , this purification is still at the level of previous similar studies ( liang et al . , 2008 ; wang et al . , 2006 ) . for confirmation , we immunoprecipitated endogenous oct4 from whole - cell extracts of untagged feeder - free e14 mouse escs in duplicate and analyzed immunoprecipitates by mass spectrometry . forty - six proteins reproducibly overlapped with the ftap data set ( table s6 ) . we detected all proteins identified in a similar experiment ( liang et al . , 2008 ) . not surprisingly , proteins that were reproducibly copurified with endogenous oct4 tended to be more abundant in our single - affinity purification data set . to address whether the interactions detected were due to coassembly of factors on chromatin , we then immunoprecipitated oct4 in the presence of dnase treatment with benzonase . western blotting showed that parp1 , a ubiquitous dna - binding protein , coimmunoprecipitates with oct4 even in the absence of dna ( figure 1c ) . preliminary purification experiments with a differently tagged oct4-ftap cell line suggested that other dna - binding proteins such as ligase 3 and topoisomerase 2a also copurify with oct4 in the absence of dna ( m.p . and s.p . this suggests that the interactions we detect are not dna mediated . summing up , over 50% of the oct4-associated proteins ( 47 of 92 ) varying in abundance across our data set have been confirmed by independent means , suggesting that the data set we provide here is a bona fide set of oct4 binding partners . to uncover general trends in the functions of the oct4-interacting proteins , we carried out computational systems - level analyses in a workflow depicted in figure 1d . we first performed a functional annotation analysis using david 2008 ( dennis et al . , 2003 ) and the panther database ( thomas et al . , 2003 ) . we found an enrichment of go terms such as nucleus , chromosome , and chromatin in the cellular component ontology ; nucleic acid binding , protein binding , transcription factor activity , in the molecular function ontology ; and transcription , regulation of gene expression , and embryonic development in the biological process ontology ( figure s2 and table s2 ) . this indicates that go terms associated to oct4 are highly represented within the list of oct4-copurifying proteins , adding consistency to the data set . twenty proteins in the data set ( 21% ) are annotated with the go term oct4 has been shown to associate with several transcription factors , and our results agree with the notion that combinatorial binding among pluripotency factors , which contributes to achieving specificity in gene regulation , may be a frequent pattern in escs ( chambers and tomlinson , 2009 ) . david analysis detected an enrichment of proteins involved in the control of gene expression by vitamin d nuclear hormone receptor , mainly members of the fact and swi / snf complexes . the data set also contains several proteins involved in the nuclear part of the wnt signaling pathway , as revealed by panther analysis . , 2007 ; sato et al . , 2004 ) , possibly by modulating levels of pluripotency factors oct4 , nanog , and sox2 ( kalmar et al . , 2009 ) . we then analyzed the domain composition of oct4-interacting proteins and detected a significant abundance of dna - binding and chromatin - related domains ( tables s3 and s4 ) . highly represented domains were dead / deah box helicase , snf2-related , phd and zinc fingers , and chromo , bromo , and homeobox domains , all of which are either involved in atp - dependent chromatin remodeling or bind dna and posttranslationally modified nucleosomes , thereby influencing gene expression . the data set was manually classified into known protein complexes and functional categories . as shown in table 1 and supported by the go and panther analyses , oct4 associates mainly with transcriptional regulators , but also with a variety of other chromatin binding proteins involved in dna replication , recombination , and repair , proteins involved in nuclear assembly and/or organization , and diverse enzymes , some of which are responsible for addition of posttranslational modifications . to gain an overall view of the previously known interactions among oct4-associated proteins , we retrieved interaction data from intact , hrpd , and mint for the data set and represented them as a protein interaction network ( figure 1e ) . repressor complexes nurd and swi / snf and dna repair and de novo dna methylation modules are apparent in the network . many known oct4 binding proteins are esc - specific factors ( wang et al . , 2008 ) . however , oct4 has also been shown to interact with more general modulators of transcription that are expressed ubiquitously , such as members of the nurd complex ( liang et al . we investigated the patterns of expression of the oct4-associated data set in cells at different stages of differentiation , including embryonic carcinoma , embryonic stem cells , embryoid bodies , and various differentiated cell types , on the basis of transcriptomics data ( campbell et al . , 2007 ) . protein abundances were fairly varied and most interactors maintained near constant expression across the samples analyzed ( figure 2 ) . this suggests that oct4 interacts mostly with proteins that are ubiquitously expressed in both differentiated and undifferentiated cells . after statistical analysis , 33 oct4-interacting proteins were found to be significantly less expressed in differentiated cells compared to escs , in correlation with oct4 's expression pattern ( figure 2 and table s6 ) . among these are the dna methylation regulatory factor dnmt3l and the developmentally important transcription factors klf4 , sall1 , and sall4 . we observed that many complexes or interacting pairs in the interaction network contained at least one member significantly downregulated upon esc differentiation ( figure 1e ) , possibly conferring an esc - specific role . regulation of gene expression involves complex dynamics employing sequence - specific dna binding proteins that form the transcriptional regulatory network ( babu et al . , 2004 ; transcription factors often operate in feedback loops , whereby the expression of a transcriptional target modulates the function of the transcription factor itself ( figure 3a ) . pluripotency factors in escs are no exception and show a high degree of transcriptional auto and interregulation ( orkin et al . , 2008 ) . we next investigated whether the promoters of the oct4-associated gene set contain binding sites for transcription factors that are central in the establishment and maintenance of esc identity . promoter binding sites for nine such transcription factors have previously been identified by chip - on - chip ( kim et al . , these include oct4 , dax1 , klf4 , c - myc , nac1 , nanog , rex1 , zfp281 , and sox2 . nine of the 92 oct4-associated proteins were found to be transcriptionally regulated by oct4 itself in mouse escs , and 51% of genes encoding oct4 partners are targets of at least one key esc transcription factor ( figure s3 ) . this concurs with findings by others on a much smaller data set ( orkin et al . , 2008 ; wang et al . , 2006 ) . to assess whether this is statistically significant the expected percentage of promoter binding by esc transcription factors was only 28% ( z = 4.45 , p < 10 ) , indicating that it is a significant trait of the data set . several genes in the interaction set are common targets of multiple transcription factors ( 20 of 92 are targets of at least three transcription factors ) , making it likely that they have central roles in pluripotency and self - renewal . ten of these are significantly downregulated in differentiation , and all but two show a downregulation trend ( table s6 ) , in agreement with the hypothesis that genes bound by multiple factors are active in escs and become repressed as cells differentiate ( chambers and tomlinson , 2009 ; kim et al . 2008 ) . we constructed a regulatory network by integrating promoter target data for the nine stem cell transcription factors with the list of oct4 binding proteins ( figure 3b ) . several of the transcription factors cluster together because of shared targets ( e.g. , sox2 , nanog , nac1 , and dax1 ) , whereas c - myc and klf4 exclusively target certain groups of oct4-interacting factors . this agrees with the genome - wide trend of the c - myc target set , which is largely distinct from the rest of the pluripotency factors ( kim et al . , we next explored the consequences of loss of oct4-interacting proteins in escs or mouse development . five oct4-interacting proteins have been identified as required for stem cell self - renewal in large scale rnai screens ( ding et al . , 2009 ; literature searches ascribed a role in esc self - renewal or pluripotency to an additional nine oct4-interacting proteins ( table s6 ) . loss - of - function phenotypes in mice were available in the mgi database for 49 oct4-associated proteins . all 49 show diverse phenotypes when absent or mutated ( figure 4 and table s5 ) . significantly , 83% ( 41 of 49 ) of the studied knockout alleles of the interaction set showed embryonic and/or perinatal lethality , with over 60% ( 30 of 49 ) being embryonic lethal ( figure 1e ) . similar analyses on random control data sets allowed us to conclude that the result is significant ( z = 7.48 , p < 10 ) . in addition , 41% ( 20 of 49 ) showed an abnormal development phenotype . these results indicate a high level of requirement for components of the oct4 network in early mouse development . although feedback loops are expected to add to the robustness of a transcriptional regulatory network , the high frequency with which mutation of single oct4 partners causes severe early developmental phenotypes suggests they are essential downstream regulatory hubs . given the extent of their part in mouse development and the current excitement about the cancer stem cell hypothesis , we next explored a possible role of oct4-associated proteins in human disease . human orthologs were identified for all oct4-associated proteins and sequence identities determined between mouse and human ( figure s4 ) . all oct4-associated proteins were found to be highly conserved , with a median sequence identity of 94% , compared to 77% genomic median . this strong sequence conservation implies that the findings reported here could be applied to human esc biology . we next investigated the involvement of the human orthologs in human disease and development of cancer interrogating the omim database and the cancer gene census , which records genes whose mutation has been causally linked to cancer . genes encoding 14 of 92 oct4-associated proteins are implicated in one or more hereditary diseases , mostly of developmental nature , with six of them predisposing to certain types of cancer ( table 2 ) . somatic mutations in eight oct4-associated proteins and oct4 itself were found to be responsible for different types of cancer , often through gene translocations , presumably affecting their regulation ( table 3 ) . statistical analysis on random sets indicated that the observed numbers of oct4-interacting proteins linked to human disease ( z = 1.06 , p < 10 ) and cancer ( z = 4.43 , p < 10 ) are significantly higher than expected . in light of the central role of oct4 in pluripotency and the cancer stem cell hypothesis , we investigated which of oct4 's physical interactors are misexpressed in cancer using the oncomine human cancer expression database . a large fraction ( 60% ) of the oct4 interactors show misexpression in at least one cancer type , providing a degree of additional support to the connection between stem cell identity and cancer . the characterization of protein - protein interactions is a very efficient strategy for understanding protein function and regulation . the development of high - affinity tags , including the tap ( rigaut et al . , 1999 ) and in vivo biotinylation tag ( de boer et al . , 2003 ) , in combination with advances in mass spectrometry that now allow protein identification with high sensitivity and accuracy , has recently produced several protein interaction network reports . however , most studies in the literature rely on cdna overexpression driven by exogenous promoters or transgenic random integration approaches . we report here an epitope - tagging strategy for the purification of protein complexes in mouse escs . we introduced the tag by recombineering into a full - length oct4-containing bac and then integrated this in a precise location in the mouse genome . this approach has the advantage of maintaining the endogenous promoter and therefore natural transcriptional regulation . the technology is amenable to high - throughput delivery , as recently demonstrated by random integration of tagged bac transgenes ( poser et al . , 2008 ) , and should greatly facilitate systematic tagging of genes and analysis of protein complexes with roles in development in different contexts , be it in stem cells , differentiated cell types , or even mouse tissue ( fernndez et al . , 2009 ) . the affinity purification method described here is rapid , with the goal of capturing weak or short - lived interactions . previous proteomic studies of oct4 protein complexes have relied on lengthy single or tandem purifications from nuclear extracts with streptavidin capture ( wang et al . , 2006 ) or anti - oct4 antibodies ( liang et al . , 2008 ) and yielded small data sets , very similar to our tandem purification data set . we identified all of the partners reported by the liang study except hdac2 , and only five oct4 partners found in the wang study were not detected in our data set , maybe because of our use of whole extracts . indeed , our approach has produced by far the most extensive analysis of oct4-associated proteins to date . by using whole extracts , thereby not restricting the analysis to the nuclear environment , our data set encompasses diverse aspects of the life of oct4 , both nuclear and nonnuclear . the broad data set puts oct4 at the center of diverse cellular processes that can have an impact on aspects of stem cell biology ( figure 5 ) , the most interesting of which are discussed below . oct4 can both activate and repress transcriptional targets in mouse and human escs ( babaie et al . , oct4 has been shown to be associated mainly with members of repressor complexes nurd and swi / snf ( liang et al . , 2008 ; nurd , a histone deacetylase complex , was the most prominent , further confirming this link . sall4 , a well - known oct4 partner , and other members of the spalt - like family of transcriptional cofactors have been shown to associate to nurd ( lauberth and rauchman , 2006 ) , raising the possibility that they may bridge the interaction between oct4 and nurd . this hypothesis is also supported by the similar amounts in which they are detected in our experiments . we also found several subunits of the swi / snf nucleosome - remodeler complex , some of which have previously been linked to nanog ( liang et al . , 2008 ) , confirming the link to this chromatin remodeling complex . also among oct4 binding proteins we found various molecules involved in positive regulation of transcription , including several activators and coactivators and chromatin - modifying enzymes such as myst2 , a histone h4 acetyltransferase ( doyon et al . , 2006 ; sterner and berger , 2000 ) . in addition , we detected ttf2 , a component of the general transcription machinery , providing evidence of a physical link between pluripotency factors and basal transcription players . the oct4 interactome included other basal dna - process - related factors such as proteins involved in dna replication , recombination , and repair . this could explain why many of the oct4-interacting proteins are ubiquitously expressed in both differentiated and undifferentiated cells . our experiments suggest that the interaction is not dna mediated , given that copurification of dna - binding proteins still occurs upon dna elimination by benzonase . ogt is responsible for posttranslational addition of o - linked n - acetylglucosamine ( o - glcnac ) , a regulatory protein modification similar to phosphorylation possibly working in concert with it ( kamemura and hart , 2003 ) . oct4 is modified by o - glcnac in human escs ( webster et al . , 2009 ) , and sp1 , one of oct4 partners , is too ( jackson and tjian , 1988 ) . a thorough analysis of o - glcnac modification in the oct4 interactome might yield important insight into dynamic modulation of stem cell factors . posttranslational modification of transcription factors and cofactors is proving to be a critical component of the regulation of gene transcription in general , and important specifically in stem cell biology ( brill et al . half of oct4-associated proteins seem to be directly regulated by transcription factors with key roles in stem cell pluripotency and/or reprogramming . this is also a characteristic of pluripotency networks derived from smaller data sets from different entry points ( orkin et al . , 2008 ; this indicates that even in the expanded and functionally diverse network , this attribute still holds true , supporting a previously unsuspected role in stem cell biology for some of the proteins we identify here . expression of oct4 decreases in a switch - like fashion as escs differentiate into lineage - specific cell types , including progenitor cells . our analysis has uncovered 33 physical interactors of oct4 that share this trend . among these are several transcription factors , such as the dna methyltransferase 3-like regulatory protein dnmt3l , which stimulates genomic imprinting in germ cells ( bourc'his et al . , 2001 ; this is consistent with a recent report demonstrating that treatment with dna methyltransferase inhibitors can improve the efficiency of the reprogramming process of differentiated cells ( mikkelsen et al . , 2008 ) . therefore , the 33 interactors upregulated in escs and the transcription factors that regulate them might be interesting candidates whose expression could be manipulated to facilitate reprogramming . we find that loss of function of most oct4-associated genes studied to date results in embryonic or perinatal lethality , suggesting that many serve crucial functions in development . interestingly , most oct4-binding proteins linked to a human hereditary disorder ( 13 of 14 ) , mostly developmental or cancer predisposition , give rise to a related phenotype when absent in the mouse . we find cancer - associated genes , either causal or predisposing , to be transcriptional regulators involved in processes relating to the cell cycle , differentiation , and dna repair , acting through chromatin remodeling , signaling , or transcription factor activity . these results implicate the orthologs of oct4-interacting proteins in roles in human development and cancer , and therefore the data presented here should be useful in elucidating their part in human disease . in summation , the extensive systems - level analyses described here compiling data sets of currently available genome - wide studies provide an integrated vision of the oct4 interactome . detailed investigation of this information should facilitate the choice of candidate factors to test for roles in esc maintenance , differentiation , and reprogramming and provide great insight into the transcriptional regulation of esc biology . full details are provided in supplemental experimental procedures . in brief , the ftap epitope tag ( 3flag-2tev - cbp ) sequence was synthesized as two dna fragments by annealing overlapping complementary oligonucleotide molecules with pcr . the two fragments were cloned into a modified version of recombineering vector pl450 ( liu et al . , 2003 ) for pctr9 creation . homology arms for recombineering were pcr amplified from the oct4 containing c57black/6j derived bac clone ( rpci 23 - 213m12 ) and cloned into the recombineering vector to create pcts1 ( figure s1 ) . the 5 homology arm creates an in - frame fusion between the oct4 c - terminal coding sequence and the ftap tag coding sequence , while deleting the stop codon . a fragment for recombineering the ftap tag sequence into the oct4-containing bac ( rpci 23 - 213m12 ) was generated by digesting clone pcts1 . correct recombination into e. coli dh10b containing bac clone rpci 23 - 213m12 was confirmed by southern analysis of bac dna with homology arm - specific dna probes for all six tagged bac clones tested . esc cultures , electroporation , and mini - southern - blot analysis of esc clones were as described previously ( ramrez - solis et al . , 1993 ) . integration of single - copy bac transgenes at the hprt locus by recombinase - mediated cassette exchange ( rmce ) has been described previously ( prosser et al . , 2008 ) . for rmce integration of tagged oct4 bac insert into hprt allele of cci18#1.6 g , cells were cotransfected with pcaggs - cre ( araki et al . , 1997 ) and the rpci 23 - 213m12 bac clone carrying an integrated copy of the ftap tag cassette and neomycin resistance gene . double - resistant colonies were isolated after successive selection with g418 ( 200 mg / ml ) and 6-tg ( 10 m ) . site - specific bac integration was very efficient , as verified by southern analysis with hprt flanking probes , with 19 of 23 double - resistant colonies showing correct single - copy integration . for removal of the selection cassette , the verified esc clones were transfected with pcaggs - flpe ( schaft et al . , 2001 ) and then selected with fiau ( 200 nm ) . transgenic clones were analyzed for expression of tagged pou5f1 by western blotting , demonstrating that 60% of clones expressed the oct4-ftap fusion protein . murine escs expressing oct4-ftap or wild - type control cells ( ab2.2 ) were separated from feeders by trypsinization and incubation on gelatin - coated plates for 60 min . whole - cell extracts were incubated with anti - flag m2 dynal beads in buffer containing 150 mm nacl and 0.1% np-40 for 90 min at 4c . anti - flag dynal beads were prepared by crosslinking m2 flag antibody ( sigma ) to protein g - dynal beads ( invitrogen ) in accordance with the manufacturer 's instructions . bound complexes were eluted with actev protease ( invitrogen ) . for tandem affinity purification , the tev eluate was incubated with calmodulin resin ( stratagene ) for 60 min at 4c . tev or egta eluates were concentrated in vivaspin 500 pes centrifugal filters ( vivascience ) , reduced with 1 mm dtt , and alkylated with 2 mm iodoacetamide prior to sample fractionation by polyacrylamide gel electrophoresis with novex nupage bis - tris 4%12% gels ( invitrogen ) . gels were stained with colloidal coomassie ( sigma ) according to rowley ( rowley et al . , 2000 ) . whole lanes were cut in 24 slices , destained completely , and digested with trypsin ( sequencing grade , roche ) . peptides were extracted with 0.5% formic acid 50% acetonitrile and dried in a speed vac ( thermo ) . oct4 complexes were immunoprecipitated with an oct4 antibody ( santa cruz ) coupled to dynal - protein g beads ( invitrogen ) . immunoprecipitates were eluted by boiling in 1 lds loading buffer ( invitrogen ) and separated by lds - page ( invitrogen ) . western blotting was carried out with antibodies from abcam ( parp1 , sall4 , and myst2 ) , bethyl laboratories ( chd4 ) , or santa cruz ( oct4 and hdac1 ) . peptides were redissolved in 0.5% formic acid and analyzed with online nanolc - ms / ms on a ltq ft mass spectrometer ( thermo fisher scientific ) coupled with an ultimate 3000 nano / capillary lc system ( dionex ) . samples were first loaded and desalted on a trap ( 0.3 mm i d 5 mm ) at 25 l / min with 0.1% formic acid for 5 min and then separated on an analytical column ( 75 m i d 15 cm ) ( both pepmap c18 , lc packings ) over a 30 min linear gradient of 4%32% ch3cn/0.1% formic acid at 300 nl / min . the survey scans ( m / z 4002000 ) were acquired on the ft - icr at a resolution of 100,000 at m / z 400 , and one microscan was acquired per spectrum . the three most abundant multiply charged ions with a minimal intensity of 1000 counts were subject to ms / ms in the linear ion trap at an isolation width of 3 th . dynamic exclusion width was set at 10 ppm for 45 s. the automatic gain control target value was regulated at 5e5 for ft and 1e4 for the ion trap , with maximum injection time at 1000 ms for ft and 200 ms for the ion trap , respectively . database searches were performed with mascot v.2.1 ( matrix science ) against the mouse ipi database ( v. january 2009 ) . the search parameters were : trypsin / p with two missed cleavages , 10 ppm mass tolerance for ms , 0.5 da tolerance for ms / ms , fixed modification carbamidomethyl ( c ) , and variable modifications of acetyl ( protein n - term ) , deamidated ( nq ) , dioxidation ( m ) , formyl ( n - term ) , gln- > pyro - glu ( n - term q ) , methyl ( e ) , and oxidation ( m ) . decoy database searches were performed at the same time as the real searches , resulting in false discovery rates under 5% . only peptides with scores above 20 protein identification required at least one high - confidence peptide ( peptide score above identity threshold , e 0.05 , length > 8 aas , precursor ion mass accuracy < 5 ppm where e 0.005 , peptide hit rank 1 , and delta peptide score > 10 ) . there is increased risk of false discovery when a protein is identified by only one peptide . thus , all peptides identifying a protein without additional support met the strict confidence requirements above and were manually verified . mascot results were clustered to 95% protein homology to collapse highly homologous sequences corresponding to the same gene , and all lists for target and control purifications were compared in parallel . external contaminants ( keratins , albumin , casein , and tev protease ) were excluded from the list . in the final list of oct4-associated proteins we report only proteins identified in all three replicates . we have chosen one representative of each protein cluster , the one with the highest number of peptide matches , meaningful gene symbol , and highest molecular weight . orthologous human proteins were identified with the g : profiler orthology search tool ( reimand et al . , 2007 ) or ncbi blastp and aligned with the needleman - wunsch algorithm . for assessment of the degree of conservation between the oct4-associated proteins and their orthologs , sequence identities of all mouse - to - human ortholog pairs of comparable sequence length in ensembl domains were identified with pfam 24.0 , and genome - wide frequencies were calculated from domain annotations in uniprotkb / swiss - prot release 15.15 ( uniprot consortium , 2010 ) . mammalian phenotype ontology annotations were obtained from the mouse genome informatics project ( blake et al . , 2009 ) , human disease associations were obtained from omim ( hamosh et al . , 2005 ) , and known cancer - causing mutations in genes were obtained from the cancer gene census ( futreal et al . student 's t test was used for assessing the significance of the observed numbers of oct4-associated genes with lethal phenotypes , disease , and cancer associations against 1000 random sets of 92 genes , in each case . chip - on - chip data were obtained for oct4 and eight other transcription factors ( kim et al . , 2008 ) . the significance of the number of oct4-associated proteins regulated by these factors was assessed against 1000 random sets of 92 genes . the protein interaction network was generated with cytoscape 2.6.3 ( cline et al . , 2007 ) , with a spring - embedded layout . for the analysis of expression at different stages of differentiation , data were obtained for 43 mouse samples in stembase ( sandie et al . , 2009 ) , originating from 16 studies with affymetrix moe430a microarray chips , as used in an oct4 expression profiling study ( campbell et al . , 2007 ) covering murine escs , embryonal carcinoma cell lines , and several early differentiated lineages . expression data was available for 70 of the 92 oct4-associated proteins . where multiple probes were available , expression was averaged . student 's t test was used for identifying genes differentially expressed in escs as compared to more differentiated cell types ( bonferroni - corrected for multiple testing ) . expression values were log2-transformed and color - coded as a gradient from blue ( more than twice the standard deviation below the global microarray mean ) via black ( microarray mean ) to yellow ( more than twice the standard deviation above the mean ) . data on significantly misexpressed genes was curated from the oncomine human cancer expression database ( rhodes et al . genes were considered mis - expressed below a p - value threshold of 10 ( bonferroni - corrected for multiple testing ) .

summarythe transcription factor oct4 is key in embryonic stem cell identity and reprogramming . insight into its partners should illuminate how the pluripotent state is established and regulated . here , we identify a considerably expanded set of oct4-binding proteins in mouse embryonic stem cells . we find that oct4 associates with a varied set of proteins including regulators of gene expression and modulators of oct4 function . half of its partners are transcriptionally regulated by oct4 itself or other stem cell transcription factors , whereas one - third display a significant change in expression upon cell differentiation . the majority of oct4-associated proteins studied to date show an early lethal phenotype when mutated . a fraction of the human orthologs is associated with inherited developmental disorders or causative of cancer . the oct4 interactome provides a resource for dissecting mechanisms of oct4 function , enlightening the basis of pluripotency and development , and identifying potential additional reprogramming factors .

Introduction Results Discussion Experimental Procedures

two characteristics define embryonic stem cells ( escs ) , self - renewal ability and pluripotency . recently , ectopic expression of combinations of transcription factors ( oct4 , nanog , sox2 , c - myc , esrrb , and klf4 ) has been shown to reprogram mouse and human fibroblasts into a pluripotent state ( kaji et al . the induced pluripotent stem cells ( ipscs ) are very similar to escs and retain the ability to self - renew and differentiate into the three germ layers and thus promise great therapeutic potential in regenerative medicine ( amabile and meissner , 2009 ; maherali et al . the pou transcription factor oct4 , also termed pou5f1 , is a central player in esc self - renewal and differentiation into specific lineages . a decrease in oct4 levels by 50% induces differentiation toward the trophectoderm lineage , whereas a 50% increase causes differentiation into mesoderm and endoderm ( niwa et al . , oct4 plays an essential role in early development given that loss of oct4 in the mouse embryo causes the failure of the inner cell mass to develop ( nichols et al . oct4 regulates transcriptional programs to maintain esc pluripotency primarily in collaboration with transcription factors sox2 and nanog ( boyer et al . to further elucidate the esc transcriptional network , we have carried out an unbiased and extensive study of oct4-associated proteins , using an affinity purification and mass spectrometry approach . in contrast with a previous similar study ( wang et al . , 2006 ) , epitope - tagged oct4 was expressed under the control of oct4 's endogenous promoter to keep the natural transcriptional regulation . our data significantly expands the current repertoire of oct4-associated proteins , thereby shedding light on the complex regulatory circuitries of escs . the oct4 interactome provides a useful resource to investigate the mechanisms of oct4 function and regulation and to explore the basic principles underlying stem cell biology . to investigate the molecular network around oct4/pou5f1 , we used an epitope - tagging affinity purification strategy . the ftap was fused at the c terminus of the oct4 coding region by recombineering into a bac clone containing full - length oct4 . we first performed three independent one - step purifications on whole - cell extracts from both oct4-ftap - expressing and control unmodified cells ( figure 1a ) . , 2009 ) mass spectrometry analysis resulted in the identification of 92 proteins ( excluding oct4 itself ) that were present in all oct4-ftap purifications , but not in controls ( table 1 ) . the identification of some of the interacting proteins was confirmed by western blotting ( figure 1b ) . we detected 13 previously identified oct4 interacting proteins in our study ( table s6 ) . these included sall4 , arid3b , zfp219 , and sp1 ( wang et al . furthermore , we also identified sox2 and nanog , two of the best characterized oct4 binding partners ( ambrosetti et al . , 2006 ) , and zfp281 , requiem / dpf2 , yy1 , rybp , dax1 , esrrb , and arid3a , recently shown to physically interact with oct4 ( donohoe et al . , 2008 ) in one or two ( arid3a and esrrb ) purifications , but because of our strict criteria of result reproducibility , we did not include them in the final data set . we also identified proteins reported to be linked to oct4 through association with some of its interactors , namely sall1 and smarcc1 ( wang et al . , 2006 ) , and zfp206 ( yu et al . , 2009 ; we next performed tandem affinity purification , although yields were not high because of the low levels of tagged oct4 and purification efficiency . we identified seven proteins of the 92 , mainly members of nurd , sall proteins , and transcription factors e3 and eb ( table s6 ) . to address whether the interactions detected were due to coassembly of factors on chromatin , we then immunoprecipitated oct4 in the presence of dnase treatment with benzonase . summing up , over 50% of the oct4-associated proteins ( 47 of 92 ) varying in abundance across our data set have been confirmed by independent means , suggesting that the data set we provide here is a bona fide set of oct4 binding partners . to uncover general trends in the functions of the oct4-interacting proteins , we carried out computational systems - level analyses in a workflow depicted in figure 1d . we found an enrichment of go terms such as nucleus , chromosome , and chromatin in the cellular component ontology ; nucleic acid binding , protein binding , transcription factor activity , in the molecular function ontology ; and transcription , regulation of gene expression , and embryonic development in the biological process ontology ( figure s2 and table s2 ) . twenty proteins in the data set ( 21% ) are annotated with the go term oct4 has been shown to associate with several transcription factors , and our results agree with the notion that combinatorial binding among pluripotency factors , which contributes to achieving specificity in gene regulation , may be a frequent pattern in escs ( chambers and tomlinson , 2009 ) . david analysis detected an enrichment of proteins involved in the control of gene expression by vitamin d nuclear hormone receptor , mainly members of the fact and swi / snf complexes . , 2004 ) , possibly by modulating levels of pluripotency factors oct4 , nanog , and sox2 ( kalmar et al . highly represented domains were dead / deah box helicase , snf2-related , phd and zinc fingers , and chromo , bromo , and homeobox domains , all of which are either involved in atp - dependent chromatin remodeling or bind dna and posttranslationally modified nucleosomes , thereby influencing gene expression . as shown in table 1 and supported by the go and panther analyses , oct4 associates mainly with transcriptional regulators , but also with a variety of other chromatin binding proteins involved in dna replication , recombination , and repair , proteins involved in nuclear assembly and/or organization , and diverse enzymes , some of which are responsible for addition of posttranslational modifications . to gain an overall view of the previously known interactions among oct4-associated proteins , we retrieved interaction data from intact , hrpd , and mint for the data set and represented them as a protein interaction network ( figure 1e ) . however , oct4 has also been shown to interact with more general modulators of transcription that are expressed ubiquitously , such as members of the nurd complex ( liang et al . we investigated the patterns of expression of the oct4-associated data set in cells at different stages of differentiation , including embryonic carcinoma , embryonic stem cells , embryoid bodies , and various differentiated cell types , on the basis of transcriptomics data ( campbell et al . this suggests that oct4 interacts mostly with proteins that are ubiquitously expressed in both differentiated and undifferentiated cells . among these are the dna methylation regulatory factor dnmt3l and the developmentally important transcription factors klf4 , sall1 , and sall4 . regulation of gene expression involves complex dynamics employing sequence - specific dna binding proteins that form the transcriptional regulatory network ( babu et al . , 2004 ; transcription factors often operate in feedback loops , whereby the expression of a transcriptional target modulates the function of the transcription factor itself ( figure 3a ) . we next investigated whether the promoters of the oct4-associated gene set contain binding sites for transcription factors that are central in the establishment and maintenance of esc identity . promoter binding sites for nine such transcription factors have previously been identified by chip - on - chip ( kim et al . , these include oct4 , dax1 , klf4 , c - myc , nac1 , nanog , rex1 , zfp281 , and sox2 . nine of the 92 oct4-associated proteins were found to be transcriptionally regulated by oct4 itself in mouse escs , and 51% of genes encoding oct4 partners are targets of at least one key esc transcription factor ( figure s3 ) . to assess whether this is statistically significant the expected percentage of promoter binding by esc transcription factors was only 28% ( z = 4.45 , p < 10 ) , indicating that it is a significant trait of the data set . several genes in the interaction set are common targets of multiple transcription factors ( 20 of 92 are targets of at least three transcription factors ) , making it likely that they have central roles in pluripotency and self - renewal . ten of these are significantly downregulated in differentiation , and all but two show a downregulation trend ( table s6 ) , in agreement with the hypothesis that genes bound by multiple factors are active in escs and become repressed as cells differentiate ( chambers and tomlinson , 2009 ; kim et al . we constructed a regulatory network by integrating promoter target data for the nine stem cell transcription factors with the list of oct4 binding proteins ( figure 3b ) . several of the transcription factors cluster together because of shared targets ( e.g. , sox2 , nanog , nac1 , and dax1 ) , whereas c - myc and klf4 exclusively target certain groups of oct4-interacting factors . , we next explored the consequences of loss of oct4-interacting proteins in escs or mouse development . five oct4-interacting proteins have been identified as required for stem cell self - renewal in large scale rnai screens ( ding et al . loss - of - function phenotypes in mice were available in the mgi database for 49 oct4-associated proteins . significantly , 83% ( 41 of 49 ) of the studied knockout alleles of the interaction set showed embryonic and/or perinatal lethality , with over 60% ( 30 of 49 ) being embryonic lethal ( figure 1e ) . these results indicate a high level of requirement for components of the oct4 network in early mouse development . given the extent of their part in mouse development and the current excitement about the cancer stem cell hypothesis , we next explored a possible role of oct4-associated proteins in human disease . human orthologs were identified for all oct4-associated proteins and sequence identities determined between mouse and human ( figure s4 ) . all oct4-associated proteins were found to be highly conserved , with a median sequence identity of 94% , compared to 77% genomic median . we next investigated the involvement of the human orthologs in human disease and development of cancer interrogating the omim database and the cancer gene census , which records genes whose mutation has been causally linked to cancer . genes encoding 14 of 92 oct4-associated proteins are implicated in one or more hereditary diseases , mostly of developmental nature , with six of them predisposing to certain types of cancer ( table 2 ) . somatic mutations in eight oct4-associated proteins and oct4 itself were found to be responsible for different types of cancer , often through gene translocations , presumably affecting their regulation ( table 3 ) . in light of the central role of oct4 in pluripotency and the cancer stem cell hypothesis , we investigated which of oct4 's physical interactors are misexpressed in cancer using the oncomine human cancer expression database . a large fraction ( 60% ) of the oct4 interactors show misexpression in at least one cancer type , providing a degree of additional support to the connection between stem cell identity and cancer . , 2008 ) , and should greatly facilitate systematic tagging of genes and analysis of protein complexes with roles in development in different contexts , be it in stem cells , differentiated cell types , or even mouse tissue ( fernndez et al . previous proteomic studies of oct4 protein complexes have relied on lengthy single or tandem purifications from nuclear extracts with streptavidin capture ( wang et al . we identified all of the partners reported by the liang study except hdac2 , and only five oct4 partners found in the wang study were not detected in our data set , maybe because of our use of whole extracts . indeed , our approach has produced by far the most extensive analysis of oct4-associated proteins to date . by using whole extracts , thereby not restricting the analysis to the nuclear environment , our data set encompasses diverse aspects of the life of oct4 , both nuclear and nonnuclear . the broad data set puts oct4 at the center of diverse cellular processes that can have an impact on aspects of stem cell biology ( figure 5 ) , the most interesting of which are discussed below . oct4 can both activate and repress transcriptional targets in mouse and human escs ( babaie et al . sall4 , a well - known oct4 partner , and other members of the spalt - like family of transcriptional cofactors have been shown to associate to nurd ( lauberth and rauchman , 2006 ) , raising the possibility that they may bridge the interaction between oct4 and nurd . we also found several subunits of the swi / snf nucleosome - remodeler complex , some of which have previously been linked to nanog ( liang et al . in addition , we detected ttf2 , a component of the general transcription machinery , providing evidence of a physical link between pluripotency factors and basal transcription players . the oct4 interactome included other basal dna - process - related factors such as proteins involved in dna replication , recombination , and repair . this could explain why many of the oct4-interacting proteins are ubiquitously expressed in both differentiated and undifferentiated cells . oct4 is modified by o - glcnac in human escs ( webster et al . , 2009 ) , and sp1 , one of oct4 partners , is too ( jackson and tjian , 1988 ) . a thorough analysis of o - glcnac modification in the oct4 interactome might yield important insight into dynamic modulation of stem cell factors . posttranslational modification of transcription factors and cofactors is proving to be a critical component of the regulation of gene transcription in general , and important specifically in stem cell biology ( brill et al . half of oct4-associated proteins seem to be directly regulated by transcription factors with key roles in stem cell pluripotency and/or reprogramming . this is also a characteristic of pluripotency networks derived from smaller data sets from different entry points ( orkin et al . , 2008 ; this indicates that even in the expanded and functionally diverse network , this attribute still holds true , supporting a previously unsuspected role in stem cell biology for some of the proteins we identify here . among these are several transcription factors , such as the dna methyltransferase 3-like regulatory protein dnmt3l , which stimulates genomic imprinting in germ cells ( bourc'his et al . , 2001 ; this is consistent with a recent report demonstrating that treatment with dna methyltransferase inhibitors can improve the efficiency of the reprogramming process of differentiated cells ( mikkelsen et al . we find that loss of function of most oct4-associated genes studied to date results in embryonic or perinatal lethality , suggesting that many serve crucial functions in development . interestingly , most oct4-binding proteins linked to a human hereditary disorder ( 13 of 14 ) , mostly developmental or cancer predisposition , give rise to a related phenotype when absent in the mouse . we find cancer - associated genes , either causal or predisposing , to be transcriptional regulators involved in processes relating to the cell cycle , differentiation , and dna repair , acting through chromatin remodeling , signaling , or transcription factor activity . these results implicate the orthologs of oct4-interacting proteins in roles in human development and cancer , and therefore the data presented here should be useful in elucidating their part in human disease . in summation , the extensive systems - level analyses described here compiling data sets of currently available genome - wide studies provide an integrated vision of the oct4 interactome . detailed investigation of this information should facilitate the choice of candidate factors to test for roles in esc maintenance , differentiation , and reprogramming and provide great insight into the transcriptional regulation of esc biology . homology arms for recombineering were pcr amplified from the oct4 containing c57black/6j derived bac clone ( rpci 23 - 213m12 ) and cloned into the recombineering vector to create pcts1 ( figure s1 ) . the 5 homology arm creates an in - frame fusion between the oct4 c - terminal coding sequence and the ftap tag coding sequence , while deleting the stop codon . esc cultures , electroporation , and mini - southern - blot analysis of esc clones were as described previously ( ramrez - solis et al . tev or egta eluates were concentrated in vivaspin 500 pes centrifugal filters ( vivascience ) , reduced with 1 mm dtt , and alkylated with 2 mm iodoacetamide prior to sample fractionation by polyacrylamide gel electrophoresis with novex nupage bis - tris 4%12% gels ( invitrogen ) . whole lanes were cut in 24 slices , destained completely , and digested with trypsin ( sequencing grade , roche ) . the survey scans ( m / z 4002000 ) were acquired on the ft - icr at a resolution of 100,000 at m / z 400 , and one microscan was acquired per spectrum . the search parameters were : trypsin / p with two missed cleavages , 10 ppm mass tolerance for ms , 0.5 da tolerance for ms / ms , fixed modification carbamidomethyl ( c ) , and variable modifications of acetyl ( protein n - term ) , deamidated ( nq ) , dioxidation ( m ) , formyl ( n - term ) , gln- > pyro - glu ( n - term q ) , methyl ( e ) , and oxidation ( m ) . in the final list of oct4-associated proteins we report only proteins identified in all three replicates . we have chosen one representative of each protein cluster , the one with the highest number of peptide matches , meaningful gene symbol , and highest molecular weight . for assessment of the degree of conservation between the oct4-associated proteins and their orthologs , sequence identities of all mouse - to - human ortholog pairs of comparable sequence length in ensembl domains were identified with pfam 24.0 , and genome - wide frequencies were calculated from domain annotations in uniprotkb / swiss - prot release 15.15 ( uniprot consortium , 2010 ) . , 2005 ) , and known cancer - causing mutations in genes were obtained from the cancer gene census ( futreal et al . student 's t test was used for assessing the significance of the observed numbers of oct4-associated genes with lethal phenotypes , disease , and cancer associations against 1000 random sets of 92 genes , in each case . the significance of the number of oct4-associated proteins regulated by these factors was assessed against 1000 random sets of 92 genes . , 2007 ) covering murine escs , embryonal carcinoma cell lines , and several early differentiated lineages . expression data was available for 70 of the 92 oct4-associated proteins .

alcoholic beverages have been consumed by humans since prehistoric times for a variety of reasons . nowadays , recreational alcohol intake is common across the globe and health and social problems resulting from alcohol consumption are becoming a concern . although moderate alcohol use is recommended , excessive alcohol consumption is the third leading cause of premature death in the united states ( behind smoking and obesity ) . among the many problems associated with heavy alcohol drinking , the relationship between alcohol intake and body weight has been extensively studied over the past years [ 3 , 4 , 5 ] . based on the fact that 1 gram of alcohol provides 7.1 kcal ( 29 kj ) and studies showing that energy consumed as alcohol is additive to that from other dietary sources , increased energy intake with alcohol use can certainly promote a positive energy balance and ultimately weight gain . however , a clear cause - and - effect association between alcohol intake and weight gain is not apparent based on the mixed and conflicting available evidence on the topic . given that both excessive alcohol intake and obesity are of public health concern , a better understanding of the association between alcohol consumption and excess body weight is warranted . therefore , the objective of this article is to provide an update on the link between alcohol intake and obesity . furthermore , factors that may explain the conflicting findings in this research area are discussed . finally , recommendations for future research are provided to promote a better understanding of the possible obesity - promoting effects of energy intake from alcohol . observational studies on the effect of alcohol intake on obesity date back almost 30 years . it has been examined across small and large cohorts , in many countries , across various ethnicities and age groups . within the large body of observational research , contradictory findings exist , which warrant further exploration [ 3 , 4 ] . among cross - sectional studies , a common trend appears to be that alcohol intake is not associated with body mass index ( bmi ) in men , while either negatively or not associated with bmi in women . indeed , several cohorts ranging from 10,482 to 138,031 individuals have shown no correlation ( or a small negative correlation ) between alcohol intake and bmi in men , and a small negative association with bmi in women [ 613 ] . other studies have found that alcohol intake is positively correlated with bmi in men or in both sexes ; however , an analysis of recent studies suggests that this may be due to differences in intake patterns [ 4 , 7 ] . for instance , several studies in adults have found that the amount or intensity of drinking per drinking occasion is positively correlated with bmi , while the frequency of drinking is negatively correlated , suggesting that frequent light drinking might offer a protective effect [ 1416 ] . furthermore , several studies have found that only excessive or heavy drinking is correlated with increased measures of adiposity . wannamethee , shaper & whincup found that in men , drinking < 20 drinks per week was not associated with higher bmi , waist circumference ( wc ) or waist - to - hip ratio ( whr ) compared to non - drinkers . these anthropometric measures were much higher than non - drinkers in men who consumed > 21 drinks per week . similarly , coulson et al . found higher bmi , percent body fat , and waist circumference in individuals drinking five or more drinks per day compared to non - drinkers . other studies have shown j - shaped curves when comparing bmi , wc , and whr among male and female drinkers , with light drinking being negatively associated with adiposity indicators compared to heavy drinking , or abstention [ 1923 ] . to test the proposal that patterns of alcohol intake may be an important influence on overweight and obesity , most recently , sheldon and knott recorded the energy intake from alcohol on the days that individuals had their highest intake . the risk of obesity was 70% higher in the heaviest drinking group compared to the lightest . these individuals had obtained 75% of their total daily energy intake from alcohol compared to the lightest group which had < 24% of their daily energy intake from alcohol on their heaviest drinking day . binge drinking has been associated with greater risk of obesity and large wc in other studies [ 19 , 25 ] . had individuals retrospectively estimate their age at peak alcohol intake and earlier reported peak alcohol intake was associated with more heavy drinking episodes and abdominal obesity . however , the retrospective nature of this study creates the potential for inaccuracies or biases in participants memory of their drinking behaviors . despite the recent body of cross - sectional evidence suggesting the benign or potentially protective effect of frequent light drinking on body weight and obesity , several studies have found conflicting results . . found a very modest positive linear relationship between alcohol intake and bmi in men . however , the bmi of the highest drinking group varied only by 0.4 points compared to non - drinkers , although the trend was statistically significant . in overweight participants this relationship was even less pronounced ( a difference of 0.2 points ) . . found that in older participants ( > 39.2 years old ) , alcohol intake was positively correlated with bmi , but no association was seen in younger participants . . found that alcohol intake was positively associated with body fat in adolescent girls , but negatively associated with body fat in adolescent boys . found a positive association between alcohol intake and risk of overweight in 1314 year olds . among 1516 year olds , however , they found a j - shape relationship , with only the heaviest drinkers having a higher risk of overweight . this suggests that there may be differences in alcohol metabolism between age groups ; however , it is important to note that this study assessed only the amount of alcohol per drinking occasion , and not drinking frequency patterns . overall , the majority of cross - sectional studies since 2005 have demonstrated that frequent light to moderate alcohol intake does not seem to be associated with obesity risk . heavy drinking and binge drinking , however , are more likely to carry such an association with excess body weight [ 16 , 19 , 2225 ] . alcohol intake may also promote overweight and higher body fat percentage in adolescents or older adults [ 20 , 28 , 29 ] . these studies are limited in their ability to demonstrate cause - and - effect relationships or changes in weight status over time . prospective studies have looked at the association between alcohol intake and adiposity gain in various populations , with follow - up periods ranging from several months to 20 years [ 4 , 30 , 31 ] . several studies have found no association or a negative association between alcohol intake and changes in weight , bmi or other measures of adiposity [ 12 , 30 , 3239 ] . other studies have found such an association only in women , while finding a positive association between obesity risk and alcohol intake in men . this study , however , did not specify the amount of alcohol intake , and did not control for participant s physical activity ( pa ) levels . conversely , one study found no association between alcohol intake and increases in wc in men , but a small positive association in women . there have also been recent studies that have found a general positive association between alcohol intake and weight gain . this study , however , did not control for pa levels and only assessed alcohol intake on a yes / no scale . changes in alcohol intake patterns have been associated with weight gain in prospective studies [ 15 , 31 , 43 ] . . found that men who increased their frequency or amount of alcohol intake over the follow - up period were more likely to experience increases in bmi and wc . mozafarrian et al . also found that changes in drinking behavior were associated with increases in weight . over four years each increase of one drink per day this relationship was strongest for beer , suggesting a possible influence for the type of alcohol ingested . finally , other recent studies have shown that heavy drinking may be more of a risk factor for weight gain than light - to - moderate drinking [ 44 , 4547 ] . macinnis et al . [ 44 ] found that lower bmi and wc were seen after 12 years of follow up in light and moderate drinkers than heavy drinkers or abstainers . sayon - orea et al . found that drinking more than seven times per week was associated with increased risk of weight gain and development of overweight and obesity . . found that male light - to - moderate beer drinkers had smaller increases in wc and weight than non - drinkers or heavy drinkers over 8.5 years of follow up . however , conversely , in women they found a dose response relationship between beer intake and weight gain and wc gain . there are many reasons as to why the relationship between alcohol and adiposity varies between men and women , involving genetic and lifestyle factors , some of which will be discussed in depth below . as with cross - sectional studies , the way by which alcohol intake is measured and categorized likely influences the interpretation of the results . several studies have grouped all levels of individual alcohol intake above 30g / day as conversely , other studies examined alcohol intake more thoroughly , considering frequency and amount per drinking day separately . measured alcohol frequency ranging from 12 times per year to every day , while estimating the number of drinks per drinking day from 136 . such an analysis allows for a more complete description of participants drinking patterns , and is important as cross - sectional studies suggest that drinking frequency and intensity influence weight differently [ 1416 ] . collectively , the most recent prospective studies suggest that light - to - moderate alcohol intake is not associated with weight gain or changes in wc [ 44 ] . heavy drinking , however , has been more consistently associated with weight gain [ 4547 ] . furthermore , increases in alcohol intake patterns appear to promote weight gain [ 15 , 31 , 43 ] . binge drinking behavior was not specifically examined in any of the recent prospective studies analyzed . among cross - sectional studies , a common trend appears to be that alcohol intake is not associated with body mass index ( bmi ) in men , while either negatively or not associated with bmi in women . indeed , several cohorts ranging from 10,482 to 138,031 individuals have shown no correlation ( or a small negative correlation ) between alcohol intake and bmi in men , and a small negative association with bmi in women [ 613 ] . other studies have found that alcohol intake is positively correlated with bmi in men or in both sexes ; however , an analysis of recent studies suggests that this may be due to differences in intake patterns [ 4 , 7 ] . for instance , several studies in adults have found that the amount or intensity of drinking per drinking occasion is positively correlated with bmi , while the frequency of drinking is negatively correlated , suggesting that frequent light drinking might offer a protective effect [ 1416 ] . furthermore , several studies have found that only excessive or heavy drinking is correlated with increased measures of adiposity . wannamethee , shaper & whincup found that in men , drinking < 20 drinks per week was not associated with higher bmi , waist circumference ( wc ) or waist - to - hip ratio ( whr ) compared to non - drinkers . these anthropometric measures were much higher than non - drinkers in men who consumed > 21 drinks per week . similarly , coulson et al . found higher bmi , percent body fat , and waist circumference in individuals drinking five or more drinks per day compared to non - drinkers . other studies have shown j - shaped curves when comparing bmi , wc , and whr among male and female drinkers , with light drinking being negatively associated with adiposity indicators compared to heavy drinking , or abstention [ 1923 ] . to test the proposal that patterns of alcohol intake may be an important influence on overweight and obesity , most recently , sheldon and knott recorded the energy intake from alcohol on the days that individuals had their highest intake . the risk of obesity was 70% higher in the heaviest drinking group compared to the lightest . these individuals had obtained 75% of their total daily energy intake from alcohol compared to the lightest group which had < 24% of their daily energy intake from alcohol on their heaviest drinking day . binge drinking has been associated with greater risk of obesity and large wc in other studies [ 19 , 25 ] . had individuals retrospectively estimate their age at peak alcohol intake and earlier reported peak alcohol intake was associated with more heavy drinking episodes and abdominal obesity . however , the retrospective nature of this study creates the potential for inaccuracies or biases in participants memory of their drinking behaviors . despite the recent body of cross - sectional evidence suggesting the benign or potentially protective effect of frequent light drinking on body weight and obesity , several studies have found conflicting results . . found a very modest positive linear relationship between alcohol intake and bmi in men . however , the bmi of the highest drinking group varied only by 0.4 points compared to non - drinkers , although the trend was statistically significant . in overweight participants this relationship was even less pronounced ( a difference of 0.2 points ) . . found that in older participants ( > 39.2 years old ) , alcohol intake was positively correlated with bmi , but no association was seen in younger participants . found that alcohol intake was positively associated with body fat in adolescent girls , but negatively associated with body fat in adolescent boys . finally , croezen et al . found a positive association between alcohol intake and risk of overweight in 1314 year olds . among 1516 year olds , however , they found a j - shape relationship , with only the heaviest drinkers having a higher risk of overweight . this suggests that there may be differences in alcohol metabolism between age groups ; however , it is important to note that this study assessed only the amount of alcohol per drinking occasion , and not drinking frequency patterns . overall , the majority of cross - sectional studies since 2005 have demonstrated that frequent light to moderate alcohol intake does not seem to be associated with obesity risk . heavy drinking and binge drinking , however , are more likely to carry such an association with excess body weight [ 16 , 19 , 2225 ] . alcohol intake may also promote overweight and higher body fat percentage in adolescents or older adults [ 20 , 28 , 29 ] . these studies are limited in their ability to demonstrate cause - and - effect relationships or changes in weight status over time . prospective studies have looked at the association between alcohol intake and adiposity gain in various populations , with follow - up periods ranging from several months to 20 years [ 4 , 30 , 31 ] . several studies have found no association or a negative association between alcohol intake and changes in weight , bmi or other measures of adiposity [ 12 , 30 , 3239 ] . other studies have found such an association only in women , while finding a positive association between obesity risk and alcohol intake in men . this study , however , did not specify the amount of alcohol intake , and did not control for participant s physical activity ( pa ) levels . conversely , one study found no association between alcohol intake and increases in wc in men , but a small positive association in women . there have also been recent studies that have found a general positive association between alcohol intake and weight gain . this study , however , did not control for pa levels and only assessed alcohol intake on a yes / no scale . changes in alcohol intake patterns have been associated with weight gain in prospective studies [ 15 , 31 , 43 ] . . found that men who increased their frequency or amount of alcohol intake over the follow - up period were more likely to experience increases in bmi and wc . mozafarrian et al . also found that changes in drinking behavior were associated with increases in weight . over four years each increase of one drink per day this relationship was strongest for beer , suggesting a possible influence for the type of alcohol ingested . finally , other recent studies have shown that heavy drinking may be more of a risk factor for weight gain than light - to - moderate drinking [ 44 , 4547 ] . [ 44 ] found that lower bmi and wc were seen after 12 years of follow up in light and moderate drinkers than heavy drinkers or abstainers . sayon - orea et al . found that drinking more than seven times per week was associated with increased risk of weight gain and development of overweight and obesity . . found that male light - to - moderate beer drinkers had smaller increases in wc and weight than non - drinkers or heavy drinkers over 8.5 years of follow up . however , conversely , in women they found a dose response relationship between beer intake and weight gain and wc gain . there are many reasons as to why the relationship between alcohol and adiposity varies between men and women , involving genetic and lifestyle factors , some of which will be discussed in depth below . as with cross - sectional studies , the way by which alcohol intake is measured and categorized likely influences the interpretation of the results . several studies have grouped all levels of individual alcohol intake above 30g / day as conversely , other studies examined alcohol intake more thoroughly , considering frequency and amount per drinking day separately . measured alcohol frequency ranging from 12 times per year to every day , while estimating the number of drinks per drinking day from 136 . such an analysis allows for a more complete description of participants drinking patterns , and is important as cross - sectional studies suggest that drinking frequency and intensity influence weight differently [ 1416 ] . collectively , the most recent prospective studies suggest that light - to - moderate alcohol intake is not associated with weight gain or changes in wc [ 44 ] . heavy drinking , however , has been more consistently associated with weight gain [ 4547 ] . furthermore , increases in alcohol intake patterns appear to promote weight gain [ 15 , 31 , 43 ] . binge drinking behavior was not specifically examined in any of the recent prospective studies analyzed . several experimental studies have been conducted to examine the short - term effect of alcohol intake on feeding behavior and appetite control [ 3 , 5 ] . a recent review summarized a number of these studies , showing that alcohol ingested before a meal has frequently been shown to have a neutral effect on intake , or to increase intake , despite the added energy that come from the alcohol preload . in these studies however , to date there have been few intervention studies conducted to experimentally examine the effects of regular alcohol intake on weight gain or obesity in humans . all of the available studies have examined moderate intake of alcohol , and the majority have reported results on beer and wine intake , but not other forms of alcohol [ 3 , 5 ] . crouse and grundy looked at the effect of adding 630 kcal / day of alcohol to the diets of 12 men in a metabolic unit . there were no significant changes in weight for normal weight participants over the four - week intervention study . they however noted that about half of the obese participants gained weight , with the largest weight gain being 1.8 kg . in a randomized crossover study , cordain et al . found that drinking two glasses of red wine ( 270 ml ) with dinner daily for six weeks did not lead to changes in weight or body fat percentage in 14 men . they noted that self - reported nutrient intake and physical activity did not differ between conditions , although there may have been dietary compensation that was not accurately reported by their 3-day food logs . similarly , cordain et al . found that 10 weeks of wine intake equal to 6 - 7% of total energy intake ( 135 ml , five times per week ) did not result in any significant change in body weight or fat percentage in 20 sedentary , overweight women . also , beulens et al . reported similar results in 34 male adults with large wc , consuming 450 ml of red wine per day for 4 weeks , compared to consuming alcohol - free wine for the same time period . the effect of beer intake was examined by romeo et al . who found that one month of daily beer consumption ( equivalent to 12g / day of alcohol for women and 24 g / day for men ) did not result in significant increases in bmi or wc compared to abstention . biceps skin fold was the only anthropometric measurement that was increased in their participants after the beer drinking condition . found that replacing 10% of total daily energy intake during a weight - loss intervention with either grape juice or white wine resulted in similar weight loss , with the white wine group showing a slightly higher ( although not statistically significant ) weight loss . in this case both diets were isoenergetic so this is not a surprising result , as the thermic effect of food was likely higher for white wine than grape juice [ 53 , 54 ] . finally , more recently , cresci et al . found that self - reported alcohol intake was not a significant predictor of success or failure in losing 5% of body weight during a 6-month weight loss intervention . overall , the available experimental evidence reviewed in this article suggests that moderate intake of alcohol does not lead to weight gain . the systematic review by bendsen et al . [ 3 ] suggests that this trend is less likely in experimental studies examining beer consumption exclusively . also , the intervention periods in the aforementioned studies ranged from 410 weeks , and therefore may not have been long enough to identify the slight changes in weight that can accumulate over time to result in overweight or obesity . a modest increase in weight of one kilogram over a 10 week period seems insignificant but over five years this could result in up to 26 kg of weight gain if no compensation takes place . to our knowledge , there does not appear to be any experimental evidence specifically testing the effects of heavy / binge drinking , or of drinking spirits or a combination of alcohol sources on weight gain / obesity . a summary of the studies examined in this article , organized by the trend between alcohol and weight gain / obesity can be found in table 1.table 1summary of trends in cross - sectional , longitudinal , and experimental studies examining the link between alcohol intake and measures of adipositytrendstudy ( measurement)menwomencross - sectional studiesno associationcolditz et al . ( whr , bmi)wakabayashi ( wc)wannamethee , shaper & whincup ( bmi , wc , whr)arif & rohrer ( obesity risk)duvigneaud et al . ( ( bmi , wc)wakabayashi ( wc)binge/ heavy drinking positively associatedarif & rohrer ( obesity rate)coulson et al . ( bmi , wc , body fat % ) lee ( wc)shelton & knott ( risk of obesity)arif & rohrer ( obesity rate)lee ( wc)shelton & knott ( risk of obesity)frequency negatively associated , intensity positively associatedbreslow & smothers ( bmi)french et al . ( weight gain of > 10lbs)drinkers gain more over follow - upbell , ge & popkins ( risk of weight gain)hou et al . ( risk of weight gain)bell , ge & popkins ( risk of weight gain)romaguera et al . ( only in african american participants , change in weight > 5kg)drinkers gain less over follow - upliu et al . ( change in weight > 5kg)experimental studiesno significant effect of alcohol intakecordain et al . ( change in weight)crouse & grundy ( normal weight participants , change in weight)romeo et al . ( weight gain , wc)positive impact of alcohol consumptioncrouse & grundy ( half of obese patients , change in weight)romeo et al . ( biceps skinfold)bmi = body mass index , wc = waist circumference , whr = waist - to - hip ratio summary of trends in cross - sectional , longitudinal , and experimental studies examining the link between alcohol intake and measures of adiposity bmi = body mass index , wc = waist circumference , whr = waist - to - hip ratio in summarizing the recent literature it appears that light - to - moderate alcohol intake is less likely to be a risk factor for obesity than heavy drinking . there are several lines of evidence suggesting the potential for alcohol to promote weight gain , and the contradictory results often seen in the literature have led to the development of alternative hypotheses regarding the influence of alcohol on body weight . a review by yeomans highlights some of the potential explanations for alcohol s influence on weight gain or obesity . first , as previously mentioned , energy from alcohol appears to be additive to energy from other sources . several studies suggest that consuming alcohol before or during a meal does not influence the amount of food eaten in that meal , despite increasing the energy density of the meal . thus , individuals do not appear to compensate for the added energy from alcohol in the short - term , and alcohol appears to have little effect on satiety . beyond adding energy to a meal of the 17 studies reviewed by yeomans , ten showed increased food intake following alcohol consumption . one explanation is that there is a learned association between alcohol and eating ; however , several experimenters disguised the presence of alcohol in their protocols and still found increased energy intake . it is unclear whether alcohol promotes food intake in the absence of hunger ; however , it has been noted that alcohol may amplify individuals perception of appetite in response to food stimuli . the results of several studies propose that alcohol may influence energy intake by inhibiting the effects of leptin , or glucagon - like peptide-1 ( glp-1 ) [ 56 , 57 ] . to date , the evidence suggests that alcohol does not appear to increase appetite through the action of peptide yy ( pyy ) , ghrelin , gastric inhibitory peptide ( gip ) , or cholecystokinin ( cck ) [ 5761 ] . . found that alcohol did not increase plasma levels of neuropeptide y ( npy ) ; however , animal models have shown that central npy levels are increased following alcohol consumption . the effects of alcohol on opioid , serotonergic , and gabaergic pathways in the brain all suggest the potential to increase appetite [ 6265 ] . given the complexity of the interplay between central and peripheral signals of satiety , more research needs to be performed in order to elucidate the precise biochemical mechanism driving food intake following alcohol consumption . a summary of the effects of alcohol on important appetite hormones and central neurological pathways in humans can be found in table 2.table 2effect of alcohol on various peripheral hormones and central neurotransmitter systems related to hunger and energy intakehormone/ neurotransmittereffect on hunger/ energy intakeeffect of alcohol intake on hormone/ neurotransmitter responsereference(s)peripheral signalsccksuppressesincreasehajnal , flores and valenzuela ; manabe et al . cck = cholecystokinin , glp-1 = glucagon - like peptide-1 , gip = gastric inhibitory peptide , pyy = peptide yy , npy = neuropeptide y , gaba = gamma - aminobutyric acid effect of alcohol on various peripheral hormones and central neurotransmitter systems related to hunger and energy intake cck = cholecystokinin , glp-1 = glucagon - like peptide-1 , gip = gastric inhibitory peptide , pyy = peptide yy , npy = neuropeptide y , gaba = gamma - aminobutyric acid aside from the immediate influence on appetite that comes from alcohol consumption , there are also effects on energy storage . alcohol inhibits fat oxidation , suggesting that frequent alcohol consumption could lead to fat sparing , and thus higher body fat in the long term . however , the results of the various cross - sectional and longitudinal studies examined in this review do not unequivocally support such a hypothesis . finally , there is also evidence to suggest that traits that predispose individuals to binge drinking may also predispose to binge eating . although there is evidence to suggest that frequent alcohol intake may predispose to weight gain or obesity over the long - term , first , it has been found that alcohol intake increases energy expenditure , likely due in part to the fact that it has a high thermogenic effect . wasted , due to the activation of the inefficient hepatic microsomal ethanol - oxidizing system ( meos ) . the meos is induced through chronic alcohol intake , and the level of induction increases with increased intake [ 54 , 67 ] . oxidation of alcohol via the meos produces less atp than oxidation via alcohol dehydrogenase , using the energy from alcohol intake primarily to enhance heat production [ 37 , 54 ] . the extent to which wasted energy from regular alcohol consumption contributes to weight gain prevention is unclear . it has also been suggested that individuals who frequently drink moderate amounts of alcohol may enjoy a moderate lifestyle in which exercise and food intake are modulated over the long term to accommodate for alcohol intake . however , studies using food logs and self - reported physical activity levels have still shown a null or negative association between moderate alcohol intake and weight gain after controlling for these and other confounders , although they may fail to truly capture the habits of participants [ 4 , 49 ] . while cross - sectional and longitudinal studies have controlled for a number of important lifestyle factors , there are many to consider when examining body weight regulation . it is highly likely that the paradoxical results seen in studies examining the effect of alcohol on weight gain and obesity are also the product of a multitude of factors beyond the individual s ingestion habits . future research must consider the other important factors that may influence the link between alcohol and obesity , some of which are discussed below . the mixed evidence with respect to alcohol s role in promoting obesity is a product of many factors [ e.g. , gender , type , frequency and amount of alcohol consumed , drinking pattern ( e.g. , binge drinking ) , physical activity level , sleeping habits , depression symptoms , psychosocial problems , chronic illness , medication use , disinhibition eating behavior trait , history of alcohol use , predisposition to gain weight , etc . ] . not taking into account some of these potential confounding factors can certainly lead to biased estimates of the relationship between alcohol intake and body weight given that large inter - individual variations exist . the association between alcohol intake and body weight is generally stronger in men than women , especially because of the amount and type of alcohol consumed by men . alcohol has been reported to account for 16% of adult drinkers total energy intake in the united states , with men consuming about three times the amount consumed by women . men are also more likely to drink beer , which is carbohydrate rich , and provides more energy than wine per standard drink . consistent with this observation are the findings from a recent systematic review showing positive associations between beer consumption and measures of abdominal adiposity ( also known as beer belly ) in men ; however , inconsistent results were found in women [ 3 ] . many epidemiologic studies fail to consider lifestyle choices such as physical activity and sedentary behaviors despite the fact that increased energy expenditure may counter increases in energy intake through alcohol consumption [ 17 , 23 , 30 , 32 , 40 , 42 ] . furthermore , beer and spirit drinkers appear to have poorer dietary habits in general than wine drinkers [ 3 ] . thus , accounting for both sides of the energy balance equation ( intake , expenditure and lifestyle habits ) is crucial to evaluate adequately the association between alcohol intake and obesity . insufficient sleep has also been shown to be associated with greater alcohol consumption and excess body weight in adults [ 69 , 70 ] . specifically , sleeping less than 6 hours per night in adults is associated with greater alcohol intake , an increased risk of exceeding the guidelines for sensible weekly alcohol intake , and higher bmi . furthermore , we showed that the combination of short sleep duration with disinhibited eating behavior is associated with greater alcohol intake and excess weight [ 69 , 71 ] . these results emphasize the need to identify high - risk individuals ( e.g. , disinhibited short sleepers ) who are in greater need of preventive strategies . genetic aspects can also play a role in the predisposition of individuals to gain weight as a result of alcohol consumption . recent results showed that genetic polymorphisms affect susceptibility to alcoholism and may affect body weight via gene - associated differences in fuel utilization . the authors found that alcohol dehydrogenase-1b ( adh1b ) genotype ( rs1229984 ) is a strong determinant of body weight in alcoholics . the more rapid ethanol elimination associated with the adh1b*2 allele may result in less efficient utilization of ethanol as an energy source . those findings thus suggest that more research is needed to unravel genetic aspects involved in the connection between alcohol intake and weight gain . overall , obesity is a multi - factorial condition and it is difficult to truly assess the independent influence of alcohol intake on obesity risk . the observational evidence is hampered by the possibility of residual confounding by unmeasured variables and the experimental evidence is limited by the short - term follow - up period and the difficulty to control for all lifestyle habits under free - living conditions . the slow development of obesity and multi - faceted nature of this condition really complicates the possibility to show a cause - and - effect association between alcohol consumption and weight gain . thus , we need to rely on short - term intervention studies and epidemiologic studies , each of which has clear limitations in showing an effect of alcohol intake on the vulnerability to gain weight . however , the preponderance of the evidence taken as a whole suggests that alcohol may be a risk factor for obesity in some individuals , especially when consumed in large quantities . alcohol consumption has probably contributed to the excess energy intake associated with weight gain in some individuals over the past years . however , the available evidence is conflicting and hampered by important limitations that preclude a strong conclusion on the effect of alcohol intake on obesity risk . moderation in drinking is still an important recommendation , together with a healthy lifestyle not conducive to weight gain .

recreational alcohol intake is a widespread activity globally and alcohol energy ( 7 kcal / g ) can be a contributing factor to weight gain if not compensated for . given that both excessive alcohol intake and obesity are of public health interest , the present paper provides an update on the association between alcohol consumption and body weight . in general , recent prospective studies show that light - to - moderate alcohol intake is not associated with adiposity gain while heavy drinking is more consistently related to weight gain . experimental evidence is also mixed and suggests that moderate intake of alcohol does not lead to weight gain over short follow - up periods . however , many factors can explain the conflicting findings and a better characterization of individuals more likely to gain weight as a result of alcohol consumption is needed . in particular , individuals who frequently drink moderate amounts of alcohol may enjoy a healthier lifestyle in general that may protect them from weight gain . in conclusion , despite the important limitations of current studies , it is reasonable to say that alcohol intake may be a risk factor for obesity in some individuals , likely based on a multitude of factors , some of which are discussed herein .

Introduction Alcohol Intake and Obesity: Observational Evidence Cross-sectional Evidence Longitudinal Evidence Alcohol Intake and Obesity: Experimental Evidence Alcohol Intake and Obesity: Potential Mechanisms Factors that may Explain the Conflicting Findings between Alcohol Intake and body Weight Conclusions

nowadays , recreational alcohol intake is common across the globe and health and social problems resulting from alcohol consumption are becoming a concern . although moderate alcohol use is recommended , excessive alcohol consumption is the third leading cause of premature death in the united states ( behind smoking and obesity ) . among the many problems associated with heavy alcohol drinking , the relationship between alcohol intake and body weight has been extensively studied over the past years [ 3 , 4 , 5 ] . based on the fact that 1 gram of alcohol provides 7.1 kcal ( 29 kj ) and studies showing that energy consumed as alcohol is additive to that from other dietary sources , increased energy intake with alcohol use can certainly promote a positive energy balance and ultimately weight gain . however , a clear cause - and - effect association between alcohol intake and weight gain is not apparent based on the mixed and conflicting available evidence on the topic . given that both excessive alcohol intake and obesity are of public health concern , a better understanding of the association between alcohol consumption and excess body weight is warranted . therefore , the objective of this article is to provide an update on the link between alcohol intake and obesity . furthermore , factors that may explain the conflicting findings in this research area are discussed . observational studies on the effect of alcohol intake on obesity date back almost 30 years . among cross - sectional studies , a common trend appears to be that alcohol intake is not associated with body mass index ( bmi ) in men , while either negatively or not associated with bmi in women . indeed , several cohorts ranging from 10,482 to 138,031 individuals have shown no correlation ( or a small negative correlation ) between alcohol intake and bmi in men , and a small negative association with bmi in women [ 613 ] . other studies have found that alcohol intake is positively correlated with bmi in men or in both sexes ; however , an analysis of recent studies suggests that this may be due to differences in intake patterns [ 4 , 7 ] . wannamethee , shaper & whincup found that in men , drinking < 20 drinks per week was not associated with higher bmi , waist circumference ( wc ) or waist - to - hip ratio ( whr ) compared to non - drinkers . other studies have shown j - shaped curves when comparing bmi , wc , and whr among male and female drinkers , with light drinking being negatively associated with adiposity indicators compared to heavy drinking , or abstention [ 1923 ] . to test the proposal that patterns of alcohol intake may be an important influence on overweight and obesity , most recently , sheldon and knott recorded the energy intake from alcohol on the days that individuals had their highest intake . had individuals retrospectively estimate their age at peak alcohol intake and earlier reported peak alcohol intake was associated with more heavy drinking episodes and abdominal obesity . despite the recent body of cross - sectional evidence suggesting the benign or potentially protective effect of frequent light drinking on body weight and obesity , several studies have found conflicting results . found a very modest positive linear relationship between alcohol intake and bmi in men . found that alcohol intake was positively associated with body fat in adolescent girls , but negatively associated with body fat in adolescent boys . found a positive association between alcohol intake and risk of overweight in 1314 year olds . this suggests that there may be differences in alcohol metabolism between age groups ; however , it is important to note that this study assessed only the amount of alcohol per drinking occasion , and not drinking frequency patterns . overall , the majority of cross - sectional studies since 2005 have demonstrated that frequent light to moderate alcohol intake does not seem to be associated with obesity risk . heavy drinking and binge drinking , however , are more likely to carry such an association with excess body weight [ 16 , 19 , 2225 ] . prospective studies have looked at the association between alcohol intake and adiposity gain in various populations , with follow - up periods ranging from several months to 20 years [ 4 , 30 , 31 ] . several studies have found no association or a negative association between alcohol intake and changes in weight , bmi or other measures of adiposity [ 12 , 30 , 3239 ] . other studies have found such an association only in women , while finding a positive association between obesity risk and alcohol intake in men . this study , however , did not specify the amount of alcohol intake , and did not control for participant s physical activity ( pa ) levels . conversely , one study found no association between alcohol intake and increases in wc in men , but a small positive association in women . there have also been recent studies that have found a general positive association between alcohol intake and weight gain . this study , however , did not control for pa levels and only assessed alcohol intake on a yes / no scale . changes in alcohol intake patterns have been associated with weight gain in prospective studies [ 15 , 31 , 43 ] . found that men who increased their frequency or amount of alcohol intake over the follow - up period were more likely to experience increases in bmi and wc . finally , other recent studies have shown that heavy drinking may be more of a risk factor for weight gain than light - to - moderate drinking [ 44 , 4547 ] . found that drinking more than seven times per week was associated with increased risk of weight gain and development of overweight and obesity . found that male light - to - moderate beer drinkers had smaller increases in wc and weight than non - drinkers or heavy drinkers over 8.5 years of follow up . however , conversely , in women they found a dose response relationship between beer intake and weight gain and wc gain . there are many reasons as to why the relationship between alcohol and adiposity varies between men and women , involving genetic and lifestyle factors , some of which will be discussed in depth below . as with cross - sectional studies , the way by which alcohol intake is measured and categorized likely influences the interpretation of the results . collectively , the most recent prospective studies suggest that light - to - moderate alcohol intake is not associated with weight gain or changes in wc [ 44 ] . heavy drinking , however , has been more consistently associated with weight gain [ 4547 ] . among cross - sectional studies , a common trend appears to be that alcohol intake is not associated with body mass index ( bmi ) in men , while either negatively or not associated with bmi in women . indeed , several cohorts ranging from 10,482 to 138,031 individuals have shown no correlation ( or a small negative correlation ) between alcohol intake and bmi in men , and a small negative association with bmi in women [ 613 ] . other studies have found that alcohol intake is positively correlated with bmi in men or in both sexes ; however , an analysis of recent studies suggests that this may be due to differences in intake patterns [ 4 , 7 ] . wannamethee , shaper & whincup found that in men , drinking < 20 drinks per week was not associated with higher bmi , waist circumference ( wc ) or waist - to - hip ratio ( whr ) compared to non - drinkers . other studies have shown j - shaped curves when comparing bmi , wc , and whr among male and female drinkers , with light drinking being negatively associated with adiposity indicators compared to heavy drinking , or abstention [ 1923 ] . to test the proposal that patterns of alcohol intake may be an important influence on overweight and obesity , most recently , sheldon and knott recorded the energy intake from alcohol on the days that individuals had their highest intake . had individuals retrospectively estimate their age at peak alcohol intake and earlier reported peak alcohol intake was associated with more heavy drinking episodes and abdominal obesity . despite the recent body of cross - sectional evidence suggesting the benign or potentially protective effect of frequent light drinking on body weight and obesity , several studies have found conflicting results . found a very modest positive linear relationship between alcohol intake and bmi in men . found that alcohol intake was positively associated with body fat in adolescent girls , but negatively associated with body fat in adolescent boys . found a positive association between alcohol intake and risk of overweight in 1314 year olds . this suggests that there may be differences in alcohol metabolism between age groups ; however , it is important to note that this study assessed only the amount of alcohol per drinking occasion , and not drinking frequency patterns . overall , the majority of cross - sectional studies since 2005 have demonstrated that frequent light to moderate alcohol intake does not seem to be associated with obesity risk . heavy drinking and binge drinking , however , are more likely to carry such an association with excess body weight [ 16 , 19 , 2225 ] . prospective studies have looked at the association between alcohol intake and adiposity gain in various populations , with follow - up periods ranging from several months to 20 years [ 4 , 30 , 31 ] . several studies have found no association or a negative association between alcohol intake and changes in weight , bmi or other measures of adiposity [ 12 , 30 , 3239 ] . other studies have found such an association only in women , while finding a positive association between obesity risk and alcohol intake in men . this study , however , did not specify the amount of alcohol intake , and did not control for participant s physical activity ( pa ) levels . conversely , one study found no association between alcohol intake and increases in wc in men , but a small positive association in women . there have also been recent studies that have found a general positive association between alcohol intake and weight gain . this study , however , did not control for pa levels and only assessed alcohol intake on a yes / no scale . changes in alcohol intake patterns have been associated with weight gain in prospective studies [ 15 , 31 , 43 ] . found that men who increased their frequency or amount of alcohol intake over the follow - up period were more likely to experience increases in bmi and wc . finally , other recent studies have shown that heavy drinking may be more of a risk factor for weight gain than light - to - moderate drinking [ 44 , 4547 ] . found that drinking more than seven times per week was associated with increased risk of weight gain and development of overweight and obesity . found that male light - to - moderate beer drinkers had smaller increases in wc and weight than non - drinkers or heavy drinkers over 8.5 years of follow up . however , conversely , in women they found a dose response relationship between beer intake and weight gain and wc gain . there are many reasons as to why the relationship between alcohol and adiposity varies between men and women , involving genetic and lifestyle factors , some of which will be discussed in depth below . as with cross - sectional studies , the way by which alcohol intake is measured and categorized likely influences the interpretation of the results . collectively , the most recent prospective studies suggest that light - to - moderate alcohol intake is not associated with weight gain or changes in wc [ 44 ] . heavy drinking , however , has been more consistently associated with weight gain [ 4547 ] . binge drinking behavior was not specifically examined in any of the recent prospective studies analyzed . a recent review summarized a number of these studies , showing that alcohol ingested before a meal has frequently been shown to have a neutral effect on intake , or to increase intake , despite the added energy that come from the alcohol preload . in these studies however , to date there have been few intervention studies conducted to experimentally examine the effects of regular alcohol intake on weight gain or obesity in humans . all of the available studies have examined moderate intake of alcohol , and the majority have reported results on beer and wine intake , but not other forms of alcohol [ 3 , 5 ] . overall , the available experimental evidence reviewed in this article suggests that moderate intake of alcohol does not lead to weight gain . to our knowledge , there does not appear to be any experimental evidence specifically testing the effects of heavy / binge drinking , or of drinking spirits or a combination of alcohol sources on weight gain / obesity . a summary of the studies examined in this article , organized by the trend between alcohol and weight gain / obesity can be found in table 1.table 1summary of trends in cross - sectional , longitudinal , and experimental studies examining the link between alcohol intake and measures of adipositytrendstudy ( measurement)menwomencross - sectional studiesno associationcolditz et al . ( weight gain of > 10lbs)drinkers gain more over follow - upbell , ge & popkins ( risk of weight gain)hou et al . ( weight gain , wc)positive impact of alcohol consumptioncrouse & grundy ( half of obese patients , change in weight)romeo et al . ( biceps skinfold)bmi = body mass index , wc = waist circumference , whr = waist - to - hip ratio summary of trends in cross - sectional , longitudinal , and experimental studies examining the link between alcohol intake and measures of adiposity bmi = body mass index , wc = waist circumference , whr = waist - to - hip ratio in summarizing the recent literature it appears that light - to - moderate alcohol intake is less likely to be a risk factor for obesity than heavy drinking . there are several lines of evidence suggesting the potential for alcohol to promote weight gain , and the contradictory results often seen in the literature have led to the development of alternative hypotheses regarding the influence of alcohol on body weight . a review by yeomans highlights some of the potential explanations for alcohol s influence on weight gain or obesity . one explanation is that there is a learned association between alcohol and eating ; however , several experimenters disguised the presence of alcohol in their protocols and still found increased energy intake . it is unclear whether alcohol promotes food intake in the absence of hunger ; however , it has been noted that alcohol may amplify individuals perception of appetite in response to food stimuli . the results of several studies propose that alcohol may influence energy intake by inhibiting the effects of leptin , or glucagon - like peptide-1 ( glp-1 ) [ 56 , 57 ] . to date , the evidence suggests that alcohol does not appear to increase appetite through the action of peptide yy ( pyy ) , ghrelin , gastric inhibitory peptide ( gip ) , or cholecystokinin ( cck ) [ 5761 ] . found that alcohol did not increase plasma levels of neuropeptide y ( npy ) ; however , animal models have shown that central npy levels are increased following alcohol consumption . a summary of the effects of alcohol on important appetite hormones and central neurological pathways in humans can be found in table 2.table 2effect of alcohol on various peripheral hormones and central neurotransmitter systems related to hunger and energy intakehormone/ neurotransmittereffect on hunger/ energy intakeeffect of alcohol intake on hormone/ neurotransmitter responsereference(s)peripheral signalsccksuppressesincreasehajnal , flores and valenzuela ; manabe et al . cck = cholecystokinin , glp-1 = glucagon - like peptide-1 , gip = gastric inhibitory peptide , pyy = peptide yy , npy = neuropeptide y , gaba = gamma - aminobutyric acid effect of alcohol on various peripheral hormones and central neurotransmitter systems related to hunger and energy intake cck = cholecystokinin , glp-1 = glucagon - like peptide-1 , gip = gastric inhibitory peptide , pyy = peptide yy , npy = neuropeptide y , gaba = gamma - aminobutyric acid aside from the immediate influence on appetite that comes from alcohol consumption , there are also effects on energy storage . although there is evidence to suggest that frequent alcohol intake may predispose to weight gain or obesity over the long - term , first , it has been found that alcohol intake increases energy expenditure , likely due in part to the fact that it has a high thermogenic effect . oxidation of alcohol via the meos produces less atp than oxidation via alcohol dehydrogenase , using the energy from alcohol intake primarily to enhance heat production [ 37 , 54 ] . the extent to which wasted energy from regular alcohol consumption contributes to weight gain prevention is unclear . it has also been suggested that individuals who frequently drink moderate amounts of alcohol may enjoy a moderate lifestyle in which exercise and food intake are modulated over the long term to accommodate for alcohol intake . however , studies using food logs and self - reported physical activity levels have still shown a null or negative association between moderate alcohol intake and weight gain after controlling for these and other confounders , although they may fail to truly capture the habits of participants [ 4 , 49 ] . while cross - sectional and longitudinal studies have controlled for a number of important lifestyle factors , there are many to consider when examining body weight regulation . it is highly likely that the paradoxical results seen in studies examining the effect of alcohol on weight gain and obesity are also the product of a multitude of factors beyond the individual s ingestion habits . future research must consider the other important factors that may influence the link between alcohol and obesity , some of which are discussed below . the mixed evidence with respect to alcohol s role in promoting obesity is a product of many factors [ e.g. , binge drinking ) , physical activity level , sleeping habits , depression symptoms , psychosocial problems , chronic illness , medication use , disinhibition eating behavior trait , history of alcohol use , predisposition to gain weight , etc . ] not taking into account some of these potential confounding factors can certainly lead to biased estimates of the relationship between alcohol intake and body weight given that large inter - individual variations exist . the association between alcohol intake and body weight is generally stronger in men than women , especially because of the amount and type of alcohol consumed by men . thus , accounting for both sides of the energy balance equation ( intake , expenditure and lifestyle habits ) is crucial to evaluate adequately the association between alcohol intake and obesity . insufficient sleep has also been shown to be associated with greater alcohol consumption and excess body weight in adults [ 69 , 70 ] . specifically , sleeping less than 6 hours per night in adults is associated with greater alcohol intake , an increased risk of exceeding the guidelines for sensible weekly alcohol intake , and higher bmi . furthermore , we showed that the combination of short sleep duration with disinhibited eating behavior is associated with greater alcohol intake and excess weight [ 69 , 71 ] . genetic aspects can also play a role in the predisposition of individuals to gain weight as a result of alcohol consumption . the authors found that alcohol dehydrogenase-1b ( adh1b ) genotype ( rs1229984 ) is a strong determinant of body weight in alcoholics . those findings thus suggest that more research is needed to unravel genetic aspects involved in the connection between alcohol intake and weight gain . overall , obesity is a multi - factorial condition and it is difficult to truly assess the independent influence of alcohol intake on obesity risk . the observational evidence is hampered by the possibility of residual confounding by unmeasured variables and the experimental evidence is limited by the short - term follow - up period and the difficulty to control for all lifestyle habits under free - living conditions . the slow development of obesity and multi - faceted nature of this condition really complicates the possibility to show a cause - and - effect association between alcohol consumption and weight gain . thus , we need to rely on short - term intervention studies and epidemiologic studies , each of which has clear limitations in showing an effect of alcohol intake on the vulnerability to gain weight . however , the preponderance of the evidence taken as a whole suggests that alcohol may be a risk factor for obesity in some individuals , especially when consumed in large quantities . alcohol consumption has probably contributed to the excess energy intake associated with weight gain in some individuals over the past years . however , the available evidence is conflicting and hampered by important limitations that preclude a strong conclusion on the effect of alcohol intake on obesity risk . moderation in drinking is still an important recommendation , together with a healthy lifestyle not conducive to weight gain .

sex hormone binding globulin ( shbg ) is the major sex hormone carrier protein in serum . under physiological conditions , approximately 70% of testosterone ( te ) is bound to shbg with high affinity , about 2030% is weakly bound to albumin , and the remaining 1 - 2% is free [ 1 , 2 ] . pubertal increases in serum testosterone in males peak at 20 years of age and remain stable until the eighth decade . after childhood , shbg declines by the age of 20 years and then remains stable until the sixth decade with a gradual , progressive rise thereafter . for many years it has been widely accepted that the primary function of shbg is to transport sex steroid hormones to target tissues where shbg modulates the level of free sex hormones that can enter target cells [ 4 , 5 ] . however , in recent years , additional roles for shbg have been identified . there is strong evidence that shbg mediates steroid hormone signal transduction at the plasma membrane and a growing body of evidence that shbg may be taken up by cells and have intracellular biological functions . a study has demonstrated that shbg is internalised through the megalin receptor in rat yolk sac cells and that megalin deficient mice display defects similar to animals treated with androgen- or estrogen - receptor antagonists . shbg internalisation has also been described in other cell types such as periventricular neurons and fibroblast cells , suggesting that it may have an intracellular role [ 8 , 9 ] . intracellular expression of shbg in mouse proximal tubule cells has been demonstrated to increase uptake of h - dihydrotestosterone ( dht ) and to prolong the expression of androgen responsive genes . this study was designed to determine whether the androgen sensitive lncap prostate cancer cell line internalises shbg ; whether shbg uptake affects te uptake ; the effects of shbg on te function and metabolism ; and the effects of shbg and te on cell growth . the androgen responsive human prostate cancer cell line lncap clone fgc ( atcc , va , usa ) was chosen for this study . cells were cultured in roswell park memorial institute ( rpmi ) medium ( sigma - aldrich , vic , australia ) supplemented with 10% fetal bovine serum ( fbs ) , 500 u / ml penicillin streptomycin ( gibco , vic , australia ) , and 5 g / ml plasmocin ( invivogen , vic , australia ) in a conventional cell culture incubator . all experiments were conducted in serum - free medium to eliminate shbg and albumin contamination from fbs and in the absence of phenol red , which can bind to steroid receptors . purified native human shbg protein was purchased from fitzgerald industries international , ma , usa . shbg was purified from the serum of pregnant women by ligand affinity chromatography with a purity of > 98% with an unspecified residual steroid content . testosterone was purchased from sigma - aldrich , vic , australia , and prepared as a 500 mm stock in ethanol . testosterone was diluted > 5 10 times in serum - free medium ( final < 0.00002% ethanol ) and used at a maximal final concentration of 100 nm in experiments . ethanol had no effects on lncap cell growth at concentrations between 0.00001% and 1% over 96 hours ( data not shown ) . unless specified all other reagents were purchased from sigma - aldrich . purified native human shbg protein was labeled using an alexa fluor 546 protein labeling kit ( life technologies , vic , australia ) . briefly , 1 ml of 2 mg / ml purified shbg protein was incubated with alexa fluor 546 reactive dye for 2 hours at room temperature . then alexa fluor 546 labeled - shbg ( alexa546-shbg ) was purified through a bio - gel p-6 gel column ( biorad , ca , usa ) . to determine the concentration of labeled shbg and the degree of fluorescent labeling , the conjugate solution was measured at 280 nm and 540 nm in a nanodrop 2000 spectrophotometer ( thermo scientific , nc , usa ) . for alexa546-shbg uptake experiments , the cells were serum - starved for 1 hour and then incubated in 125 nm alexa546-shbg 25 nm te in serum - free rpmi for 1 hour at room temperature cells were washed with phosphate buffered saline ( pbs ) , fixed in 2% paraformaldehyde , and permeabilised in 0.2% triton x-100 in pbs . cells were costained with alexa488-phalloidin to highlight the -actin skeleton and nuclei were stained with dapi . images were captured using a deltavision deconvolution microscope ( applied precision , washington dc , usa ) . uptake of alexa546-shbg was confirmed by costaining internalised alexa546-shbg with a monoclonal anti - shbg antibody ( sigma , vic , australia ) conjugated to an alexa488 labeled secondary antibody . the colocalisation of alexa546 ( red ) and alexa488 ( green ) discriminated alexa - shbg from endogenous shbg , which was stained with alexa488 only . treatments with serum - free medium only and alexa546 dye only were used as negative controls . shbg secretion from lncap cells was not detectable ( data not shown ) by western blot or unicel immunoassay ( lower limit of detection < 2 nm ) , despite cell culture medium being concentrated 12-fold ; therefore endogenous levels of shbg should have little effect on our experiments using 125 nm shbg . cells were cultured in 6-well plates and serum - starved for 4 hours prior to uptake experiments . cells were then incubated in serum - free uptake medium containing 25 nm ( 30 pmoles / well ) of te 125 nm shbg for 1 , 4 , and 24 hours . cell monolayers were washed carefully with 2 ml pbs ( ph 7.3 ) and then cells were lysed with pierce m - per mammalian protein extraction reagent ( thermo fisher scientific ) to extract soluble protein . further lysis with pierce ripa lysis and extraction buffer ( thermo fisher scientific ) was used to extract remaining proteins from the membrane and nuclei . te was measured in the original uptake medium ( 25 nm ) , cell culture medium , and cell lysates using tandem mass spectrometry . te measured in all fractions was expressed as a percentage of the te in the original uptake medium . cellular te includes all te measured in both lysates including cytosol , membrane , and nuclei . liquid chromatography followed by tandem mass spectrometry ( lc msms ) was used to measure te on a waters ultra performance liquid chromatography ( uplc ) acquity system . briefly , samples ( in 2 ml 96 deep well plates ) were precipitated with 3 volume equivalents of 50 mm zinc sulphate/40% methanol containing an internal standard ( testosterone d2 ) . after protein precipitation , the samples were centrifuged and the plate was transferred to an autosampler . an aliquot of 50 l was injected onto a waters online sample manager ( osm ) . the extract was concentrated onto the masstrak xbridge c18 osm 10 m cartridge , washed with 30% methanol , and then switched into line and eluted directly into the uplc system . the analytes were separated on an acquity beh c18 2.1 50 mm column . the column eluent was directed ( without stream splitting ) into the ion source of a waters xevo tq - d tandem quadrupole ms , operated in positive esi mode . the following quantifier and qualifier transitions were utilised : testosterone ( 289.1 > 97.0 , 289.1 > 109.0 ) and testosterone d2 ( 291.1 > 111.0 ) . the gradient was returned to initial conditions in preparation for the next sample , which is being prepared in parallel by the osm . nmol / l with an interrun imprecision of 9.8% for testosterone at 0.9 nmol / l . measurement of te - glucuronide formation and efflux into medium was determined by digestion of cell culture medium with e. coli -glucuronidase ( sigma , vic , australia ) , followed by measurement of te by lc msms . enzymatic deconjugation of te - glucuronide was optimised by using two different concentrations of -glucuronidase ( 200 u / ml and 400 u / ml ) , two different dilutions of sample ( culture medium neat and diluted 1 : 10 ) for 24 hours incubation time . maximal deconjugation was achieved in neat samples incubated with 200 u / ml -glucuronidase for 24 hours at 37c . te - glucuronide was not detectable in cell lysates ; therefore only effluxed te - glucuronide in cell culture medium was measured . after culturing cells with 25 nm ( 30 pmoles / well ) te 125 nm shbg for 4 or 24 hours , culture medium was diluted in an equal volume of 0.25 m potassium phosphate buffer , ph 6.9 200 units / ml of e. coli -glucuronidase ( sigma , vic , australia ) and incubated at 37c for 24 hours . to quantitate the amount of te - glucuronide the quantity of te in each sample was measured by lc msms and expressed as a percentage of te in the original uptake medium . both unconjugated and previously glucuronidated te were measured ; therefore the value for unconjugated te was subtracted from this in order to quantify the glucuronidated fraction . total rna was isolated using an rneasy mini kit ( qiagen , vic , australia ) . rna concentrations and quality were determined using a nanodrop-3000 spectrophotometer ( nanodrop technologies , inc . four micrograms of total rna was reverse - transcribed with 200 units of superscript iii reverse transcriptase ( invitrogen life technologies , ca , usa ) and 0.5 g of oligo ( dt ) 15 primers ( roche diagnostics , mannheim , germany ) in a reaction volume of 20 l . rt - pcr was performed using 3 l of cdna ( represented 6 ng rna ) , 0.5 m of each primer ( table 1 ) , and faststart sybr green pcr master ( roche diagnostics , mannheim , germany ) in a rotor gene rg-6000 ( corbett research , nsw , australia ) . to quantify the gene expression profile in each sample , data were presented as ratio of the amount of targeted mrna ( arbitrary units ) normalised with the house - keeping gene beta 2-microglobulin ( 2 m ) lncap cells ( ~80,000 cells / well ) were cultured on 24-well plates for 48 hours . following serum starvation , cells were incubated with combinations of alexa546-shbg te and imaged using an incucyte zoom live cell - imaging system ( essen bioscience , mi , usa ) with phase and red channels and scanned at 3-hour intervals for a total of 30 hours . at the end of the incubation period , cells were washed in an acid solution ( 200 mm glycine , 150 mm nacl , ph 2.5 ) to remove surface bound alexa546-shbg . incucyte zoom software was used to quantify the red objects in 9 nonoverlapping images per well . lncap cells were cultured on 24-well plates with ~35,000 cells per well . after serum starvation the cells were treated with various combinations of te shbg and incubated in an incucyte zoom set to scan every 3 hours for up to 86 hours . the kinetic measurements of proliferation and data analysis were performed using incucyte zoom software according to manufacturer 's instructions . all experiments were repeated at least three times on separate cell cultures and performed in triplicate for each treatment group . firstly , after incubation with alexa546-shbg , the cells were imaged using deltavision deconvolution microscopy ( figure 1(a ) ) . internalisation of alexa546-shbg was confirmed by generating an orthogonal view and a series of z - stack images . the red fluorescent signal representing alex546 labeled shbg was observed in the cells incubated with alexa546-shbg ( figure 1(a)(3 ) ) . there was no red signal observed in the negative controls incubated with unconjugated alexa546 dye ( figure 1(a)(2 ) ) , confirming that alexa-546 shbg and not the alexa dye itself was internalised by the cells . additionally the internalised alexa546-shbg ( red ) colocalised with an alexa488 ( green ) labeled anti - shbg antibody ( figure 1(b ) ) further proving that shbg was internalised while still being conjugated to alexa546 dye . endogenous shbg is stained only with the alexa488 anti - shbg antibody ( green ) . secondly , to quantitate internalised alexa546-shbg incucyte zoom technology was used . addition of te had no significant effect on alexa - shbg uptake ( figure 1(d ) ) . very weak signal was detected in alexa546 dye treated culture and after the acid wash , no signal was detected . incubation with 25 nm te for 1 , 4 , and 24 hours resulted in rapid uptake of te followed by a decline in cellular levels ( figure 2(a ) ) . cellular te levels peaked at one hour ( 17.84 0.31% ) and then declined to 14.31 0.93% and 5.95 0.31% by 4 and 24 hours , respectively ( figure 2(a ) ) . in contrast , in the presence of 125 nm shbg , cellular te levels were lower at 1 hour ( 1.10 0.31% ) and steadily increased to 1.32 0.003% and 2.64 0.04% by 4 and 24 hours . at 24 hours , cellular te levels in cells incubated with te plus figure 2(b ) shows the total te measured in both the cell lysates and cell culture medium . when lncap cells were cultured in te alone , 93 2.80% of te from the original uptake medium was measured at 1 hour but decreased to 55.50 4.36% and then 16.79 0.93% by 4 and 24 hours , respectively . when cells were cultured in te plus shbg , te measurements did not change significantly over the three time points ( 72.69 0.62% , 77.31 0.93% , and 74.23 0.93% at 1 , 4 , and 24 hours , resp . ) . cells were exposed to 25 nm te 125 nm shbg for 4 and 24 hours ; then cell culture medium was collected and treated with -glucuronidase . when lncap cells were cultured in te for 4 hours , 50.73 3.51% of te was glucuronidated and effluxed and only 11.12 1.06% of te was unconjugated ( figure 2(c ) ) . however , when shbg was also added , only 4.41 2.1% of te was glucuronidated and 60.84 1.93% of te was unconjugated . following 24 hours in culture with te , lncap cells had glucuronidated and effluxed 67.07 5.48% of te and only 3.8 1.59% remained unconjugated . again , less te was glucuronidated when cells were incubated with te and shbg for 24 hours with 13.19 4.93% of te glucuronidated compared to 67.11 11.74% unconjugated . lncap cells were seeded at initial 20% confluence and after 38 hours of culture cell confluency reached about 3540% with no differences between treatment groups ( figure 3(a)(a ) ) . exposure to only shbg did not alter cell growth . however after 38 hours in culture , growth declined in a dose dependent manner in those cells treated with 1 , 25 , and 100 nm te . addition of either 25 or 125 nm shbg canceled the inhibitory effects of te in a dose dependent fashion ( figures 3(b ) and 3(c ) ) . moreover , cells cultured in te alone displayed morphological changes in that cells became rounded rather than spread out and detached from the culture plate ( figure 3(b)(b ) ) . control cells and those incubated with te plus shbg did not have these morphological changes ( figures 3(b)(a ) and 3(b)(c ) ) . figure 4(a ) displays a paired comparison of treatment of lncap cells with 0 , 1 , 10 , and 25 nm te alone and with the addition of 25 nm of shbg for 24 hours . surprisingly , addition of shbg , despite having previously been shown to reduce te uptake , significantly enhanced te upregulation of psa mrna expression at te concentrations of 10 and 25 nm ( p < 0.0001 ) ( figure 4(a ) ) . when cells were incubated with 25 nm shbg plus 0 , 1 , 5 , 10 , and 25 nm te ( figure 4(b ) ) , expression of psa was increased by te ( p < 0.0001 ) . when cells were incubated with 10 nm te with 0 , 10 , 25 , or 50 nm shbg , psa mrna expression increased with increasing amounts of shbg to a maximum at 25 nm shbg ( p < 0.001 ) ( figure 4(c ) ) . in the same samples , androgen receptor ( ar ) mrna expression was decreased by te and expression was further decreased by the addition of 25 or 50 nm shbg ( figure 4(d ) ) . shbg mrna expression was significantly decreased by te ( p < 0.0001 ) ( figure 4(e ) ) . additionally , shbg further decreased the te downregulation of shbg expression at all shbg concentrations ; however this did not reach statistical significance . the primary role of hepatically produced shbg as a serum carrier of sex hormones is well documented [ 5 , 15 ] . shbg expression has also been demonstrated in sex hormone target tissues such as human prostate [ 16 , 17 ] , lncap cells , testes , duodenum , ovary , placenta , proximal tubule epithelial cells ( ptec ) , and cerebral cortex as well as several cancers [ 10 , 15 , 1921 ] . higher expression of shbg in prostate cancer tissue is associated with poor clinicopathological features suggesting a role in prostate cancer progression , perhaps due to prolonged expression of androgen responsive genes . it has also been proposed that locally regulated and produced shbg may be destined to participate in signalling at the prostate cell membrane . using a variety of technical approaches we have confirmed that lncap cells take up shbg in a dose dependent but not androgen dependent manner . it is well known that shbg reduces cellular uptake of te ; however we have demonstrated that shbg may also maintain stable levels of biofunctional te and physiologically relevant intracellular te levels by reducing te glucuronidation and efflux . another study has also demonstrated that lncap cells incubated in 30 nm dht glucuronidated 85% of the dht in 5 days . upon addition of 140 nm shbg , 95% of dht was not taken up by cells and remained in the medium . this rapid inactivation of te by glucuronidation has been demonstrated in human clinical studies , where a single oral dose of te did not alter serum total te levels but rapidly and significantly increased levels of conjugated te in serum . however we acknowledge that the level of free te in healthy men is much lower ( 174729 pmol / l ) than that used in this study , since in vivo shbg is always present . when shbg is reduced or absent , excess free te may enter cells and oversaturate the ars since it has been reported that ar saturation occurs at approximately 2 - 3 nm in prostate . the lncap cells may respond to this by rapidly glucuronidating and effluxing the excess te . the steroid content of shbg ( fitzgerald industries international ) was not specified ; however it was purified from the serum of pregnant women which contains significantly lower te than that of adult males [ 3 , 27 ] . even if all of the serum te were carried over , the te at the maximum shbg concentrations used ( 125 nm ) would be much less than 1 nm , significantly less than that used in our experiments . importantly , shbg treatment had no significant effect on gene expression when compared to untreated controls . to further explore the intracellular biological actions of shbg the effects of shbg on te responsive genes were investigated . as expected te upregulated psa mrna expression . however , despite the significantly lower intracellular te in the presence of shbg , addition of shbg in combination with te further increased the expression of psa mrna . similarly , downregulation of ar mrna expression by te was also further enhanced when cells were incubated with shbg and te . addition of shbg also further decreased the te downregulation of shbg mrna although , under these conditions , the reduction did not reach statistical significance . in this experiment the cellular te level was lower in the presence of shbg than when te was added alone ; however the effects on gene expression were much greater . moreover , maximal effects on gene expression were reached at a lower concentration of te when shbg was present . intracellular expression of shbg in mouse proximal tubule cells has also been shown to prolong the expression of androgen responsive genes and overexpression of shbg in lncap cells has been shown to influence the expression of estrogen and androgen responsive genes . this would suggest that both endogenously expressed shbg and internalised shbg have similar positive stimulatory effects on androgen sensitive gene expression . in our in vitro study , te decreased shbg gene expression . we speculate that , in pathological conditions where serum shbg is reduced , for example , in overweight and obese men [ 3033 ] , te uptake , glucuronidation , and efflux would be increased potentially exacerbating te deficiency . in the present study te treatment did not promote cell growth , and in fact a dose dependent inhibition of growth was demonstrated after 38 hours in culture . coincubation with shbg prevented the te inhibition of cell growth . a biphasic effect on cell proliferation has previously been described where proliferation was increased up to 0.3 nm dht and then progressively decreased when concentrations were raised above 0.3 nm dht [ 23 , 34 ] . the inhibitory effect of dht was abrogated by addition of human serum or shbg . the effect of the synthetic androgen methyltrienolone ( r1881 ) , which binds poorly to shbg , was not affected by addition of shbg . other synthetic androgens displayed a similar inhibitory effect in lncap cells resulting in morphological changes and inhibition of colony formation ; however these effects were not reproduced in the androgen receptor - negative prostate cells lines pc-3 , du145 , and mrc-5 . furthermore , addition of antiandrogen drugs ( 6-chloro-6-dehydro-17x - acetoxy - hla , cyproterone acetate , and hydroxyflutamide ) restored lncap cell growth . all of these studies suggest that inhibition is through an ar pathway . in the present study the striking changes in cell morphology suggest that the cells may be stressed , perhaps due to the requirement to glucuronidate high levels of intracellular te ; however previous studies have not included descriptions of cell morphology in order to make a comparison . the mechanism by which te inhibits cell growth in lncap cells after 38 hours is unknown . regardless of the percentage of cell confluence at the beginning of treatment , te always caused morphological changes ( data not shown ) . this perhaps suggests that te had an effect on cell morphology and attachment rather than directly on cell proliferation . in this study we have demonstrated that in the absence of shbg large amounts of te rapidly enter the cell where they are inactivated by conjugation to glucuronic acid and effluxed . in the presence of shbg however , while there is reduced te uptake , glucuronidation and efflux of te are also reduced and the effects of te on androgen responsive genes are enhanced . this in vitro study of the role of shbg may have significant clinical relevance , and as such , the assessment of androgen deficiency in vivo should simply rely not only on measurements of total te alone but also on the evaluation of serum shbg levels . these findings clearly demonstrate that shbg plays an important regulatory and intracellular role to modify te metabolism and function and to promote cell growth . if a consistent effect is demonstrated , it will significantly change our current understanding of the role of shbg in health and disease .

sex hormone binding globulin ( shbg ) is the major serum carrier of sex hormones . however , growing evidence suggests that shbg is internalised and plays a role in regulating intracellular hormone action . this study was to determine whether shbg plays a role in testosterone uptake , metabolism , and action in the androgen sensitive lncap prostate cancer cell line . internalisation of shbg and testosterone , the effects of shbg on testosterone uptake , metabolism , regulation of androgen responsive genes , and cell growth were assessed . lncap cells internalised shbg by a testosterone independent process . testosterone was rapidly taken up and effluxed as testosterone - glucuronide ; however this effect was reduced by the presence of shbg . addition of shbg , rather than reducing testosterone bioavailability , further increased testosterone - induced expression of prostate specific antigen and enhanced testosterone - induced reduction of androgen receptor mrna expression . following 38 hours of testosterone treatment cell morphology changed and growth declined ; however , cotreatment with shbg abrogated these inhibitory effects . these findings clearly demonstrate that internalised shbg plays an important regulatory and intracellular role in modifying testosterone action and this has important implications for the role of shbg in health and disease .

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

sex hormone binding globulin ( shbg ) is the major sex hormone carrier protein in serum . under physiological conditions , approximately 70% of testosterone ( te ) is bound to shbg with high affinity , about 2030% is weakly bound to albumin , and the remaining 1 - 2% is free [ 1 , 2 ] . after childhood , shbg declines by the age of 20 years and then remains stable until the sixth decade with a gradual , progressive rise thereafter . for many years it has been widely accepted that the primary function of shbg is to transport sex steroid hormones to target tissues where shbg modulates the level of free sex hormones that can enter target cells [ 4 , 5 ] . however , in recent years , additional roles for shbg have been identified . there is strong evidence that shbg mediates steroid hormone signal transduction at the plasma membrane and a growing body of evidence that shbg may be taken up by cells and have intracellular biological functions . a study has demonstrated that shbg is internalised through the megalin receptor in rat yolk sac cells and that megalin deficient mice display defects similar to animals treated with androgen- or estrogen - receptor antagonists . shbg internalisation has also been described in other cell types such as periventricular neurons and fibroblast cells , suggesting that it may have an intracellular role [ 8 , 9 ] . intracellular expression of shbg in mouse proximal tubule cells has been demonstrated to increase uptake of h - dihydrotestosterone ( dht ) and to prolong the expression of androgen responsive genes . this study was designed to determine whether the androgen sensitive lncap prostate cancer cell line internalises shbg ; whether shbg uptake affects te uptake ; the effects of shbg on te function and metabolism ; and the effects of shbg and te on cell growth . the androgen responsive human prostate cancer cell line lncap clone fgc ( atcc , va , usa ) was chosen for this study . cells were cultured in roswell park memorial institute ( rpmi ) medium ( sigma - aldrich , vic , australia ) supplemented with 10% fetal bovine serum ( fbs ) , 500 u / ml penicillin streptomycin ( gibco , vic , australia ) , and 5 g / ml plasmocin ( invivogen , vic , australia ) in a conventional cell culture incubator . all experiments were conducted in serum - free medium to eliminate shbg and albumin contamination from fbs and in the absence of phenol red , which can bind to steroid receptors . testosterone was purchased from sigma - aldrich , vic , australia , and prepared as a 500 mm stock in ethanol . testosterone was diluted > 5 10 times in serum - free medium ( final < 0.00002% ethanol ) and used at a maximal final concentration of 100 nm in experiments . ethanol had no effects on lncap cell growth at concentrations between 0.00001% and 1% over 96 hours ( data not shown ) . to determine the concentration of labeled shbg and the degree of fluorescent labeling , the conjugate solution was measured at 280 nm and 540 nm in a nanodrop 2000 spectrophotometer ( thermo scientific , nc , usa ) . for alexa546-shbg uptake experiments , the cells were serum - starved for 1 hour and then incubated in 125 nm alexa546-shbg 25 nm te in serum - free rpmi for 1 hour at room temperature cells were washed with phosphate buffered saline ( pbs ) , fixed in 2% paraformaldehyde , and permeabilised in 0.2% triton x-100 in pbs . the colocalisation of alexa546 ( red ) and alexa488 ( green ) discriminated alexa - shbg from endogenous shbg , which was stained with alexa488 only . treatments with serum - free medium only and alexa546 dye only were used as negative controls . shbg secretion from lncap cells was not detectable ( data not shown ) by western blot or unicel immunoassay ( lower limit of detection < 2 nm ) , despite cell culture medium being concentrated 12-fold ; therefore endogenous levels of shbg should have little effect on our experiments using 125 nm shbg . cells were cultured in 6-well plates and serum - starved for 4 hours prior to uptake experiments . cells were then incubated in serum - free uptake medium containing 25 nm ( 30 pmoles / well ) of te 125 nm shbg for 1 , 4 , and 24 hours . te was measured in the original uptake medium ( 25 nm ) , cell culture medium , and cell lysates using tandem mass spectrometry . te measured in all fractions was expressed as a percentage of the te in the original uptake medium . cellular te includes all te measured in both lysates including cytosol , membrane , and nuclei . after protein precipitation , the samples were centrifuged and the plate was transferred to an autosampler . the extract was concentrated onto the masstrak xbridge c18 osm 10 m cartridge , washed with 30% methanol , and then switched into line and eluted directly into the uplc system . the following quantifier and qualifier transitions were utilised : testosterone ( 289.1 > 97.0 , 289.1 > 109.0 ) and testosterone d2 ( 291.1 > 111.0 ) . the gradient was returned to initial conditions in preparation for the next sample , which is being prepared in parallel by the osm . measurement of te - glucuronide formation and efflux into medium was determined by digestion of cell culture medium with e. coli -glucuronidase ( sigma , vic , australia ) , followed by measurement of te by lc msms . enzymatic deconjugation of te - glucuronide was optimised by using two different concentrations of -glucuronidase ( 200 u / ml and 400 u / ml ) , two different dilutions of sample ( culture medium neat and diluted 1 : 10 ) for 24 hours incubation time . te - glucuronide was not detectable in cell lysates ; therefore only effluxed te - glucuronide in cell culture medium was measured . to quantitate the amount of te - glucuronide the quantity of te in each sample was measured by lc msms and expressed as a percentage of te in the original uptake medium . both unconjugated and previously glucuronidated te were measured ; therefore the value for unconjugated te was subtracted from this in order to quantify the glucuronidated fraction . rt - pcr was performed using 3 l of cdna ( represented 6 ng rna ) , 0.5 m of each primer ( table 1 ) , and faststart sybr green pcr master ( roche diagnostics , mannheim , germany ) in a rotor gene rg-6000 ( corbett research , nsw , australia ) . to quantify the gene expression profile in each sample , data were presented as ratio of the amount of targeted mrna ( arbitrary units ) normalised with the house - keeping gene beta 2-microglobulin ( 2 m ) lncap cells ( ~80,000 cells / well ) were cultured on 24-well plates for 48 hours . incucyte zoom software was used to quantify the red objects in 9 nonoverlapping images per well . lncap cells were cultured on 24-well plates with ~35,000 cells per well . after serum starvation the cells were treated with various combinations of te shbg and incubated in an incucyte zoom set to scan every 3 hours for up to 86 hours . firstly , after incubation with alexa546-shbg , the cells were imaged using deltavision deconvolution microscopy ( figure 1(a ) ) . internalisation of alexa546-shbg was confirmed by generating an orthogonal view and a series of z - stack images . the red fluorescent signal representing alex546 labeled shbg was observed in the cells incubated with alexa546-shbg ( figure 1(a)(3 ) ) . there was no red signal observed in the negative controls incubated with unconjugated alexa546 dye ( figure 1(a)(2 ) ) , confirming that alexa-546 shbg and not the alexa dye itself was internalised by the cells . additionally the internalised alexa546-shbg ( red ) colocalised with an alexa488 ( green ) labeled anti - shbg antibody ( figure 1(b ) ) further proving that shbg was internalised while still being conjugated to alexa546 dye . endogenous shbg is stained only with the alexa488 anti - shbg antibody ( green ) . addition of te had no significant effect on alexa - shbg uptake ( figure 1(d ) ) . incubation with 25 nm te for 1 , 4 , and 24 hours resulted in rapid uptake of te followed by a decline in cellular levels ( figure 2(a ) ) . in contrast , in the presence of 125 nm shbg , cellular te levels were lower at 1 hour ( 1.10 0.31% ) and steadily increased to 1.32 0.003% and 2.64 0.04% by 4 and 24 hours . at 24 hours , cellular te levels in cells incubated with te plus figure 2(b ) shows the total te measured in both the cell lysates and cell culture medium . when lncap cells were cultured in te alone , 93 2.80% of te from the original uptake medium was measured at 1 hour but decreased to 55.50 4.36% and then 16.79 0.93% by 4 and 24 hours , respectively . when cells were cultured in te plus shbg , te measurements did not change significantly over the three time points ( 72.69 0.62% , 77.31 0.93% , and 74.23 0.93% at 1 , 4 , and 24 hours , resp . ) cells were exposed to 25 nm te 125 nm shbg for 4 and 24 hours ; then cell culture medium was collected and treated with -glucuronidase . when lncap cells were cultured in te for 4 hours , 50.73 3.51% of te was glucuronidated and effluxed and only 11.12 1.06% of te was unconjugated ( figure 2(c ) ) . however , when shbg was also added , only 4.41 2.1% of te was glucuronidated and 60.84 1.93% of te was unconjugated . following 24 hours in culture with te , lncap cells had glucuronidated and effluxed 67.07 5.48% of te and only 3.8 1.59% remained unconjugated . lncap cells were seeded at initial 20% confluence and after 38 hours of culture cell confluency reached about 3540% with no differences between treatment groups ( figure 3(a)(a ) ) . exposure to only shbg did not alter cell growth . however after 38 hours in culture , growth declined in a dose dependent manner in those cells treated with 1 , 25 , and 100 nm te . addition of either 25 or 125 nm shbg canceled the inhibitory effects of te in a dose dependent fashion ( figures 3(b ) and 3(c ) ) . moreover , cells cultured in te alone displayed morphological changes in that cells became rounded rather than spread out and detached from the culture plate ( figure 3(b)(b ) ) . figure 4(a ) displays a paired comparison of treatment of lncap cells with 0 , 1 , 10 , and 25 nm te alone and with the addition of 25 nm of shbg for 24 hours . surprisingly , addition of shbg , despite having previously been shown to reduce te uptake , significantly enhanced te upregulation of psa mrna expression at te concentrations of 10 and 25 nm ( p < 0.0001 ) ( figure 4(a ) ) . when cells were incubated with 25 nm shbg plus 0 , 1 , 5 , 10 , and 25 nm te ( figure 4(b ) ) , expression of psa was increased by te ( p < 0.0001 ) . when cells were incubated with 10 nm te with 0 , 10 , 25 , or 50 nm shbg , psa mrna expression increased with increasing amounts of shbg to a maximum at 25 nm shbg ( p < 0.001 ) ( figure 4(c ) ) . in the same samples , androgen receptor ( ar ) mrna expression was decreased by te and expression was further decreased by the addition of 25 or 50 nm shbg ( figure 4(d ) ) . shbg mrna expression was significantly decreased by te ( p < 0.0001 ) ( figure 4(e ) ) . additionally , shbg further decreased the te downregulation of shbg expression at all shbg concentrations ; however this did not reach statistical significance . the primary role of hepatically produced shbg as a serum carrier of sex hormones is well documented [ 5 , 15 ] . shbg expression has also been demonstrated in sex hormone target tissues such as human prostate [ 16 , 17 ] , lncap cells , testes , duodenum , ovary , placenta , proximal tubule epithelial cells ( ptec ) , and cerebral cortex as well as several cancers [ 10 , 15 , 1921 ] . higher expression of shbg in prostate cancer tissue is associated with poor clinicopathological features suggesting a role in prostate cancer progression , perhaps due to prolonged expression of androgen responsive genes . using a variety of technical approaches we have confirmed that lncap cells take up shbg in a dose dependent but not androgen dependent manner . it is well known that shbg reduces cellular uptake of te ; however we have demonstrated that shbg may also maintain stable levels of biofunctional te and physiologically relevant intracellular te levels by reducing te glucuronidation and efflux . another study has also demonstrated that lncap cells incubated in 30 nm dht glucuronidated 85% of the dht in 5 days . upon addition of 140 nm shbg , 95% of dht was not taken up by cells and remained in the medium . this rapid inactivation of te by glucuronidation has been demonstrated in human clinical studies , where a single oral dose of te did not alter serum total te levels but rapidly and significantly increased levels of conjugated te in serum . however we acknowledge that the level of free te in healthy men is much lower ( 174729 pmol / l ) than that used in this study , since in vivo shbg is always present . when shbg is reduced or absent , excess free te may enter cells and oversaturate the ars since it has been reported that ar saturation occurs at approximately 2 - 3 nm in prostate . the lncap cells may respond to this by rapidly glucuronidating and effluxing the excess te . the steroid content of shbg ( fitzgerald industries international ) was not specified ; however it was purified from the serum of pregnant women which contains significantly lower te than that of adult males [ 3 , 27 ] . even if all of the serum te were carried over , the te at the maximum shbg concentrations used ( 125 nm ) would be much less than 1 nm , significantly less than that used in our experiments . to further explore the intracellular biological actions of shbg the effects of shbg on te responsive genes were investigated . as expected te upregulated psa mrna expression . however , despite the significantly lower intracellular te in the presence of shbg , addition of shbg in combination with te further increased the expression of psa mrna . similarly , downregulation of ar mrna expression by te was also further enhanced when cells were incubated with shbg and te . addition of shbg also further decreased the te downregulation of shbg mrna although , under these conditions , the reduction did not reach statistical significance . in this experiment the cellular te level was lower in the presence of shbg than when te was added alone ; however the effects on gene expression were much greater . intracellular expression of shbg in mouse proximal tubule cells has also been shown to prolong the expression of androgen responsive genes and overexpression of shbg in lncap cells has been shown to influence the expression of estrogen and androgen responsive genes . this would suggest that both endogenously expressed shbg and internalised shbg have similar positive stimulatory effects on androgen sensitive gene expression . we speculate that , in pathological conditions where serum shbg is reduced , for example , in overweight and obese men [ 3033 ] , te uptake , glucuronidation , and efflux would be increased potentially exacerbating te deficiency . in the present study te treatment did not promote cell growth , and in fact a dose dependent inhibition of growth was demonstrated after 38 hours in culture . coincubation with shbg prevented the te inhibition of cell growth . the inhibitory effect of dht was abrogated by addition of human serum or shbg . the effect of the synthetic androgen methyltrienolone ( r1881 ) , which binds poorly to shbg , was not affected by addition of shbg . other synthetic androgens displayed a similar inhibitory effect in lncap cells resulting in morphological changes and inhibition of colony formation ; however these effects were not reproduced in the androgen receptor - negative prostate cells lines pc-3 , du145 , and mrc-5 . furthermore , addition of antiandrogen drugs ( 6-chloro-6-dehydro-17x - acetoxy - hla , cyproterone acetate , and hydroxyflutamide ) restored lncap cell growth . in the present study the striking changes in cell morphology suggest that the cells may be stressed , perhaps due to the requirement to glucuronidate high levels of intracellular te ; however previous studies have not included descriptions of cell morphology in order to make a comparison . the mechanism by which te inhibits cell growth in lncap cells after 38 hours is unknown . regardless of the percentage of cell confluence at the beginning of treatment , te always caused morphological changes ( data not shown ) . this perhaps suggests that te had an effect on cell morphology and attachment rather than directly on cell proliferation . in this study we have demonstrated that in the absence of shbg large amounts of te rapidly enter the cell where they are inactivated by conjugation to glucuronic acid and effluxed . in the presence of shbg however , while there is reduced te uptake , glucuronidation and efflux of te are also reduced and the effects of te on androgen responsive genes are enhanced . this in vitro study of the role of shbg may have significant clinical relevance , and as such , the assessment of androgen deficiency in vivo should simply rely not only on measurements of total te alone but also on the evaluation of serum shbg levels . these findings clearly demonstrate that shbg plays an important regulatory and intracellular role to modify te metabolism and function and to promote cell growth . if a consistent effect is demonstrated , it will significantly change our current understanding of the role of shbg in health and disease .

a recent systematic review has indicated that the prevalence of pathologic myopia ( pm ) is 1%3% in adults , and that 5%11% of patients with pm develop choroidal neovascularization ( cnv).1 several of the phenotypic features of pm are associated with an increased risk of myopic cnv ; these include lacquer cracks , patchy atrophy , thinning of the choriocapillaris and choroid,2,3 and cnv in the fellow eye.4 furthermore , in 2003 , ohno - matsui et al showed that after the initial presentation of myopic cnv , cnv develops in the fellow eye in 35% of patients within 8 years.2 because of the poor natural history of myopic cnv , different treatment approaches have been investigated . thermal laser photocoagulation , transpupillary thermotherapy ( ttt ) , and photodynamic therapy ( pdt ) with verteporfin have shown some benefits , but long - term efficacy and safety outcomes are disappointing.58 anti - vascular endothelial growth factor ( anti - vegf ) agents such as bevacizumab , ranibizumab , and aflibercept are widely used for the treatment of many retinal diseases . bevacizumab is a recombinant humanized monoclonal antibody that binds all isoforms of vegf - a,9 while ranibizumab is an affinity - matured antigen - binding fragment derived from bevacizumab.9 aflibercept is a soluble decoy receptor fusion protein that binds vegf - a , vegf - b , and placental growth factor.9,10 randomized clinical trials have demonstrated the efficacy of ranibizumab and aflibercept in wet age - related macular degeneration,1113 macular edema secondary to central retinal vein occlusion,1417 and diabetic macular edema.18,19 intravitreal ranibizumab and aflibercept are generally well tolerated in patients of different ages with different retinal diseases , and these drugs have similar safety profiles.1119 based on data from a prospective open - label phase 2 trial20,21 as well as the randomized controlled phase 3 radiance trial,22 ranibizumab was recently approved in the european union and japan for the treatment of visual impairment due to cnv secondary to pm . myrror , a phase 3 study , evaluates the efficacy and safety of intravitreal aflibercept in eyes with cnv due to pm . interim study results were presented in 2013 by ohno - matsui et al , and showed greater visual and anatomic improvements in eyes treated with intravitreal aflibercept compared with sham.23 the purpose of the current pilot study was to investigate the efficacy of intravitreal injection of aflibercept in patients with cnv associated with pm . consecutive patients who presented to the laser department of the state institution the filatov institute of eye diseases and tissue therapy of the nams of ukraine with signs and symptoms consistent with cnv associated with pm were examined and screened for entering the study . inclusion criteria were high myopia ( defined as myopia of 6.00 d and/or axial length of 26.5 mm ) , new onset of cnv associated with pm < 2 months , age 18 years , myopic retinopathy ( one or more of the following signs : staphyloma , lacquer cracks , fuchs spot , and myopic chorioretinal atrophy ) , and absence of inflammation at screening . ocular exclusion criteria included any other cause of cnv , intraocular or periocular inflammation , known glaucoma or clinical suspicion of glaucoma on presentation , ocular hypertension ( intraocular pressure [ iop ] > 24 mmhg ) , or fundus observation complicated by opacity of optic media . eligible subjects were given a detailed explanation concerning their disease and the proposed study and treatment . they were then provided the opportunity to consider study participation , and only then , their signed and informed consent was obtained . this study was approved by the state institution the filatov institute of eye diseases and tissue therapy of the nams of ukraine research ethics board before it was commenced . all procedures were performed in accordance with the 1964 declaration of helsinki and good clinical practice guidelines . two obligatory loading injections of intravitreal aflibercept were administered at baseline and at month 1 . retreatment criteria included persistence of cnv or macular edema or recurrence of cnv or macular edema ( increase in retinal thickness in the foveal area > 50 m ) , sub- or intraretinal fluid and/or intraretinal cysts on optical coherence tomography ( oct ) , or subretinal hemorrhage observed during dilated fundus examination . all enrolled patients underwent baseline ophthalmologic examination and investigations including measurements of best - corrected visual acuity ( bcva ) using the shevalev chart ( a soviet analog to the early treatment of diabetic retinopathy study chart ) ; iop measurements ; pupillary , biomicroscopic , and dilated fundus examination ; as well as spectral domain oct ( spectralis ; heidelberg engineering , heidelberg , germany ) of the macular area , color fundus photography , and fluorescein angiography ( fa ) ( trc-50ex ; topcon , tokyo , japan ) . the area of hyperfluorescence was measured in the early phase of fa images using multispec computer software . hyperfluorescence before and after treatment was compared and was described as cnv closure ( absence of hyperfluorescence ) or cnv persistence ( presence of hyperfluorescence ) . staining from fibrosis was differentiated from cnv persistence by late hyperfluorescence resulting from the accumulation of fluorescein dye into the subretinal scar . follow - up examinations involved ophthalmologic examination including bcva measurements , iop measurements , and dilated fundus examination . oct and color fundus photography fa was performed in the case of decrease in bcva and/or increase of macular edema or suspicion of cnv recurrence . in addition to all study visits , a telephone safety check was performed 1 day after each intervention to monitor for complications related to the intraocular injection . specifically , the following adverse events were ruled out : elevated iop , endophthalmitis , retinal tear , retinal detachment , vitreous hemorrhage , uveitis , and corneal abrasion . the secondary outcomes included change in central retinal thickness ( crt ) in the foveal area on oct from baseline to month 12 , neovascularization activity on fa , the number of aflibercept injections administered , and safety . using a last observation carried forward strategy for the replacement of missing data , baseline and final outcome measurements for the primary and secondary outcome variables were compared utilizing a repeated measures analysis of variance design with one between - subjects factor . any statistically significant baseline differences between the groups were entered as covariates in the model . baseline characteristics were compared with student s t - test , , or fisher s exact test , as appropriate . consecutive patients who presented to the laser department of the state institution the filatov institute of eye diseases and tissue therapy of the nams of ukraine with signs and symptoms consistent with cnv associated with pm were examined and screened for entering the study . inclusion criteria were high myopia ( defined as myopia of 6.00 d and/or axial length of 26.5 mm ) , new onset of cnv associated with pm < 2 months , age 18 years , myopic retinopathy ( one or more of the following signs : staphyloma , lacquer cracks , fuchs spot , and myopic chorioretinal atrophy ) , and absence of inflammation at screening . ocular exclusion criteria included any other cause of cnv , intraocular or periocular inflammation , known glaucoma or clinical suspicion of glaucoma on presentation , ocular hypertension ( intraocular pressure [ iop ] > 24 mmhg ) , or fundus observation complicated by opacity of optic media . eligible subjects were given a detailed explanation concerning their disease and the proposed study and treatment . they were then provided the opportunity to consider study participation , and only then , their signed and informed consent was obtained . this study was approved by the state institution the filatov institute of eye diseases and tissue therapy of the nams of ukraine research ethics board before it was commenced . all procedures were performed in accordance with the 1964 declaration of helsinki and good clinical practice guidelines . two obligatory loading injections of intravitreal aflibercept were administered at baseline and at month 1 . retreatment criteria included persistence of cnv or macular edema or recurrence of cnv or macular edema ( increase in retinal thickness in the foveal area > 50 m ) , sub- or intraretinal fluid and/or intraretinal cysts on optical coherence tomography ( oct ) , or subretinal hemorrhage observed during dilated fundus examination . all enrolled patients underwent baseline ophthalmologic examination and investigations including measurements of best - corrected visual acuity ( bcva ) using the shevalev chart ( a soviet analog to the early treatment of diabetic retinopathy study chart ) ; iop measurements ; pupillary , biomicroscopic , and dilated fundus examination ; as well as spectral domain oct ( spectralis ; heidelberg engineering , heidelberg , germany ) of the macular area , color fundus photography , and fluorescein angiography ( fa ) ( trc-50ex ; topcon , tokyo , japan ) . the area of hyperfluorescence was measured in the early phase of fa images using multispec computer software . hyperfluorescence before and after treatment was compared and was described as cnv closure ( absence of hyperfluorescence ) or cnv persistence ( presence of hyperfluorescence ) . staining from fibrosis was differentiated from cnv persistence by late hyperfluorescence resulting from the accumulation of fluorescein dye into the subretinal scar . follow - up examinations involved ophthalmologic examination including bcva measurements , iop measurements , and dilated fundus examination . oct and color fundus photography fa was performed in the case of decrease in bcva and/or increase of macular edema or suspicion of cnv recurrence . in addition to all study visits , a telephone safety check was performed 1 day after each intervention to monitor for complications related to the intraocular injection . specifically , the following adverse events were ruled out : elevated iop , endophthalmitis , retinal tear , retinal detachment , vitreous hemorrhage , uveitis , and corneal abrasion . the secondary outcomes included change in central retinal thickness ( crt ) in the foveal area on oct from baseline to month 12 , neovascularization activity on fa , the number of aflibercept injections administered , and safety . using a last observation carried forward strategy for the replacement of missing data , baseline and final outcome measurements for the primary and secondary outcome variables were compared utilizing a repeated measures analysis of variance design with one between - subjects factor . any statistically significant baseline differences between the groups were entered as covariates in the model . baseline characteristics were compared with student s t - test , , or fisher s exact test , as appropriate . the mean ( standard deviation [ sd ] ) age of patients was 43.0 ( 12.4 ) years , and 25 ( 83.0% ) were female . seven ( 23% ) patients had a past medical history of arterial hypertension and two ( 6.7% ) were smokers . all patients had a high myopia with myopic retinopathy ( staphyloma , lacquer cracks , fuchs spot , and myopic chorioretinal atrophy ) . a total of seven ( 23% ) cases of cnv were juxtafoveal and 24 ( 77% ) were subfoveal . mean ( sd ) baseline retinal thickness in the foveal area was 285 ( 61.6 ) m . mean bcva improved from 0.2 ( 0.1 ) at baseline to 0.35 ( 0.16 ) at month 12 ( figure 1 ) . the greatest improvement in bcva was seen within the first 2 months ( p=0.01 ) . , an improvement in bcva of 3 lines was seen in five ( 16% ) eyes . fourteen ( 45% ) eyes improved by 2 but <3 lines , and eight ( 26% ) eyes improved by 1 but < 2 lines . mean ( sd ) crt on oct showed a reduction from 285.0 ( 62 ) m at baseline to 227.0 ( 42 ) m ( p=0.01 ) at month 12 ( figure 2 ) . there was a continuous decrease in mean crt on oct over time ( table 1 ) . mean ( sd ) area of hyperfluorescence decreased from 0.41 ( 0.38 ) mm at baseline to 0.32 ( 0.22 ) mm at 12 months ( p=0.04 ) . the mean ( sd ) number of injections was 2.2 ( 0.5 ) and 2.6 ( 0.9 ) at months 3 and 12 , respectively . a total of 18 patients ( 18 eyes ) received only two loading injections and did not require additional injections . the maximum number of injections ( 5.0 ) was administered to three patients ( three eyes ) . three patients ( three eyes ) showed recurrence of cnv , which was successfully treated with additional injections . figure 3 provides representative examples of anatomic improvements following intravitreal aflibercept injections . as seen in the fundus photographs ( figure 3a and b ) , this patient experienced regression in cnv lesion size , reduction of angiographic leakage on fa ( figure 3c and d ) , and decrease in crt ( figure 3e and f ) after two intravitreal injections . side effects observed after intravitreal injections included eye pain ( 8/81 injections , 10% ) , subconjunctival hemorrhage ( 6/81 injections , 7% ) , and vitreous reflux ( 14/81 injections , 17% ) . no cases of endophthalmitis or of uveitis , stroke , or retinal detachment were noted . mean bcva improved from 0.2 ( 0.1 ) at baseline to 0.35 ( 0.16 ) at month 12 ( figure 1 ) . the greatest improvement in bcva was seen within the first 2 months ( p=0.01 ) . , an improvement in bcva of 3 lines was seen in five ( 16% ) eyes . fourteen ( 45% ) eyes improved by 2 but <3 lines , and eight ( 26% ) eyes improved by 1 but < 2 lines . mean ( sd ) crt on oct showed a reduction from 285.0 ( 62 ) m at baseline to 227.0 ( 42 ) m ( p=0.01 ) at month 12 ( figure 2 ) . there was a continuous decrease in mean crt on oct over time ( table 1 ) . mean ( sd ) area of hyperfluorescence decreased from 0.41 ( 0.38 ) mm at baseline to 0.32 ( 0.22 ) mm at 12 months ( p=0.04 ) . the mean ( sd ) number of injections was 2.2 ( 0.5 ) and 2.6 ( 0.9 ) at months 3 and 12 , respectively . a total of 18 patients ( 18 eyes ) received only two loading injections and did not require additional injections . the maximum number of injections ( 5.0 ) was administered to three patients ( three eyes ) . three patients ( three eyes ) showed recurrence of cnv , which was successfully treated with additional injections . figure 3 provides representative examples of anatomic improvements following intravitreal aflibercept injections . as seen in the fundus photographs ( figure 3a and b ) , this patient experienced regression in cnv lesion size , reduction of angiographic leakage on fa ( figure 3c and d ) , and decrease in crt ( figure 3e and f ) after two intravitreal injections . side effects observed after intravitreal injections included eye pain ( 8/81 injections , 10% ) , subconjunctival hemorrhage ( 6/81 injections , 7% ) , and vitreous reflux ( 14/81 injections , 17% ) . no cases of endophthalmitis or of uveitis , stroke , or retinal detachment were noted . before the introduction of anti - vegf therapy , laser photocoagulation , ttt , pdt with verteporfin , and different surgical techniques were used for the treatment of myopic cnv ; however , these treatment approaches were typically unsatisfactory in that they failed to improve visual acuity to a clinically relevant extent in the mid to long term . laser photocoagulation of well - defined subfoveal cnv secondary to age - related macular degeneration has been described as successful in stopping cnv activity , but the ensuing damage to the overlying neurosensory retina compromised visual acuity.24,25 in myopic patients , laser photocoagulation of extrafoveal cnv has poor long - term results because of atrophic progression even in eyes free of cnv recurrence.8,26,27 in a comparison of laser photocoagulation with the natural history of myopic cnv , laser - treated eyes had statistically better bcva at 2 years , but this difference was insignificant after 5 years.24,26 in a study of eyes with extrafoveal cnv secondary to pm and treated with laser photocoagulation , jalkh et al27 found that bcva improved in only 11% of eyes , remained unchanged in 21% , and worsened in 68% ( average posttreatment follow - up period 29.2 months ) . although ttt seems to stabilize myopic cnv both clinically and angiographically , with a significant decrease in the activity of lesions , this technique did not result in any significant change in bcva over a 1-year observation period.7 furthermore , adequate energy levels have not been clearly defined . pdt with verteporfin was advocated as a safe and well - tolerated approach for patients with myopic cnv . report no 3 of the verteporfin in photodynamic therapy study group is the largest study to analyze the efficacy and safety of pdt as the treatment for subfoveal myopic cnv . at the month 12 examination , 72% of the verteporfin - treated patients lost fewer than eight letters in bcva , compared with 44% of the placebo - treated patients . in addition , 32% of the verteporfin - treated patients versus 15% of the placebo - treated patients improved by at least five letters . finally , 86% of the verteporfin - treated patients lost fewer than 15 letters , compared with 67% of the placebo - treated patients . results at 2 years were less promising and demonstrated no statistical significance between the eyes treated with pdt and the control groups.6,28 since the advent of intravitreal anti - vegf agents , it has been possible not only to preserve but also to improve visual acuity in patients suffering from various types of cnv , including myopic cnv . many case series on the off - label use of bevacizumab suggest visual improvement with a limited number of injections and an acceptable safety profile . however , there is no highest level of evidence from clinical trials and no regulatory approval for bevacizumab in this indication . in an open - label study of 17 patients , arias et al29 found that at the 6-month follow - up , after receiving an average of 1.5 intravitreal bevacizumab injections , mean visual acuity improved by 8.4 letters . a total of 41% of patients gained at least 1 line of visual acuity , and 17% gained at least 6 lines . laud et al30 found a mean 1.5 lines improvement in eyes with myopic cnv treated with intravitreal bevacizumab , with a mean follow - up of 7.3 months . in their 24-month study , baba et al5 showed that intravitreal bevacizumab is more effective than pdt in eyes with myopic cnv . in a case series of 63 eyes with cnv secondary to pm , ikuno et al31 reported improvements in mean bcva from 0.570.43 logarithm to the minimal angle of resolution ( logmar ) ( 0.27 decimal equivalent ) to 0.330.34 logmar ( 0.47 decimal equivalent ) with about one to six bevacizumab injections during the 12 months of observation ( mean 2.4 injections ) . after 12 months , improvement in bcva of more than 3 lines was noted in 25 ( 40% ) eyes , worsening of more than 3 lines was observed in three ( 5% ) eyes , and vision was unchanged in 35 ( 56% ) eyes . fluorescein leakage from the cnv ceased in 30 ( 48% ) eyes , diminished in 28 ( 44% ) eyes , and was unchanged in five ( 8% ) eyes . ranibizumab has recently received approval in the european union for the treatment of myopic cnv . this decision was made based on results from the radiance sham - controlled , double - masked randomized clinical trial , which established the superiority of intravitreal ranibizumab over pdt.22 radiance and the open - label repair study20,21 showed that around 40% of patients treated with ranibizumab gained 15 or more letters of visual acuity at 3 months , compared with 15% of pdt - treated patients . mean visual acuity gain was approximately 14 letters at 1 year after a median of 2.0 ranibizumab injections.2022 a number of small prospective and retrospective studies have reported favorable results with intravitreal ranibizumab for the treatment of myopic cnv . for example , in a series of 16 consecutive patients with myopic cnv , lai et al32 demonstrated a mean improvement in bcva of 3 lines at 12 months , with 12 ( 75.0% ) eyes experiencing an improvement of 2 or more lines . a total of 15 ( 93.8% ) eyes had angiographic cnv closure at 3 months , while two ( 12.5% ) eyes had recurrence of cnv and required retreatment between 3 and 9 months . oct showed a significant reduction in the mean central foveal thickness after treatment with ranibizumab . in another case series , silva et al33 found that at the 12-month follow - up , the mean visual acuity improved from 20/100 to 20/63 after a mean of 3.6 intravitreal ranibizumab injections . no cases of severe visual acuity loss occurred , and no systemic or ocular side effects were registered during the follow - up . however , of the 32 patients with cnv secondary to pm at baseline , only nine patients completed 6 months of follow - up . the results of the myrror study of intravitreal afliber - cept in myopic cnv were published in 2015 . the authors reported a mean change of + 13.5 letters in bcva in aflibercept - treated patients compared with + 3.9 letters in patients receiving sham treatment ( and receiving aflibercept injections at week 24 ) and maintenance of gains in visual acuity through week 48 . a direct comparison of bcva results between the current study and myrror is not possible given the different ways in which vision was measured ( decimal vs early treatment diabetic research study chart letters ) ; however , both studies demonstrated significant improvements in bcva in eyes treated with aflibercept . the myrror study showed significant decreases from 349.7 m at baseline to 263.5 m at week 48 . in our study , crt decreased significantly from 285 m at baseline to 227 m at month 12 . special mention should be made of the schedule of injections . in our study , patients received two mandatory loading injections ( at baseline and week 4 ) followed by prn retreatment . patients received a mean of 2.6 injections overall , 2.0 injections in the first 4 weeks , and 0.6 injection during the remainder of the study ( 48 weeks ) . in the myrror study the median number of injections was 2.0 in the first study quarter and 0 in the remaining quarters . in most studies of ranibizumab , the mean baseline bcva was similar ( 0.20.3 in decimal equivalent)22,3133 and in all studies , improvement in mean bcva of 23 lines was noted . however , in studies with 12 months of follow - up , there was a difference in the mean number of injections ( from one to four).22,3133 the results from the literature regarding ranibizumab and bevacizumab treatment are in accordance with the findings from the current study , which demonstrated that intravitreal aflibercept is a safe and efficacious treatment option in myopic cnv . mean bcva significantly improved by almost 3 lines in logmar equivalent in conjunction with decreasing crt . patients maintained visual and anatomic improvements after 12 months with an average of 2.6 intravitreal aflibercept injections . the number of injections needed to achieve visual and anatomic improvements is also consistent with the literature about other anti - vegf drugs in patients with myopic cnv . the myrror study group reported that serious adverse events occurred in seven patients in the intravitreal aflibercept treatment group . of these , only one ocular serious adverse event ( macular hole ) was reported . the most frequently reported adverse events were eye pain ( 7.7% ) and conjunctival hemorrhage ( 11.0%).23 in the current study , there were no serious adverse events . the rates of eye pain and subconjunctival hemorrhage were similar compared with myrror ( 10% and 7% , respectively ) . the higher incidence of serious adverse events in the myrror study may be related to the larger sample size ( four times as many patients as in our study ) . most serious adverse events are rare and may not occur in a smaller treatment group such as ours . clinical trials of ranibizumab for myopic cnv ( radiance and repair ) also demonstrated low rates of serious adverse events and ocular adverse events.21,22 in the repair study , one case of culture - negative endophthalmitis considered by investigators to be related to the injection was reported.21 the strength of this study is its ability to show the significant visual and anatomical improvements that can be achieved in patients with myopic cnv treated with intravitreal aflibercept in routine clinical practice . the inability to directly compare results with the myrror study due to differences in follow - up period and bcva measurement is a limitation . the 12-month results of the current study of intravitreal aflibercept for myopic cnv using a prn regimen were positive . a total of 27 ( 87% ) eyes experienced improvement of 1 line in bcva , with a gain of 3 lines in five ( 16% ) eyes . intravitreal aflibercept retreatment guided by disease activity criteria based on oct and bcva was able to provide visual acuity benefits without requiring fa . overall , intravitreal aflibercept was well tolerated in patients with myopic cnv over 12 months in our study .

purposeto determine the efficacy of intravitreal aflibercept injections for the treatment of patients with choroidal neovascularization ( cnv ) associated with pathologic myopia.methodsin this uncontrolled , prospective cohort study , 31 eyes of 30 consecutive patients affected by cnv associated with pathologic myopia were treated with intravitreal aflibercept ( 2 mg ) as needed following two initial monthly doses and observed over a 12-month follow - up period . the primary endpoint was change in best - corrected visual acuity ( bcva ) at month 12 , while central retinal thickness ( crt ) on optical coherence tomography ( oct ) , neovascularization activity on fluorescein angiography , the number of aflibercept injections administered , and safety were examined as secondary endpoints.resultspatients received a mean of 2.6 intravitreal aflibercept injections over the 12-month study period . compared with baseline , bcva improved significantly at all time points ( p<0.05 ) . mean ( standard deviation [ sd ] ) decimal bcva was 0.2 ( 0.1 ) at baseline and 0.35 ( 0.16 ) at month 12 . the greatest improvement in bcva was seen within the first 2 months ( p=0.01 ) . mean ( sd ) crt on oct decreased from 285 ( 62 ) m at baseline to 227 ( 42 ) m ( p=0.01 ) at month 12 . there was a continuous decrease in mean crt on oct over time . no cases of endophthalmitis , uveitis , stroke , or retinal detachment were noted . no patient demonstrated an intraocular pressure > 20 mmhg during any study visit.conclusionthe 12-month results of intravitreal aflibercept for myopic cnv using an as - needed regimen were positive , showing benefits in visual and anatomic outcomes and an acceptable tolerability profile .

Introduction Patients and methods Protocol Intervention Study visits Outcome measures Analysis Results Primary and secondary outcomes Complications Discussion Strength and limitations Conclusion

a recent systematic review has indicated that the prevalence of pathologic myopia ( pm ) is 1%3% in adults , and that 5%11% of patients with pm develop choroidal neovascularization ( cnv).1 several of the phenotypic features of pm are associated with an increased risk of myopic cnv ; these include lacquer cracks , patchy atrophy , thinning of the choriocapillaris and choroid,2,3 and cnv in the fellow eye.4 furthermore , in 2003 , ohno - matsui et al showed that after the initial presentation of myopic cnv , cnv develops in the fellow eye in 35% of patients within 8 years.2 because of the poor natural history of myopic cnv , different treatment approaches have been investigated . thermal laser photocoagulation , transpupillary thermotherapy ( ttt ) , and photodynamic therapy ( pdt ) with verteporfin have shown some benefits , but long - term efficacy and safety outcomes are disappointing.58 anti - vascular endothelial growth factor ( anti - vegf ) agents such as bevacizumab , ranibizumab , and aflibercept are widely used for the treatment of many retinal diseases . bevacizumab is a recombinant humanized monoclonal antibody that binds all isoforms of vegf - a,9 while ranibizumab is an affinity - matured antigen - binding fragment derived from bevacizumab.9 aflibercept is a soluble decoy receptor fusion protein that binds vegf - a , vegf - b , and placental growth factor.9,10 randomized clinical trials have demonstrated the efficacy of ranibizumab and aflibercept in wet age - related macular degeneration,1113 macular edema secondary to central retinal vein occlusion,1417 and diabetic macular edema.18,19 intravitreal ranibizumab and aflibercept are generally well tolerated in patients of different ages with different retinal diseases , and these drugs have similar safety profiles.1119 based on data from a prospective open - label phase 2 trial20,21 as well as the randomized controlled phase 3 radiance trial,22 ranibizumab was recently approved in the european union and japan for the treatment of visual impairment due to cnv secondary to pm . myrror , a phase 3 study , evaluates the efficacy and safety of intravitreal aflibercept in eyes with cnv due to pm . interim study results were presented in 2013 by ohno - matsui et al , and showed greater visual and anatomic improvements in eyes treated with intravitreal aflibercept compared with sham.23 the purpose of the current pilot study was to investigate the efficacy of intravitreal injection of aflibercept in patients with cnv associated with pm . consecutive patients who presented to the laser department of the state institution the filatov institute of eye diseases and tissue therapy of the nams of ukraine with signs and symptoms consistent with cnv associated with pm were examined and screened for entering the study . inclusion criteria were high myopia ( defined as myopia of 6.00 d and/or axial length of 26.5 mm ) , new onset of cnv associated with pm < 2 months , age 18 years , myopic retinopathy ( one or more of the following signs : staphyloma , lacquer cracks , fuchs spot , and myopic chorioretinal atrophy ) , and absence of inflammation at screening . ocular exclusion criteria included any other cause of cnv , intraocular or periocular inflammation , known glaucoma or clinical suspicion of glaucoma on presentation , ocular hypertension ( intraocular pressure [ iop ] > 24 mmhg ) , or fundus observation complicated by opacity of optic media . two obligatory loading injections of intravitreal aflibercept were administered at baseline and at month 1 . retreatment criteria included persistence of cnv or macular edema or recurrence of cnv or macular edema ( increase in retinal thickness in the foveal area > 50 m ) , sub- or intraretinal fluid and/or intraretinal cysts on optical coherence tomography ( oct ) , or subretinal hemorrhage observed during dilated fundus examination . all enrolled patients underwent baseline ophthalmologic examination and investigations including measurements of best - corrected visual acuity ( bcva ) using the shevalev chart ( a soviet analog to the early treatment of diabetic retinopathy study chart ) ; iop measurements ; pupillary , biomicroscopic , and dilated fundus examination ; as well as spectral domain oct ( spectralis ; heidelberg engineering , heidelberg , germany ) of the macular area , color fundus photography , and fluorescein angiography ( fa ) ( trc-50ex ; topcon , tokyo , japan ) . follow - up examinations involved ophthalmologic examination including bcva measurements , iop measurements , and dilated fundus examination . specifically , the following adverse events were ruled out : elevated iop , endophthalmitis , retinal tear , retinal detachment , vitreous hemorrhage , uveitis , and corneal abrasion . the secondary outcomes included change in central retinal thickness ( crt ) in the foveal area on oct from baseline to month 12 , neovascularization activity on fa , the number of aflibercept injections administered , and safety . using a last observation carried forward strategy for the replacement of missing data , baseline and final outcome measurements for the primary and secondary outcome variables were compared utilizing a repeated measures analysis of variance design with one between - subjects factor . consecutive patients who presented to the laser department of the state institution the filatov institute of eye diseases and tissue therapy of the nams of ukraine with signs and symptoms consistent with cnv associated with pm were examined and screened for entering the study . inclusion criteria were high myopia ( defined as myopia of 6.00 d and/or axial length of 26.5 mm ) , new onset of cnv associated with pm < 2 months , age 18 years , myopic retinopathy ( one or more of the following signs : staphyloma , lacquer cracks , fuchs spot , and myopic chorioretinal atrophy ) , and absence of inflammation at screening . ocular exclusion criteria included any other cause of cnv , intraocular or periocular inflammation , known glaucoma or clinical suspicion of glaucoma on presentation , ocular hypertension ( intraocular pressure [ iop ] > 24 mmhg ) , or fundus observation complicated by opacity of optic media . two obligatory loading injections of intravitreal aflibercept were administered at baseline and at month 1 . retreatment criteria included persistence of cnv or macular edema or recurrence of cnv or macular edema ( increase in retinal thickness in the foveal area > 50 m ) , sub- or intraretinal fluid and/or intraretinal cysts on optical coherence tomography ( oct ) , or subretinal hemorrhage observed during dilated fundus examination . all enrolled patients underwent baseline ophthalmologic examination and investigations including measurements of best - corrected visual acuity ( bcva ) using the shevalev chart ( a soviet analog to the early treatment of diabetic retinopathy study chart ) ; iop measurements ; pupillary , biomicroscopic , and dilated fundus examination ; as well as spectral domain oct ( spectralis ; heidelberg engineering , heidelberg , germany ) of the macular area , color fundus photography , and fluorescein angiography ( fa ) ( trc-50ex ; topcon , tokyo , japan ) . follow - up examinations involved ophthalmologic examination including bcva measurements , iop measurements , and dilated fundus examination . specifically , the following adverse events were ruled out : elevated iop , endophthalmitis , retinal tear , retinal detachment , vitreous hemorrhage , uveitis , and corneal abrasion . the secondary outcomes included change in central retinal thickness ( crt ) in the foveal area on oct from baseline to month 12 , neovascularization activity on fa , the number of aflibercept injections administered , and safety . using a last observation carried forward strategy for the replacement of missing data , baseline and final outcome measurements for the primary and secondary outcome variables were compared utilizing a repeated measures analysis of variance design with one between - subjects factor . the mean ( standard deviation [ sd ] ) age of patients was 43.0 ( 12.4 ) years , and 25 ( 83.0% ) were female . mean ( sd ) baseline retinal thickness in the foveal area was 285 ( 61.6 ) m . mean bcva improved from 0.2 ( 0.1 ) at baseline to 0.35 ( 0.16 ) at month 12 ( figure 1 ) . the greatest improvement in bcva was seen within the first 2 months ( p=0.01 ) . , an improvement in bcva of 3 lines was seen in five ( 16% ) eyes . mean ( sd ) crt on oct showed a reduction from 285.0 ( 62 ) m at baseline to 227.0 ( 42 ) m ( p=0.01 ) at month 12 ( figure 2 ) . there was a continuous decrease in mean crt on oct over time ( table 1 ) . mean ( sd ) area of hyperfluorescence decreased from 0.41 ( 0.38 ) mm at baseline to 0.32 ( 0.22 ) mm at 12 months ( p=0.04 ) . the mean ( sd ) number of injections was 2.2 ( 0.5 ) and 2.6 ( 0.9 ) at months 3 and 12 , respectively . as seen in the fundus photographs ( figure 3a and b ) , this patient experienced regression in cnv lesion size , reduction of angiographic leakage on fa ( figure 3c and d ) , and decrease in crt ( figure 3e and f ) after two intravitreal injections . no cases of endophthalmitis or of uveitis , stroke , or retinal detachment were noted . mean bcva improved from 0.2 ( 0.1 ) at baseline to 0.35 ( 0.16 ) at month 12 ( figure 1 ) . the greatest improvement in bcva was seen within the first 2 months ( p=0.01 ) . , an improvement in bcva of 3 lines was seen in five ( 16% ) eyes . mean ( sd ) crt on oct showed a reduction from 285.0 ( 62 ) m at baseline to 227.0 ( 42 ) m ( p=0.01 ) at month 12 ( figure 2 ) . there was a continuous decrease in mean crt on oct over time ( table 1 ) . mean ( sd ) area of hyperfluorescence decreased from 0.41 ( 0.38 ) mm at baseline to 0.32 ( 0.22 ) mm at 12 months ( p=0.04 ) . the mean ( sd ) number of injections was 2.2 ( 0.5 ) and 2.6 ( 0.9 ) at months 3 and 12 , respectively . as seen in the fundus photographs ( figure 3a and b ) , this patient experienced regression in cnv lesion size , reduction of angiographic leakage on fa ( figure 3c and d ) , and decrease in crt ( figure 3e and f ) after two intravitreal injections . no cases of endophthalmitis or of uveitis , stroke , or retinal detachment were noted . before the introduction of anti - vegf therapy , laser photocoagulation , ttt , pdt with verteporfin , and different surgical techniques were used for the treatment of myopic cnv ; however , these treatment approaches were typically unsatisfactory in that they failed to improve visual acuity to a clinically relevant extent in the mid to long term . laser photocoagulation of well - defined subfoveal cnv secondary to age - related macular degeneration has been described as successful in stopping cnv activity , but the ensuing damage to the overlying neurosensory retina compromised visual acuity.24,25 in myopic patients , laser photocoagulation of extrafoveal cnv has poor long - term results because of atrophic progression even in eyes free of cnv recurrence.8,26,27 in a comparison of laser photocoagulation with the natural history of myopic cnv , laser - treated eyes had statistically better bcva at 2 years , but this difference was insignificant after 5 years.24,26 in a study of eyes with extrafoveal cnv secondary to pm and treated with laser photocoagulation , jalkh et al27 found that bcva improved in only 11% of eyes , remained unchanged in 21% , and worsened in 68% ( average posttreatment follow - up period 29.2 months ) . although ttt seems to stabilize myopic cnv both clinically and angiographically , with a significant decrease in the activity of lesions , this technique did not result in any significant change in bcva over a 1-year observation period.7 furthermore , adequate energy levels have not been clearly defined . report no 3 of the verteporfin in photodynamic therapy study group is the largest study to analyze the efficacy and safety of pdt as the treatment for subfoveal myopic cnv . at the month 12 examination , 72% of the verteporfin - treated patients lost fewer than eight letters in bcva , compared with 44% of the placebo - treated patients . results at 2 years were less promising and demonstrated no statistical significance between the eyes treated with pdt and the control groups.6,28 since the advent of intravitreal anti - vegf agents , it has been possible not only to preserve but also to improve visual acuity in patients suffering from various types of cnv , including myopic cnv . many case series on the off - label use of bevacizumab suggest visual improvement with a limited number of injections and an acceptable safety profile . in an open - label study of 17 patients , arias et al29 found that at the 6-month follow - up , after receiving an average of 1.5 intravitreal bevacizumab injections , mean visual acuity improved by 8.4 letters . a total of 41% of patients gained at least 1 line of visual acuity , and 17% gained at least 6 lines . laud et al30 found a mean 1.5 lines improvement in eyes with myopic cnv treated with intravitreal bevacizumab , with a mean follow - up of 7.3 months . after 12 months , improvement in bcva of more than 3 lines was noted in 25 ( 40% ) eyes , worsening of more than 3 lines was observed in three ( 5% ) eyes , and vision was unchanged in 35 ( 56% ) eyes . ranibizumab has recently received approval in the european union for the treatment of myopic cnv . this decision was made based on results from the radiance sham - controlled , double - masked randomized clinical trial , which established the superiority of intravitreal ranibizumab over pdt.22 radiance and the open - label repair study20,21 showed that around 40% of patients treated with ranibizumab gained 15 or more letters of visual acuity at 3 months , compared with 15% of pdt - treated patients . mean visual acuity gain was approximately 14 letters at 1 year after a median of 2.0 ranibizumab injections.2022 a number of small prospective and retrospective studies have reported favorable results with intravitreal ranibizumab for the treatment of myopic cnv . for example , in a series of 16 consecutive patients with myopic cnv , lai et al32 demonstrated a mean improvement in bcva of 3 lines at 12 months , with 12 ( 75.0% ) eyes experiencing an improvement of 2 or more lines . in another case series , silva et al33 found that at the 12-month follow - up , the mean visual acuity improved from 20/100 to 20/63 after a mean of 3.6 intravitreal ranibizumab injections . no cases of severe visual acuity loss occurred , and no systemic or ocular side effects were registered during the follow - up . however , of the 32 patients with cnv secondary to pm at baseline , only nine patients completed 6 months of follow - up . the authors reported a mean change of + 13.5 letters in bcva in aflibercept - treated patients compared with + 3.9 letters in patients receiving sham treatment ( and receiving aflibercept injections at week 24 ) and maintenance of gains in visual acuity through week 48 . the myrror study showed significant decreases from 349.7 m at baseline to 263.5 m at week 48 . in our study , crt decreased significantly from 285 m at baseline to 227 m at month 12 . in our study , patients received two mandatory loading injections ( at baseline and week 4 ) followed by prn retreatment . patients received a mean of 2.6 injections overall , 2.0 injections in the first 4 weeks , and 0.6 injection during the remainder of the study ( 48 weeks ) . in the myrror study the median number of injections was 2.0 in the first study quarter and 0 in the remaining quarters . in most studies of ranibizumab , the mean baseline bcva was similar ( 0.20.3 in decimal equivalent)22,3133 and in all studies , improvement in mean bcva of 23 lines was noted . however , in studies with 12 months of follow - up , there was a difference in the mean number of injections ( from one to four).22,3133 the results from the literature regarding ranibizumab and bevacizumab treatment are in accordance with the findings from the current study , which demonstrated that intravitreal aflibercept is a safe and efficacious treatment option in myopic cnv . patients maintained visual and anatomic improvements after 12 months with an average of 2.6 intravitreal aflibercept injections . the number of injections needed to achieve visual and anatomic improvements is also consistent with the literature about other anti - vegf drugs in patients with myopic cnv . clinical trials of ranibizumab for myopic cnv ( radiance and repair ) also demonstrated low rates of serious adverse events and ocular adverse events.21,22 in the repair study , one case of culture - negative endophthalmitis considered by investigators to be related to the injection was reported.21 the strength of this study is its ability to show the significant visual and anatomical improvements that can be achieved in patients with myopic cnv treated with intravitreal aflibercept in routine clinical practice . the inability to directly compare results with the myrror study due to differences in follow - up period and bcva measurement is a limitation . the 12-month results of the current study of intravitreal aflibercept for myopic cnv using a prn regimen were positive . intravitreal aflibercept retreatment guided by disease activity criteria based on oct and bcva was able to provide visual acuity benefits without requiring fa .

multiple sclerosis ( ms ) is a chronic progressive disease that destroys the central nervous system myelin , thereby affecting the sensory and motor function and can be determined in 85 to 90% of patients with periods of worsening ( exacerbation of symptoms ) and recovery . this disease is one of the most common neurological diseases in humans and the most common cause of disability in young people and one of the most important life - changing diseases , because it damages the best time of the individuals life and leads them gradually to inability . the most common age affected by this disease is from 20 to 40 years and the prevalence is 1.1 million people in the world.3 the number of people with this disease in iran is about 40,000 people . the prognosis of this disease is uncertain and the patients experience various physical and mental disorders that strongly influence their daily performance , social and family life , functional independence , and individual planning for the future , and in relation to a high level of uncertainty associated with negative emotional consequences , such as depression and emotional distress that needs to be coped with . on the other hand , the cost of treatment for any patient with multiple sclerosis in iran is estimated to be $ 1,000 monthly , and much of the costs is imposed on patients , together with physical and mental disabilities caused by the disease which require more coping with the disease . since multiple sclerosis has a wide range of debilitating effects , patients encounter confusion in assessment of its impacts on their lives , which requires coping with the disease to improve the quality of life . in addition , iranians are faced with some problems that are specific to the iranian context . therefore , these issues attract the attention of the patients in the country to cope with the increasing challenges of the disease and investigate the affecting antecedents . moreover , investigating the antecedents affecting coping , based on the experiences of the patients , can be effective in the transfer from mere medical focus to using individual experiences for improving coping and the quality of life . antecedents coping with the disease are the underlying factors that help the patients with multiple sclerosis to cope with the disease , achieve better quality of life , and also positive outcomes . on the other hand , since patients with multiple sclerosis , compared with other diseases , experience a higher prevalence of affective disorders and are faced with various physical and psychological coping needs , investigating the affecting antecedents based on the participants experiences can help to improve and reduce the need for drug therapies . although quantitative studies can well explain causal relationships , in rich data creation which is the result of deep understanding of the phenomenon , they are powerless . being present in natural environment , qualitative research tried to interpret the phenomena and is the best method to describe experiences ; therefore , studying and explaining the antecedents of coping based on the experiences of patients who live with the disease is a major help for the promotion of coping with the disease . based on the conducted surveys , a study with this subject was not found , and also qualitative studies conducted mostly the emphasis is on coping process and how the patients cope . to the best of our knowledge , no study has examined the factors affecting coping based on the experiences of multiple sclerosis patients . thus , a deep understanding of the antecedents of coping in patients who experience this crisis is essential . therefore , this study was conducted to investigate the experiences of patients with multiple sclerosis in the context of antecedents affecting coping with the disease using a qualitative approach . this study is a qualitative research with conventional content analysis approach which was performed from april to december 2015 , ( about nine months ) in iran ms society in tehran , iran . their age ranged from 24 to 46 years and their disease experience varied from 1 to 20 years . patients were selected using purposive sampling based on the willingness and ability to provide experience with an attempt to observe maximum variations in terms of demographic characteristics . also , primary participants were selected from multiple sclerosis patients whom the researcher knew . inclusion criteria for the participants were ( a ) definite diagnosis of multiple sclerosis disease , ( b ) willingness to participate in the study , and ( c ) ability to express experiences . exclusion criteria for participation were mental illness confirmed by a physician or the individual patient and lack of willingness to participate in the study . data collection was continued until saturation which occurred when a new category did not appear and until the existing categories were enriched . all interviews were conducted in a peaceful environment and with prior agreement of the participants . explain briefly about the nature of the illness and its problems , what did you do when you were diagnosed with this disease ? , what factors help you to better cope with your illness ? , how did you come along with this disease ? , and all interviews were audio - recorded with the permission of the participants . in analyzing the data , qualitative content analysis by conventional method and graneheim and lundman model were used , with the following steps : ( a ) implementing the interview conducted immediately after it was done , ( b ) reading the entire text to understand its general content , ( c ) determining the units of meaning and initial codes , and ( d ) classifying the initial codes in more comprehensive categories . in this approach thus , after each interview , the text was transcribed and typed verbatim in microsoft word and the initial codes were extracted after several times of listening . then , the sub - categories and categories were formed based on the similarities of the extracted codes . in the beginning , initial codes were analyzed by ad ; then , the list of categories and sub - categories was reviewed and re - categorized by the study team . cases of disagreement were discussed with the research team to reach a final consensus . to provide rigor and reliable data concerning credibility , the researcher had a long and close acquaintance with the participants and spent a long time in the field searching for data and enough time to gather and analyze the data . dependability was established by using the experts comments , and revision was done by the participants and co - workers . sampling mode , questions development , the method of coding and category extraction and modification were recorded to obtain sufficient conformability . the present study was approved by the ethics committee of tehran university of medical sciences in iran ( code of ethics ir.tums.rec.1394.1171 ) . before data collection , the researchers obtained the oral and written informed consent to ensure the confidentiality of the names of people , privacy and an emphasis on voluntary participation . at the beginning of the interviews , research goals and method this study is a qualitative research with conventional content analysis approach which was performed from april to december 2015 , ( about nine months ) in iran ms society in tehran , iran . their age ranged from 24 to 46 years and their disease experience varied from 1 to 20 years . patients were selected using purposive sampling based on the willingness and ability to provide experience with an attempt to observe maximum variations in terms of demographic characteristics . also , primary participants were selected from multiple sclerosis patients whom the researcher knew . inclusion criteria for the participants were ( a ) definite diagnosis of multiple sclerosis disease , ( b ) willingness to participate in the study , and ( c ) ability to express experiences . exclusion criteria for participation were mental illness confirmed by a physician or the individual patient and lack of willingness to participate in the study . data collection was continued until saturation which occurred when a new category did not appear and until the existing categories were enriched . all interviews were conducted in a peaceful environment and with prior agreement of the participants . explain briefly about the nature of the illness and its problems , what did you do when you were diagnosed with this disease ? all interviews were audio - recorded with the permission of the participants . in analyzing the data , qualitative content analysis by conventional method and graneheim and lundman model were used , with the following steps : ( a ) implementing the interview conducted immediately after it was done , ( b ) reading the entire text to understand its general content , ( c ) determining the units of meaning and initial codes , and ( d ) classifying the initial codes in more comprehensive categories . in this approach , thus , after each interview , the text was transcribed and typed verbatim in microsoft word and the initial codes were extracted after several times of listening . then , the sub - categories and categories were formed based on the similarities of the extracted codes . in the beginning , initial codes were analyzed by ad ; then , the list of categories and sub - categories was reviewed and re - categorized by the study team . cases of disagreement were discussed with the research team to reach a final consensus . to provide rigor and reliable data concerning credibility , the researcher had a long and close acquaintance with the participants and spent a long time in the field searching for data and enough time to gather and analyze the data . dependability was established by using the experts comments , and revision was done by the participants and co - workers . sampling mode , questions development , the method of coding and category extraction and modification were recorded to obtain sufficient conformability . the present study was approved by the ethics committee of tehran university of medical sciences in iran ( code of ethics ir.tums.rec.1394.1171 ) . before data collection , the researchers obtained the oral and written informed consent to ensure the confidentiality of the names of people , privacy and an emphasis on voluntary participation . at the beginning of the interviews , research goals and method the mean age of the participants was 33.2 years and the mean duration of the disease was 10.5 years . based on content analysis , five main categories were revealed : ( 1 ) social support , ( 2 ) lenience , ( 3 ) reliance on faith , ( 4 ) knowledge of multiple sclerosis and modeling , and ( 5 ) economic and environmental situation . each category had several different sub - categories , as shown in table 1 . categories and subcategories of antecedents of coping with the disease among iranian multiple sclerosis patients many participants believed that social support including support from family , spouse , peer friends with multiple sclerosis and also professional organizations and the multiple sclerosis society are the factors affecting coping with the disease . participants expressed that financial , emotional , mental , and spiritual support can alleviate their symptoms and improve their adherence to treatment and life expectancy . the participants also believed that social support decreased their level of stress , enabled them to tolerate and manage their problems more easily , and improved their physical and psychological well - being . the following examples highlight this point : having the support of my family at all times helped me a lot ; my father says that they are always ready to help me another one commented : having my husband supports really helped me for coping with disease . he pays for my drugs although i know that he does nt have a high income , but tells me not to worry about the cost of drugs a participant told about the support of friends : the whole time that i was hospitalized , my friends were there for me , from morning to night , they were in hospital so that hospital nurses said your friends eat breakfast and dinner at hospital ( participant # 6 , female ) . the participants believed that organizational support like ministry of health or multiple sclerosis society decreased their level of stress , made them feel relaxed and able to bear their problems more easily , limited the symptoms of the disease , and improved their physical and psychological condition . a participant said : the ministry of health or multiple sclerosis society and or other authorities can help us to cope with the disease ; for example , they can cover our medicines by insurance or provide regular programs for the control and treatment of such patients ( participant # 8 , female ) . this category includes the following sub - categories of the underestimation of multiple sclerosis compared to other diseases , acceptance of disease symptom , distraction from one s attention , positive attitude , self - confidence and high morale , and hope for recovery . they believed that in this way they were more easily able to cope with the disease . the majority of the participants underestimated multiple sclerosis when compared with other diseases , such as cancer , and they were satisfied they were not afflicted with a worse disease . most of them thought that their situation was much better than that of other patients like cancer or incurable diseases . a participant said : if i was healthy , i might get caught in a worse situation such as cancer or a crippling disease , so thanks god that i have multiple sclerosis ( participant # 11 , male ) . i always compare myself with a cancer patient or a worse condition and then i have much better feeling ( participant # 8 , female ) . the participants also try accepting the current situation and symptoms of the disease and are indifferent to its complications . for example : one said : the fact is that i have to cope with it ; disease has happened and nothing can be done about it . for this reason , i accepted the existence of this symptom with me and do nt take it hard ( participant # 7 , female ) . patients were also trying through different ways , forget their illness and symptoms and free their mind from negative and destructive thoughts . in this regard , a participant said : i try to spend my time in going to park and mountaineering , shopping , and going out with friends to free myself from loneliness and think less about it . the participants believed that one s attitude to disease is very important in coping with it ; they also expressed that their own attitudes about ms and how they deal with them are effective in coping . the participants tried to have a positive attitude toward their problems and disease . in this regard , a participant said : everything depends on one s attitude ; many tell me that my disease will be cured , god s willing . participants commented one of the factors that help them cope with disease , indicating that self - confidence and high morale were among such factors . a participant said : positive morale can be a great help to cope with the disease ; for example , mr . he says we have multiple sclerosis and we should cope with it . ( participant # 2 , female ) . another stated : accepting my disease was not that difficult because i have high morale . i was a little depressed in the beginning , but i did not become demoralized for coping with disease , the participants also believed one should always keep hopeful and that hopelessness equals death . i always say that the definitive treatment of multiple sclerosis will be found one day , because medical science will have much progress , so why lose hope ? this category includes the following sub - categories : religious beliefs and acceptance of disease as a divine test . most participants believed that their disease was their fate , chosen by god and thought that changing the condition was impossible . they believed that affliction with multiple sclerosis had increased their closeness to allah . a male participant stated : this was my destiny . i believe god would restore my health ( participant # 3 , female ) . the majority of the participants believed that their disease was a kind of divine test , which they had to accomplish with pride . indeed , belief in god s help and acceptance of the disease as a divine test were the affecting antecedents in coping . for instance , a participant said : every problem in life is a divine test . i did not want to have this disease , but this is a divine test and i need to come along with it . most patients used religious beliefs to support them in coping and living with multiple sclerosis . this category includes the sub - categories of gathering information about the disease , community awareness about it , and observing and considering the behavior of peers . most participants believed that the patient s and community members proper knowledge about the symptoms , problems and disease treatment is effective in coping . it decreased the participants fear , anxiety , and stress , which allowed them to feel relaxed . having information about the causes of the disease created a greater sense of confidence and greater independence in them . one said : i always get information about my disease from different sources , such as physicians , books , etc .. this information showed me how i should behave with the disease ; this reduces my stress.(participant # 3 , male ) . another patient stated : if i knew more about the disease and my information was more complete , i would more conveniently cope and live with multiple sclerosis the participants believed that providing appropriate information for members of the community provides the opportunity to cope better with multiple sclerosis and its challenges in patients ; most of the participants suffered from community low information about the disease . when they see us in the streets , they do not deal properly with us and do nt understand our conditions . a participant said : there are families that leave their patients at sanitarium . if members of the community know the disease , then it can help us . the most important problem of multiple sclerosis is that nobody knows anything about it . patients also tried to model the behavior of other people with multiple sclerosis in order to model the positive and negative behaviors for better coping . they tried to be a model of patients who are successful in management and coping with the disease . a participant said : i model some of the things i see in my friends ; for example , i observe some of the patients who mope and have stress , or i model some positive things i see in them , such as being happy , speaking and laughing . this category includes the sub - categories of suitable living environment , stressful and stress - free environment , loss of employment and exorbitant cost of drugs . most of the participants said that having access to supplies , elevators and toilets , and also a quiet environment free from the stress can be effective in coping . from the participant s perspective , appropriate environmental situation leads to improvement of psychological state , relaxation and more coping of patients . for instance : i must rent a house that has elevator and a western toilet , because i feel more comfortable . a female participant said : i have designed my house so that everything is available participants commented that stress and stressful environment made their symptoms worse and the believed that it might lead to an exacerbation . one said : once , i took part in a ceremony and got immediately sick and was needed to be hospitalized for corticosteroid therapy . i go out in quiet environments weekly to be away from stress and home concerns . economic and financial state was also another antecedent identified effective in coping with multiple sclerosis . most patients mentioned that loss of employment and exorbitant cost of drugs due to degenerative nature of the disease are dramatically effective in coping . the participants also mentioned that inadequate financial resources , income reduction and disability prevented them from using rehabilitation services and assistive devices such as more effective drugs or regular physiotherapy and caused a lot of stress , anxiety and problem . several participants indicated that they left work because of the potential adverse consequences of stress . as to loss of employment , a participant said : it is about 4 years that i can not go to work for my severe dizziness ; i am unemployed and financially in crisis ; i do nt know what to do . i get betaferon early in the disease , but because of rising prices , lack of insurance coverage and financial budget drop , i had to get ziferon ; it is not as effective as betaferon , but i have to . many participants believed that social support including support from family , spouse , peer friends with multiple sclerosis and also professional organizations and the multiple sclerosis society are the factors affecting coping with the disease . participants expressed that financial , emotional , mental , and spiritual support can alleviate their symptoms and improve their adherence to treatment and life expectancy . the participants also believed that social support decreased their level of stress , enabled them to tolerate and manage their problems more easily , and improved their physical and psychological well - being . the following examples highlight this point : having the support of my family at all times helped me a lot ; my father says that they are always ready to help me another one commented : having my husband supports really helped me for coping with disease . he pays for my drugs although i know that he does nt have a high income , but tells me not to worry about the cost of drugs a participant told about the support of friends : the whole time that i was hospitalized , my friends were there for me , from morning to night , they were in hospital so that hospital nurses said your friends eat breakfast and dinner at hospital the participants believed that organizational support like ministry of health or multiple sclerosis society decreased their level of stress , made them feel relaxed and able to bear their problems more easily , limited the symptoms of the disease , and improved their physical and psychological condition . a participant said : the ministry of health or multiple sclerosis society and or other authorities can help us to cope with the disease ; for example , they can cover our medicines by insurance or provide regular programs for the control and treatment of such patients ( participant # 8 , female ) . this category includes the following sub - categories of the underestimation of multiple sclerosis compared to other diseases , acceptance of disease symptom , distraction from one s attention , positive attitude , self - confidence and high morale , and hope for recovery . they believed that in this way they were more easily able to cope with the disease . the majority of the participants underestimated multiple sclerosis when compared with other diseases , such as cancer , and they were satisfied they were not afflicted with a worse disease . most of them thought that their situation was much better than that of other patients like cancer or incurable diseases . a participant said : if i was healthy , i might get caught in a worse situation such as cancer or a crippling disease , so thanks god that i have multiple sclerosis ( participant # 11 , male ) . i always compare myself with a cancer patient or a worse condition and then i have much better feeling ( participant # 8 , female ) . the participants also try accepting the current situation and symptoms of the disease and are indifferent to its complications . for example : one said : the fact is that i have to cope with it ; disease has happened and nothing can be done about it . for this reason , i accepted the existence of this symptom with me and do nt take it hard ( participant # 7 , female ) . patients were also trying through different ways , forget their illness and symptoms and free their mind from negative and destructive thoughts . in this regard , a participant said : i try to spend my time in going to park and mountaineering , shopping , and going out with friends to free myself from loneliness and think less about it . the participants believed that one s attitude to disease is very important in coping with it ; they also expressed that their own attitudes about ms and how they deal with them are effective in coping . in this regard , a participant said : everything depends on one s attitude ; many tell me that my disease will be cured , god s willing . participants commented one of the factors that help them cope with disease , indicating that self - confidence and high morale were among such factors . a participant said : positive morale can be a great help to cope with the disease ; for example , mr . he says we have multiple sclerosis and we should cope with it . ( participant # 2 , female ) . another stated : accepting my disease was not that difficult because i have high morale . i was a little depressed in the beginning , but i did not become demoralized ( participant # 11 , male ) . for coping with disease , the participants also believed one should always keep hopeful and that hopelessness equals death . i always say that the definitive treatment of multiple sclerosis will be found one day , because medical science will have much progress , so why lose hope ? this category includes the following sub - categories : religious beliefs and acceptance of disease as a divine test . most participants believed that their disease was their fate , chosen by god and thought that changing the condition was impossible . they believed that affliction with multiple sclerosis had increased their closeness to allah . a male participant stated : this was my destiny . i believe god would restore my health ( participant # 3 , female ) . the majority of the participants believed that their disease was a kind of divine test , which they had to accomplish with pride . indeed , belief in god s help and acceptance of the disease as a divine test were the affecting antecedents in coping . for instance , a participant said : every problem in life is a divine test . i did not want to have this disease , but this is a divine test and i need to come along with it . most patients used religious beliefs to support them in coping and living with multiple sclerosis . this category includes the sub - categories of gathering information about the disease , community awareness about it , and observing and considering the behavior of peers . most participants believed that the patient s and community members proper knowledge about the symptoms , problems and disease treatment is effective in coping . awareness and information about the disease could have a good effect . it decreased the participants fear , anxiety , and stress , which allowed them to feel relaxed . having information about the causes of the disease created a greater sense of confidence and greater independence in them . one said : i always get information about my disease from different sources , such as physicians , books , etc .. this information showed me how i should behave with the disease ; this reduces my stress.(participant # 3 , male ) . another patient stated : if i knew more about the disease and my information was more complete , i would more conveniently cope and live with multiple sclerosis ( participant # 4 , male ) . the participants believed that providing appropriate information for members of the community provides the opportunity to cope better with multiple sclerosis and its challenges in patients ; most of the participants suffered from community low information about the disease . when they see us in the streets , they do not deal properly with us and do nt understand our conditions . a participant said : there are families that leave their patients at sanitarium . if members of the community know the disease , then it can help us . the most important problem of multiple sclerosis is that nobody knows anything about it . patients also tried to model the behavior of other people with multiple sclerosis in order to model the positive and negative behaviors for better coping . they tried to be a model of patients who are successful in management and coping with the disease . a participant said : i model some of the things i see in my friends ; for example , i observe some of the patients who mope and have stress , or i model some positive things i see in them , such as being happy , speaking and laughing . this category includes the sub - categories of suitable living environment , stressful and stress - free environment , loss of employment and exorbitant cost of drugs . most of the participants said that having access to supplies , elevators and toilets , and also a quiet environment free from the stress can be effective in coping . from the participant s perspective , appropriate environmental situation leads to improvement of psychological state , relaxation and more coping of patients . for instance : i must rent a house that has elevator and a western toilet , because i feel more comfortable . a female participant said : i have designed my house so that everything is available participants commented that stress and stressful environment made their symptoms worse and the believed that it might lead to an exacerbation . one said : once , i took part in a ceremony and got immediately sick and was needed to be hospitalized for corticosteroid therapy . i go out in quiet environments weekly to be away from stress and home concerns . economic and financial state was also another antecedent identified effective in coping with multiple sclerosis . most patients mentioned that loss of employment and exorbitant cost of drugs due to degenerative nature of the disease are dramatically effective in coping . the participants also mentioned that inadequate financial resources , income reduction and disability prevented them from using rehabilitation services and assistive devices such as more effective drugs or regular physiotherapy and caused a lot of stress , anxiety and problem . several participants indicated that they left work because of the potential adverse consequences of stress . as to loss of employment , a participant said : it is about 4 years that i can not go to work for my severe dizziness ; i am unemployed and financially in crisis ; i do nt know what to do . i get betaferon early in the disease , but because of rising prices , lack of insurance coverage and financial budget drop , i had to get ziferon ; it is not as effective as betaferon , but i have to . ( participant # 8 , female ) . according to the results of this study , antecedents that lead to better coping or failure to cope with multiple sclerosis are identified in five categories of social support , lenience , reliance on faith , knowledge of multiple sclerosis and modeling , economic and environmental situation . social support was the first category obtained from the data , and the participants believe that it is a crucial factor in maintaining and restoring mental peace and coping with the disease . they used the social network including family , spouse , friends , peer patients , employees , and relevant professional organizations as a source of support so that their appropriate support can reduce negative effects caused by chronic disease and help the patients to better cope with their disease . the results of a study also showed that emotional support helps the patients to rely on family support and feel that they support them at this difficult and critical situation ; thus , it is easier to cope with the disease . one study also found out that better emotional , mental , and psychosocial support from family and friends is directly associated with improved mental health and patients coping with multiple sclerosis . this study emphasized the power of the family in patients emotional support , which should not be ignored . on the other hand , patients who have less social support are more prone to mental health problems , especially stress , and the severity of the disease is higher ; this can have effects on the patients coping and quality of life . in addition to support from family , support from peers is an effective source in coping with stressful experiences and obtaining information . preliminary studies show that the impact of peer support groups on people with chronic diseases creates an opportunity to share experiential knowledge , helps the members explore their approaches that help them cope effectively with the stress caused by the disease . moreover , support groups help the patients validate personal experiences , increase awareness of therapeutic areas and cope with the chronic diseases . in addition , various studies have shown the impact of positive experiences of peer patients on coping with chronic diseases . most participants thought that their condition was better than some other diseases such as cancer and were trying to underestimate their disease , so they felt relaxed and coped with the disease easier . some participants in another study said : if i was not afflicted with multiple sclerosis , i would have been involved in a worse threatening condition and disease such as cancer ; why should i think that i will be a wheelchair user or paralyzed ; none of this will happen to me . in another study , this antecedent and its impact on coping on the other hand , patients who show that their disease is greater than what it is , always mope , and constantly look for the cause of the disease are experiencing social isolation and depression , their symptoms increase , and the process of control and recovery can be impaired . acceptance of the situation and disease symptom are frequently reported in relation to other threatening illnesses . by distracting one s attention studies have shown that patients who are experiencing life - threatening illnesses frequently distract their attention from the situation . positive attitude of patients toward the disease was the sub - category obtained from the data which can also influence the patients coping , so positive and negative attitudes lead to coping and lack of coping , respectively . the results of a study showed that patients with relapsing forms of the disease have a more positive attitude toward their duties and disease control as compared to patients with a progressive form of the disease . researchers also expressed that the individual s attitude to multiple sclerosis had a significant role in coping with it . the present study revealed that having self - confidence and high morale played an important role in helping the patients to re - engage with life and cope with the disease . these results are in the same line with the data reported by dalvandi et al . , who revealed the importance of self - confidence in re - connecting with life for those surviving after a stroke . the patients hope is the factor that can help maintain optimism and coping with multiple sclerosis ; the hope that their disease will be treated in the future and medicines will be discovered to control the disease can help them cope with the disease . the participants viewed that reliance on their faith helped them to cope with multiple sclerosis and overcome limitations . therefore , in several studies , the positive role of religion and hope on eternal power of god in coping has been mentioned . a lot of patients get more religious than in the past during the illness , when adjustment mechanisms do not have the necessary performance to cope with the disease and at this time , prayer is an effective adjustment mechanism that creates the feeling of worth and hope in the individual by reducing feelings of loneliness and pain . the results of a study in iran on chronic diseases showed that spiritual beliefs not only are effective in coping with the disease , but also play an important role in patients lifestyle , and create a sense of purposefulness , increase the patients motivation to the treatment regimen and are used as a sedative agent by patients . about patients with heart failure , the results of a study also showed that relationship with god , religious values , and communicating with others are essential components of the participants who survived from heart attacks and spirituality helped patients cope with their stress and fears . knowledge of multiple sclerosis and modeling are the fourth category obtained from the data that was identified as an antecedent affecting the coping . sufficient knowledge ( of individual and community ) about multiple sclerosis , including facts such as treatment and symptoms of the disease and how these symptoms affect their lives , as well as the quality of information and how this information was presented at the time of disease diagnosis , can enable people to understand more and use active coping and assessment processes . the participants in this study tried to cope with their disease by gathering information about the disease and its treatment and outcomes . learning about the disease and its treatment modalities gradually reduces the patients fear and enables them to cope with the conditions . therefore , cognitive impairment in early stages of the disease can prevent multiple sclerosis patients from using the coping resources . another study also showed that individual and community knowledge of the disease was effective in coping . however , some participants in the study conducted by farsi et al . believed that although overall information was often helpful , information about the mortality rates and treatment side - effects increased their stress and worries . modeling the positive and negative behaviors of peer patients is of the antecedents effective in coping with the disease from the perspective of the participants , who were in the above category . patients believed that observing and modeling the emotions and behaviors of multiple sclerosis patients who were successful in managing their disease can be effective in coping with the disease . the study results also showed that observing others emotional status and behavior is effective in arousing feelings for modeling ; thus , both positive and negative behaviors can be emulated to be effectively used by patients . the fifth category obtained from the data was the environmental - economic situation that participants frequently mentioned . in this regard , patients expressed that they chose a place to live where their access to daily supplies , elevators and toilets were provided . several studies have emphasized the relationship between environmental conditions and physical health , since environmental characteristics and factors associated with it were effective on fatigue , severity of the illness , physical activity , recreation and commuting ; they can also affect the patients quality of life and ultimately coping with the disease . previous studies have provided evidence showing that proper environmental conditions are important factors in the physical and mental health of patients with chronic diseases . a meta - analysis study findings also showed that the living environment and environmental conditions contributed to the development of many gastrointestinal and neurological disorders , such as multiple sclerosis , parkinson , etc . , and have unfavorable effects on physical and mental health and adjustment of individuals . in another meta - analysis study , also , stressors are related to the sense of health and lower self - efficacy . on the other hand , using relaxation techniques and providing a stress - free environment can help the patients to cope with the disease . economic problems resulting from the disease including loss of employment and exorbitant cost of drugs were the other antecedents causing discomfort . due to the progressive nature of the disease , many patients lose their employment leading to lower productivity and incomes of life and have effects on the patient s coping . in addition , income reduction resulting from disability as well as the high cost of treatment and medication are associated with reduced quality of life for these patients . therefore , the largest portion of direct costs in multiple sclerosis is related to the cost of the drug , consultation , and nursing , respectively . in iran these costly drugs with other relevant problems , including loss of employment , seriously affect the patients coping . therefore , the economic problem in iranian patients with multiple sclerosis is very important and requires serious attention due to the lack of easy access to drugs , lack of support in terms of rehabilitation and treatment costs , and incomplete insurance coverage of the drugs . the results of this study are limited to the antecedents affecting coping with the disease in the culture and context of iran ; thus , further studies in different cultures have to be done for further use of the findings . restricting the field of study to members of the ms society was another limitation ; thus , it is recommended that , in future studies , experiences of patients who are nt a member of the ms society should be considered . the findings of this study showed that coping with multiple sclerosis is a complex , multidimensional and contextual concept that is affected by various factors such as social support , lenience , reliance on faith , knowledge of multiple sclerosis and modeling , and economic and environmental situation . hence , identification of these factors increases our knowledge and help the nurses and other health - care professionals as well as patients and families to improve coping with the disease .

abstractbackground : due to many physical and mental disorders that occur in multiple sclerosis patients , identifying the factors affecting coping based on the experiences of patients using qualitative study is essential to improve their quality of life . this study was conducted to explore the antecedents of coping with the disease in patients with multiple sclerosis . methods : this is a qualitative study conducted on 11 patients with multiple sclerosis in 2015 in tehran , iran . these patients were selected based on purposive sampling . data were collected using semi - structured and in - depth interviews and coded . these data were analyzed using the conventional content analysis . the rigor of qualitative data using the criteria proposed by guba and lincoln were assessed.results:five main categories were revealed : ( 1 ) social support , ( 2 ) lenience , ( 3 ) reliance on faith , ( 4 ) knowledge of multiple sclerosis and modeling , and ( 5 ) economic and environmental situation . each category had several distinct sub - categories . conclusions : the results of this study showed that coping with multiple sclerosis is a complex , multidimensional and contextual concept that is affected by various factors in relation to the context of iran . the findings of the study can provide the healthcare professionals with deeper recognition and understanding of these antecedents to improve successful coping in iranian patients suffering from multiple sclerosis .

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multiple sclerosis ( ms ) is a chronic progressive disease that destroys the central nervous system myelin , thereby affecting the sensory and motor function and can be determined in 85 to 90% of patients with periods of worsening ( exacerbation of symptoms ) and recovery . the prognosis of this disease is uncertain and the patients experience various physical and mental disorders that strongly influence their daily performance , social and family life , functional independence , and individual planning for the future , and in relation to a high level of uncertainty associated with negative emotional consequences , such as depression and emotional distress that needs to be coped with . on the other hand , the cost of treatment for any patient with multiple sclerosis in iran is estimated to be $ 1,000 monthly , and much of the costs is imposed on patients , together with physical and mental disabilities caused by the disease which require more coping with the disease . since multiple sclerosis has a wide range of debilitating effects , patients encounter confusion in assessment of its impacts on their lives , which requires coping with the disease to improve the quality of life . therefore , these issues attract the attention of the patients in the country to cope with the increasing challenges of the disease and investigate the affecting antecedents . moreover , investigating the antecedents affecting coping , based on the experiences of the patients , can be effective in the transfer from mere medical focus to using individual experiences for improving coping and the quality of life . antecedents coping with the disease are the underlying factors that help the patients with multiple sclerosis to cope with the disease , achieve better quality of life , and also positive outcomes . on the other hand , since patients with multiple sclerosis , compared with other diseases , experience a higher prevalence of affective disorders and are faced with various physical and psychological coping needs , investigating the affecting antecedents based on the participants experiences can help to improve and reduce the need for drug therapies . although quantitative studies can well explain causal relationships , in rich data creation which is the result of deep understanding of the phenomenon , they are powerless . being present in natural environment , qualitative research tried to interpret the phenomena and is the best method to describe experiences ; therefore , studying and explaining the antecedents of coping based on the experiences of patients who live with the disease is a major help for the promotion of coping with the disease . based on the conducted surveys , a study with this subject was not found , and also qualitative studies conducted mostly the emphasis is on coping process and how the patients cope . to the best of our knowledge , no study has examined the factors affecting coping based on the experiences of multiple sclerosis patients . thus , a deep understanding of the antecedents of coping in patients who experience this crisis is essential . therefore , this study was conducted to investigate the experiences of patients with multiple sclerosis in the context of antecedents affecting coping with the disease using a qualitative approach . this study is a qualitative research with conventional content analysis approach which was performed from april to december 2015 , ( about nine months ) in iran ms society in tehran , iran . patients were selected using purposive sampling based on the willingness and ability to provide experience with an attempt to observe maximum variations in terms of demographic characteristics . also , primary participants were selected from multiple sclerosis patients whom the researcher knew . inclusion criteria for the participants were ( a ) definite diagnosis of multiple sclerosis disease , ( b ) willingness to participate in the study , and ( c ) ability to express experiences . , and all interviews were audio - recorded with the permission of the participants . in analyzing the data , qualitative content analysis by conventional method and graneheim and lundman model were used , with the following steps : ( a ) implementing the interview conducted immediately after it was done , ( b ) reading the entire text to understand its general content , ( c ) determining the units of meaning and initial codes , and ( d ) classifying the initial codes in more comprehensive categories . then , the sub - categories and categories were formed based on the similarities of the extracted codes . in the beginning , initial codes were analyzed by ad ; then , the list of categories and sub - categories was reviewed and re - categorized by the study team . dependability was established by using the experts comments , and revision was done by the participants and co - workers . at the beginning of the interviews , research goals and method this study is a qualitative research with conventional content analysis approach which was performed from april to december 2015 , ( about nine months ) in iran ms society in tehran , iran . patients were selected using purposive sampling based on the willingness and ability to provide experience with an attempt to observe maximum variations in terms of demographic characteristics . also , primary participants were selected from multiple sclerosis patients whom the researcher knew . inclusion criteria for the participants were ( a ) definite diagnosis of multiple sclerosis disease , ( b ) willingness to participate in the study , and ( c ) ability to express experiences . in analyzing the data , qualitative content analysis by conventional method and graneheim and lundman model were used , with the following steps : ( a ) implementing the interview conducted immediately after it was done , ( b ) reading the entire text to understand its general content , ( c ) determining the units of meaning and initial codes , and ( d ) classifying the initial codes in more comprehensive categories . then , the sub - categories and categories were formed based on the similarities of the extracted codes . in the beginning , initial codes were analyzed by ad ; then , the list of categories and sub - categories was reviewed and re - categorized by the study team . based on content analysis , five main categories were revealed : ( 1 ) social support , ( 2 ) lenience , ( 3 ) reliance on faith , ( 4 ) knowledge of multiple sclerosis and modeling , and ( 5 ) economic and environmental situation . each category had several different sub - categories , as shown in table 1 . categories and subcategories of antecedents of coping with the disease among iranian multiple sclerosis patients many participants believed that social support including support from family , spouse , peer friends with multiple sclerosis and also professional organizations and the multiple sclerosis society are the factors affecting coping with the disease . the participants also believed that social support decreased their level of stress , enabled them to tolerate and manage their problems more easily , and improved their physical and psychological well - being . the participants believed that organizational support like ministry of health or multiple sclerosis society decreased their level of stress , made them feel relaxed and able to bear their problems more easily , limited the symptoms of the disease , and improved their physical and psychological condition . a participant said : the ministry of health or multiple sclerosis society and or other authorities can help us to cope with the disease ; for example , they can cover our medicines by insurance or provide regular programs for the control and treatment of such patients ( participant # 8 , female ) . this category includes the following sub - categories of the underestimation of multiple sclerosis compared to other diseases , acceptance of disease symptom , distraction from one s attention , positive attitude , self - confidence and high morale , and hope for recovery . they believed that in this way they were more easily able to cope with the disease . the majority of the participants underestimated multiple sclerosis when compared with other diseases , such as cancer , and they were satisfied they were not afflicted with a worse disease . the participants also try accepting the current situation and symptoms of the disease and are indifferent to its complications . a participant said : positive morale can be a great help to cope with the disease ; for example , mr . most patients used religious beliefs to support them in coping and living with multiple sclerosis . this category includes the sub - categories of gathering information about the disease , community awareness about it , and observing and considering the behavior of peers . one said : i always get information about my disease from different sources , such as physicians , books , etc .. this information showed me how i should behave with the disease ; this reduces my stress. another patient stated : if i knew more about the disease and my information was more complete , i would more conveniently cope and live with multiple sclerosis the participants believed that providing appropriate information for members of the community provides the opportunity to cope better with multiple sclerosis and its challenges in patients ; most of the participants suffered from community low information about the disease . the most important problem of multiple sclerosis is that nobody knows anything about it . patients also tried to model the behavior of other people with multiple sclerosis in order to model the positive and negative behaviors for better coping . they tried to be a model of patients who are successful in management and coping with the disease . from the participant s perspective , appropriate environmental situation leads to improvement of psychological state , relaxation and more coping of patients . economic and financial state was also another antecedent identified effective in coping with multiple sclerosis . most patients mentioned that loss of employment and exorbitant cost of drugs due to degenerative nature of the disease are dramatically effective in coping . many participants believed that social support including support from family , spouse , peer friends with multiple sclerosis and also professional organizations and the multiple sclerosis society are the factors affecting coping with the disease . the participants also believed that social support decreased their level of stress , enabled them to tolerate and manage their problems more easily , and improved their physical and psychological well - being . he pays for my drugs although i know that he does nt have a high income , but tells me not to worry about the cost of drugs a participant told about the support of friends : the whole time that i was hospitalized , my friends were there for me , from morning to night , they were in hospital so that hospital nurses said your friends eat breakfast and dinner at hospital the participants believed that organizational support like ministry of health or multiple sclerosis society decreased their level of stress , made them feel relaxed and able to bear their problems more easily , limited the symptoms of the disease , and improved their physical and psychological condition . a participant said : the ministry of health or multiple sclerosis society and or other authorities can help us to cope with the disease ; for example , they can cover our medicines by insurance or provide regular programs for the control and treatment of such patients ( participant # 8 , female ) . this category includes the following sub - categories of the underestimation of multiple sclerosis compared to other diseases , acceptance of disease symptom , distraction from one s attention , positive attitude , self - confidence and high morale , and hope for recovery . the majority of the participants underestimated multiple sclerosis when compared with other diseases , such as cancer , and they were satisfied they were not afflicted with a worse disease . the participants also try accepting the current situation and symptoms of the disease and are indifferent to its complications . participants commented one of the factors that help them cope with disease , indicating that self - confidence and high morale were among such factors . a participant said : positive morale can be a great help to cope with the disease ; for example , mr . this category includes the following sub - categories : religious beliefs and acceptance of disease as a divine test . they believed that affliction with multiple sclerosis had increased their closeness to allah . indeed , belief in god s help and acceptance of the disease as a divine test were the affecting antecedents in coping . i did not want to have this disease , but this is a divine test and i need to come along with it . most patients used religious beliefs to support them in coping and living with multiple sclerosis . this category includes the sub - categories of gathering information about the disease , community awareness about it , and observing and considering the behavior of peers . another patient stated : if i knew more about the disease and my information was more complete , i would more conveniently cope and live with multiple sclerosis ( participant # 4 , male ) . the participants believed that providing appropriate information for members of the community provides the opportunity to cope better with multiple sclerosis and its challenges in patients ; most of the participants suffered from community low information about the disease . if members of the community know the disease , then it can help us . the most important problem of multiple sclerosis is that nobody knows anything about it . patients also tried to model the behavior of other people with multiple sclerosis in order to model the positive and negative behaviors for better coping . they tried to be a model of patients who are successful in management and coping with the disease . most of the participants said that having access to supplies , elevators and toilets , and also a quiet environment free from the stress can be effective in coping . economic and financial state was also another antecedent identified effective in coping with multiple sclerosis . most patients mentioned that loss of employment and exorbitant cost of drugs due to degenerative nature of the disease are dramatically effective in coping . according to the results of this study , antecedents that lead to better coping or failure to cope with multiple sclerosis are identified in five categories of social support , lenience , reliance on faith , knowledge of multiple sclerosis and modeling , economic and environmental situation . social support was the first category obtained from the data , and the participants believe that it is a crucial factor in maintaining and restoring mental peace and coping with the disease . the results of a study also showed that emotional support helps the patients to rely on family support and feel that they support them at this difficult and critical situation ; thus , it is easier to cope with the disease . one study also found out that better emotional , mental , and psychosocial support from family and friends is directly associated with improved mental health and patients coping with multiple sclerosis . this study emphasized the power of the family in patients emotional support , which should not be ignored . on the other hand , patients who have less social support are more prone to mental health problems , especially stress , and the severity of the disease is higher ; this can have effects on the patients coping and quality of life . preliminary studies show that the impact of peer support groups on people with chronic diseases creates an opportunity to share experiential knowledge , helps the members explore their approaches that help them cope effectively with the stress caused by the disease . some participants in another study said : if i was not afflicted with multiple sclerosis , i would have been involved in a worse threatening condition and disease such as cancer ; why should i think that i will be a wheelchair user or paralyzed ; none of this will happen to me . in another study , this antecedent and its impact on coping on the other hand , patients who show that their disease is greater than what it is , always mope , and constantly look for the cause of the disease are experiencing social isolation and depression , their symptoms increase , and the process of control and recovery can be impaired . acceptance of the situation and disease symptom are frequently reported in relation to other threatening illnesses . positive attitude of patients toward the disease was the sub - category obtained from the data which can also influence the patients coping , so positive and negative attitudes lead to coping and lack of coping , respectively . the results of a study showed that patients with relapsing forms of the disease have a more positive attitude toward their duties and disease control as compared to patients with a progressive form of the disease . the patients hope is the factor that can help maintain optimism and coping with multiple sclerosis ; the hope that their disease will be treated in the future and medicines will be discovered to control the disease can help them cope with the disease . the participants viewed that reliance on their faith helped them to cope with multiple sclerosis and overcome limitations . a lot of patients get more religious than in the past during the illness , when adjustment mechanisms do not have the necessary performance to cope with the disease and at this time , prayer is an effective adjustment mechanism that creates the feeling of worth and hope in the individual by reducing feelings of loneliness and pain . the results of a study in iran on chronic diseases showed that spiritual beliefs not only are effective in coping with the disease , but also play an important role in patients lifestyle , and create a sense of purposefulness , increase the patients motivation to the treatment regimen and are used as a sedative agent by patients . about patients with heart failure , the results of a study also showed that relationship with god , religious values , and communicating with others are essential components of the participants who survived from heart attacks and spirituality helped patients cope with their stress and fears . knowledge of multiple sclerosis and modeling are the fourth category obtained from the data that was identified as an antecedent affecting the coping . sufficient knowledge ( of individual and community ) about multiple sclerosis , including facts such as treatment and symptoms of the disease and how these symptoms affect their lives , as well as the quality of information and how this information was presented at the time of disease diagnosis , can enable people to understand more and use active coping and assessment processes . learning about the disease and its treatment modalities gradually reduces the patients fear and enables them to cope with the conditions . therefore , cognitive impairment in early stages of the disease can prevent multiple sclerosis patients from using the coping resources . another study also showed that individual and community knowledge of the disease was effective in coping . modeling the positive and negative behaviors of peer patients is of the antecedents effective in coping with the disease from the perspective of the participants , who were in the above category . patients believed that observing and modeling the emotions and behaviors of multiple sclerosis patients who were successful in managing their disease can be effective in coping with the disease . several studies have emphasized the relationship between environmental conditions and physical health , since environmental characteristics and factors associated with it were effective on fatigue , severity of the illness , physical activity , recreation and commuting ; they can also affect the patients quality of life and ultimately coping with the disease . previous studies have provided evidence showing that proper environmental conditions are important factors in the physical and mental health of patients with chronic diseases . a meta - analysis study findings also showed that the living environment and environmental conditions contributed to the development of many gastrointestinal and neurological disorders , such as multiple sclerosis , parkinson , etc . , and have unfavorable effects on physical and mental health and adjustment of individuals . on the other hand , using relaxation techniques and providing a stress - free environment can help the patients to cope with the disease . due to the progressive nature of the disease , many patients lose their employment leading to lower productivity and incomes of life and have effects on the patient s coping . in addition , income reduction resulting from disability as well as the high cost of treatment and medication are associated with reduced quality of life for these patients . therefore , the largest portion of direct costs in multiple sclerosis is related to the cost of the drug , consultation , and nursing , respectively . therefore , the economic problem in iranian patients with multiple sclerosis is very important and requires serious attention due to the lack of easy access to drugs , lack of support in terms of rehabilitation and treatment costs , and incomplete insurance coverage of the drugs . the results of this study are limited to the antecedents affecting coping with the disease in the culture and context of iran ; thus , further studies in different cultures have to be done for further use of the findings . restricting the field of study to members of the ms society was another limitation ; thus , it is recommended that , in future studies , experiences of patients who are nt a member of the ms society should be considered . the findings of this study showed that coping with multiple sclerosis is a complex , multidimensional and contextual concept that is affected by various factors such as social support , lenience , reliance on faith , knowledge of multiple sclerosis and modeling , and economic and environmental situation . hence , identification of these factors increases our knowledge and help the nurses and other health - care professionals as well as patients and families to improve coping with the disease .

the zika virus ( zikv ) is a mosquito - borne flavivirus with single - stranded positive rna , which is related to dengue , yellow fever , and japanese encephalitis virus . zikv was first isolated from rhesus monkeys in the zika forest of uganda in 1947 , and later , the virus was named after that place . it is potentially transmitted through sex ( heterosexual or homosexual transmission ) , blood , direct contact , or mother - to - fetus . detected zikv in blood , semen , amniotic fluid , saliva , breast milk , and cerebrospinal fluid provided further evidence of its transmission route and sent an alert to public health . it was first detected in humans in 1954 , with classic and mild clinical manifestations such as rash , conjunctivitis , fever , arthralgia , and arthritis . the disease was sporadically found in asia and africa , and then there were outbreaks in micronesia ( yap island ) , french polynesia , and brazil in 2007 , 2013 , 2015 , respectively . in early 2015 , physicians in northeast brazil first found patients with a dengue - like syndrome , and zikv was later identified as the cause . high transmissibility was found for zikv , with a range of 26.6 among the basic reproduction number , especially among those in a high migration flow . this virus can adapt to harsh conditions , even can keep stable structure in the temperature of 40c . microcephaly is a rare condition defined as a smaller head size , compared to other fetuses / babies of the same age / sex / ethnicity , often with prognosis of intellectual disabilities . there is still no uniform and universally accepted diagnostic standard of microcephaly because of its complex and diverse etiology . the possible etiology of microcephaly includes genetic cause , perinatal brain injury , postnatal brain injury , or craniosynostosis . no cause for microcephaly was found in 40% of the child victims . in november 2015 , physicians in brazil began to report that there was a surge of the number of microcephaly among newborns , which was possibly linked to zikv infection during the mothers pregnancy . a public health emergency of international concern was dispatched to explore the suspected association between zikv and microcephaly . we reviewed the published literature to search for hints of further research to clarify or verify the association . the potential association between zikv and microcephaly was reported in brazil , french polynesia , columbia , america , and slovenia [ table 1 ] . the studies were mainly conducted from 2013 to 2016 . the cases in the above studies included both resident and nonresident patients . published literature on zikv and microcephaly zikv : zika virus ; rt - pcr : reverse transcription - polymerase chain reaction ; nr : no report ; ci : confidence interval ; hbv : hepatitis b virus ; sds : standard deviations ; rr : relative risk ; ct : computed tomography . we did a comprehensive literature review with the keywords zika and/or microcephaly from inception to may 27 , 2016 , with pubmed . no publication date , language , location , or age restrictions were employed . in this review - based viewpoint , studies were included if they met all of the following criteria : probable or confirmed infant microcephaly and probable or confirmed zikv infection among mothers or infants . the information from selected articles was extracted and entered into microsoft excel 2010 ( microsoft corporation , redmond , washington , usa ) by one reviewer and separately reviewed by another independent reviewer according to the selection criteria . we extracted the following variables : title of the study , study type , reporting place , probable infection place , study period , study description , maternal zikv identification , other maternal infections exception , fetal zikv identification , other fetus infections exception , microcephaly identification , and sample size [ table 1 ] . the characteristics of the included studies are 4 ecological studies , 19 case reports , 2 cohort studies ( 1 in brazil and 1 in columbia ) , and 1 modeling study . in total , 25 articles covered 26 studies : 16 in brazil , 5 in france , 2 in columbia , 2 in usa , and 1 in slovenia . at present , what we know about zikv is limited . a few studies in process are trying to uncover the mystery and gain a better understanding . the disease often presents as mild and nonlethal among symptomatic infections and the self - limiting disease usually lasts about 1 week , while the incubation period varies from several days to 2 weeks . the characteristics of zikv make the infection process unnoticeable due to no obvious symptoms / signs . people do not care about the self - limiting disease with mild symptoms / signs . such characteristics hindered the diagnosis and ecological studies in brazil made the diagnosis imprecise by mainly relying on self - reported symptoms / signs from the patients . the clinical manifestations of zikv infections are similar to dengue or chikungunya fever and are even called a dengue - like illness . nonisolated patients without symptoms or with mild symptoms may be a potential source of infection while mosquitos with zikv are another infection source . specific and clear clinical manifestations of zikv are keys to identify probable patients although not unique . further monitoring should be conducted among the fetus if the patients are pregnant . however , unapparent clinical manifestations hinder the detection of zikv patients , not to mention monitoring among pregnant women . the virus was often misdiagnosed as dengue virus because of a high cross - reactivity rate between them . during the early stage of the zikv outbreak of yap in micronesia , the laboratory test results from the dengue igm kit mistakenly identified the dengue virus . now , the frequently used laboratory testing methods to confirm zikv include both antibody and rna detection in serum , plasma , urine , saliva , amniotic fluid specimens , cerebrospinal fluid specimens , tissues of autopsy and placenta , and conception products . a recent testing technique in the final validation may differentiate the three viruses : zika , dengue , and chikungunya in early of infection , when a physician can not confirm the infection from symptoms / signs only . although laboratory zikv testing methods are advanced , at moment , no treatment is pharmaceutically effective to reduce viral load . interferon may be an option to treat and control acute zikv infections and prevent zikv pass the placenta . until , microcephaly is diagnosed by ultrasonography , and women often had an inaccurate recall of their mild symptoms and signs dating back several weeks , even months . zika - confirmation through the viral load in the acute phase and antibody testing in the convalescence period was absent . there are so many zikv - like symptoms which might occur in diverse diseases including zika , that even latently infected women are still potentially relevant to fetal microcephaly . scientists have realized that many other infections should be ruled out to explore the association between zikv and microcephaly . in the future , we should study and determine how often patients should be tested to timely identify infection status and have a better response to the virus . it is interesting to explore why the virus was recently reported to be related to microcephaly . some viruses directly pass through the placenta to infect the fetus and some viruses cause a placental inflammatory reaction and cause further damage . previous case studies reported that zikv was detected in amniotic fluids and fetal brain tissues though blood / urine was negative for the zikv . zikv identified in amniotic fluids and the fetal brain showed that the viruses can get through the placental barrier . microscopic placental examinations showed calcific and focal chorionic villi or ultrasonography showed an abnormal placenta . animal experimental models in mice showed that zikv had the capability to destroy the central nervous system and had severe pathological changes in mice . zikv may impact the survival and growth of human brain cells by restricting the growth of neurospheres and brain organoids . more appropriate animal models may be needed to solve the mystery between maternal zikv infection and microcephaly . it is necessary to find what happened to the fetus and how zikv generated a smaller head circumference after maternal infection . the operational definition of microcephaly is a head circumference below the value of the mean 2 standard deviations or < 2% of the population of the same race , age , and sex . in brazil , the value was changed from 33 to 32 cm in all full - term newborns to estimate the number of microcephalics at birth . microcephaly diagnosis standard in brazil after december 8 , 2015 , showed a higher specificity and an equal sensitivity than before . a questioned surge in microcephaly prevalence occurred in 2015 ( about 20 cases per 10,000 births ) , compared to that in previous years ( about 1 case per 10,000 births ) . considering the uniform diagnostic standard , brazil now suffering a severe economic recession is facing a challenge to solve the rising unemployment among young people . with no or little income , the youth of child - bearing age are in an economic - disadvantaged situation and this directly affects their nutritional status . the reproductive process study of the dutch famine in 19441945 showed that head circumference declined 2.7% during the famine , and rose 2.4% afterward . randomized controlled studies showed that adequate maternal and fetal nutrition improved prenatal head growth , the difference still existed 7 weeks after birth ( corrected value ) , but catch - up growth at 36 weeks narrowed the gap to borderline significance ( p = 0.046 ) . the economic condition may contribute to a false diagnosis of microcephaly , and postdiagnosis follow - up is necessary to make a definite diagnosis or complete correct diagnosis , considering the limitation of the operational definition in the size of head circumference . while microcephaly is often identified after 20 weeks of gestation by ultrasonography false diagnosis of microcephaly may report inexact relationship between zikv and microcephaly : exaggerated , shrink , or even reverse the links . ecological studies in brazil reported a higher prevalence of microcephaly in 2015 than previous years . a higher prevalence was also found in 15 states among the 19 states having zikv infections as reported by laboratory confirmation . nevertheless , the study can not generate causal reference because it does not rule out unknown and uncontrolled confounders . . 's cohort study showed a positive association between zikv and microcephaly , 4/42 versus 0/16 among the exposure group and the nonexposure group , respectively . the limited - sample size study had a control group with a rash during pregnancy , but the cause of the rash was not clearly diagnosed or analyzed . an american case report showed zikv infections happened to pregnant women in the second and third trimester , who still delivered healthy babies . based on the nature of retrospective surveys , zikv exposure seemed to occur earlier than microcephaly diagnosis and a temporal relation was established in published articles . however , it should be taken with caution because there was either no laboratory zikv confirmation or laboratory exclusion for other infections relevant to microcephaly in the studies . in addition , there were some gaps between exposure and disease confirmation reported in the studies to verify the potential association between zikv and microcephaly [ table 2 ] . the interval between the time when the fetus was first diagnosed as microcephaly and the occurrence of maternal clinical manifestations was on average 13 weeks , with a broad range of 327 weeks . the zikv outbreak may predict a remarkable increase in the number of microcephaly cases 510 months later in brazil . as a nonspecific cause for microcephaly , we need to exclude confounders other than zikv exposure . an accurate microcephaly diagnosis will benefit the causal reference between zikv exposure and outcome of microcephaly . the cerebrospinal fluid test for zikv should be performed to verify the effectiveness of the description by the possibly infected fetus . the interval between zikv infection and diagnosis of microcephaly , literature review an ongoing cohort study in colombia will attempt to confirm zikv infection and rule out many confounders or other infections . the available etiological studies and case studies provide incomplete evidence for the confirmation of a potential association between zikv and microcephaly . however , ongoing cohort studies in brazil and columbia are expected to generate robust evidence for hypothesis verification . a dose - response relationship was observed in three trimesters , with a higher risk in the first trimester in a model based on data from french polynesia . in future , if possible , multicenter study should be performed to explore the dose - response relationship in different trimesters of pregnancy . the estimated rna mutation rate was 0.0010.002 nt year lower than the severe acute respiratory syndrome ( sars ) mutation rate ( 0.003 nt year ) , and far higher than the human dna mutation rate . perhaps this disease can shed light on the virological study of zikv strains isolated in brazil . a study showed that the isolated strains presented a 99% identification with a strain in french polynesia in 2013 ( kj776791 ) , but were much closer to the cambodian strain in 2010 . zikv strains in brazil coming from an asian lineage had 615 amino acids changes , compared to a strain in french polynesia with a 1947 preepidemic prototype . a new clade emergence of chikungunya with mutations caused outbreaks in china and india . can zikv outbreaks in yap of micronesia , french polynesia , and countries of the americas with an asian lineage be attributed to micro - mutations of the viruses ? what kind of changes happened to the virus from sporadic to outbreak ? what about an african lineage ? can the changes of the virus affect the toxicity , replication , and hosting environment ? what is the survival time in blood , amniotic fluids , brain , and other tissues ? some studies reported that the viruses had a prolonged life in amniotic fluids and fetal brain . which genes cause the neurotropism ? can a developing brain structure provide a more hospitable micro - environment for the virus ? what is the full spectrum of defects caused by congenital zikv infection ? sars research may shed light on zikv evolution at different stages in terms of nucleotide and amino acid mutation . hu et al . found that china rhinolophus was the natural host of the sars coronavirus - like virus , rather than palm civets until 2013 , and this was 10 years after the first outbreak of sars . this result was consistent with the fact that few coronavirus - like viruses were separated from palm civets . the present review - based viewpoint claims that it is too early to come up with any explanation or conclusion of the zikv infection . a few studies in process are trying to uncover the mystery and gain a better understanding . the disease often presents as mild and nonlethal among symptomatic infections and the self - limiting disease usually lasts about 1 week , while the incubation period varies from several days to 2 weeks . the characteristics of zikv make the infection process unnoticeable due to no obvious symptoms / signs . people do not care about the self - limiting disease with mild symptoms / signs . such characteristics hindered the diagnosis and ecological studies in brazil made the diagnosis imprecise by mainly relying on self - reported symptoms / signs from the patients . the clinical manifestations of zikv infections are similar to dengue or chikungunya fever and are even called a dengue - like illness . nonisolated patients without symptoms or with mild symptoms may be a potential source of infection while mosquitos with zikv are another infection source . specific and clear clinical manifestations of zikv are keys to identify probable patients although not unique . however , unapparent clinical manifestations hinder the detection of zikv patients , not to mention monitoring among pregnant women . the virus was often misdiagnosed as dengue virus because of a high cross - reactivity rate between them . during the early stage of the zikv outbreak of yap in micronesia , the laboratory test results from the dengue igm kit mistakenly identified the dengue virus . now , the frequently used laboratory testing methods to confirm zikv include both antibody and rna detection in serum , plasma , urine , saliva , amniotic fluid specimens , cerebrospinal fluid specimens , tissues of autopsy and placenta , and conception products . a recent testing technique in the final validation may differentiate the three viruses : zika , dengue , and chikungunya in early of infection , when a physician can not confirm the infection from symptoms / signs only . although laboratory zikv testing methods are advanced , at moment , no treatment is pharmaceutically effective to reduce viral load . interferon may be an option to treat and control acute zikv infections and prevent zikv pass the placenta . until , microcephaly is diagnosed by ultrasonography , and women often had an inaccurate recall of their mild symptoms and signs dating back several weeks , even months . zika - confirmation through the viral load in the acute phase and antibody testing in the convalescence period was absent . there are so many zikv - like symptoms which might occur in diverse diseases including zika , that even latently infected women are still potentially relevant to fetal microcephaly . scientists have realized that many other infections should be ruled out to explore the association between zikv and microcephaly . in the future , we should study and determine how often patients should be tested to timely identify infection status and have a better response to the virus . it is interesting to explore why the virus was recently reported to be related to microcephaly . some viruses directly pass through the placenta to infect the fetus and some viruses cause a placental inflammatory reaction and cause further damage . previous case studies reported that zikv was detected in amniotic fluids and fetal brain tissues though blood / urine was negative for the zikv . zikv identified in amniotic fluids and the fetal brain showed that the viruses can get through the placental barrier . microscopic placental examinations showed calcific and focal chorionic villi or ultrasonography showed an abnormal placenta . animal experimental models in mice showed that zikv had the capability to destroy the central nervous system and had severe pathological changes in mice . zikv may impact the survival and growth of human brain cells by restricting the growth of neurospheres and brain organoids . more appropriate animal models may be needed to solve the mystery between maternal zikv infection and microcephaly . it is necessary to find what happened to the fetus and how zikv generated a smaller head circumference after maternal infection . the operational definition of microcephaly is a head circumference below the value of the mean 2 standard deviations or < 2% of the population of the same race , age , and sex . in brazil , the value was changed from 33 to 32 cm in all full - term newborns to estimate the number of microcephalics at birth . microcephaly diagnosis standard in brazil after december 8 , 2015 , showed a higher specificity and an equal sensitivity than before . a questioned surge in microcephaly prevalence occurred in 2015 ( about 20 cases per 10,000 births ) , compared to that in previous years ( about 1 case per 10,000 births ) . considering the uniform diagnostic standard , brazil now suffering a severe economic recession is facing a challenge to solve the rising unemployment among young people . with no or little income , the youth of child - bearing age are in an economic - disadvantaged situation and this directly affects their nutritional status . the reproductive process study of the dutch famine in 19441945 showed that head circumference declined 2.7% during the famine , and rose 2.4% afterward . randomized controlled studies showed that adequate maternal and fetal nutrition improved prenatal head growth , the difference still existed 7 weeks after birth ( corrected value ) , but catch - up growth at 36 weeks narrowed the gap to borderline significance ( p = 0.046 ) . the economic condition may contribute to a false diagnosis of microcephaly , and postdiagnosis follow - up is necessary to make a definite diagnosis or complete correct diagnosis , considering the limitation of the operational definition in the size of head circumference . while microcephaly is often identified after 20 weeks of gestation by ultrasonography , it 's much earlier than the possible catch - up opportunity . false diagnosis of microcephaly may report inexact relationship between zikv and microcephaly : exaggerated , shrink , or even reverse the links . ecological studies in brazil reported a higher prevalence of microcephaly in 2015 than previous years . a higher prevalence was also found in 15 states among the 19 states having zikv infections as reported by laboratory confirmation . nevertheless , the study can not generate causal reference because it does not rule out unknown and uncontrolled confounders . brasil et al . 's cohort study showed a positive association between zikv and microcephaly , 4/42 versus 0/16 among the exposure group and the nonexposure group , respectively . the limited - sample size study had a control group with a rash during pregnancy , but the cause of the rash was not clearly diagnosed or analyzed . an american case report showed zikv infections happened to pregnant women in the second and third trimester , who still delivered healthy babies . based on the nature of retrospective surveys , zikv exposure seemed to occur earlier than microcephaly diagnosis and a temporal relation was established in published articles . however , it should be taken with caution because there was either no laboratory zikv confirmation or laboratory exclusion for other infections relevant to microcephaly in the studies . in addition , there were some gaps between exposure and disease confirmation reported in the studies to verify the potential association between zikv and microcephaly [ table 2 ] . the interval between the time when the fetus was first diagnosed as microcephaly and the occurrence of maternal clinical manifestations was on average 13 weeks , with a broad range of 327 weeks . the zikv outbreak may predict a remarkable increase in the number of microcephaly cases 510 months later in brazil . as a nonspecific cause for microcephaly , we need to exclude confounders other than zikv exposure . an accurate microcephaly diagnosis will benefit the causal reference between zikv exposure and outcome of microcephaly . the cerebrospinal fluid test for zikv should be performed to verify the effectiveness of the description by the possibly infected fetus . the interval between zikv infection and diagnosis of microcephaly , literature review an ongoing cohort study in colombia will attempt to confirm zikv infection and rule out many confounders or other infections . the available etiological studies and case studies provide incomplete evidence for the confirmation of a potential association between zikv and microcephaly . however , ongoing cohort studies in brazil and columbia are expected to generate robust evidence for hypothesis verification . a dose - response relationship was observed in three trimesters , with a higher risk in the first trimester in a model based on data from french polynesia . in future , if possible , multicenter study should be performed to explore the dose - response relationship in different trimesters of pregnancy . the estimated rna mutation rate was 0.0010.002 nt year lower than the severe acute respiratory syndrome ( sars ) mutation rate ( 0.003 nt year ) , and far higher than the human dna mutation rate . perhaps this disease can shed light on the virological study of zikv strains isolated in brazil . a study showed that the isolated strains presented a 99% identification with a strain in french polynesia in 2013 ( kj776791 ) , but were much closer to the cambodian strain in 2010 . zikv strains in brazil coming from an asian lineage had 615 amino acids changes , compared to a strain in french polynesia with a 1947 preepidemic prototype . a new clade emergence of chikungunya with mutations caused outbreaks in china and india . can zikv outbreaks in yap of micronesia , french polynesia , and countries of the americas with an asian lineage be attributed to micro - mutations of the viruses ? what kind of changes happened to the virus from sporadic to outbreak ? ? can the changes of the virus affect the toxicity , replication , and hosting environment ? what is the survival time in blood , amniotic fluids , brain , and other tissues ? some studies reported that the viruses had a prolonged life in amniotic fluids and fetal brain . which genes cause the neurotropism ? can a developing brain structure provide a more hospitable micro - environment for the virus ? what is the full spectrum of defects caused by congenital zikv infection ? sars research may shed light on zikv evolution at different stages in terms of nucleotide and amino acid mutation . hu et al . found that china rhinolophus was the natural host of the sars coronavirus - like virus , rather than palm civets until 2013 , and this was 10 years after the first outbreak of sars . this result was consistent with the fact that few coronavirus - like viruses were separated from palm civets . the present review - based viewpoint claims that it is too early to come up with any explanation or conclusion of the zikv infection . it is a prerequisite to explore the association of zikv and microcephaly from the perspective of the global health . zikv - like clinical manifestations can corroborate the diagnosis , but maternal and fetal laboratory confirmation , especially autopsy or detection of miscarriage products , could provide more objective evidence . microcephaly diagnosis relies on ultrasonography during pregnancy , physical birth examination , and/or autopsy of still - birth or miscarriage products , and postdelivery follow - up . observational studies , especially in economic - advantaged social environment , should follow the above to ensure an accurate outcome . in addition , animal experiments can disclose the possible mechanism , but robust evidence on gestational human being is overwhelming . bioinformatics techniques and findings on similar damages of central nervous system diseases might shed light on the choice of an appropriate animal model , which will be better than mice . this work was supported by the grant from 2016 presidential fund of capital medical university . this work was supported by the grant from 2016 presidential fund of capital medical university .

objective : to clarify the possible association between the zika virus ( zikv ) and microcephaly and understand where we are in terms of research and the debate on the causation between mild maternal clinical features and severe fetal microcephaly.data sources : we did a comprehensive literature review with the keywords zika and/or microcephaly from inception to may 27 , 2016 , with pubmed.study selection : studies were included and analyzed if they met all of the following criteria : probable or confirmed infant microcephaly and probable or confirmed zikv infection among mothers or infants.results:we emphasize the diagnosis of zikv infection , including maternal clinical manifestations , maternal and fetal laboratory confirmation , and possible autopsy if need . other confounders that may lead to microcephaly should be excluded from the study . we presented the results from clinical manifestations of zikv infection , testing methods evolving but the mechanism of microcephaly uncertain , flexible definition challenging the diagnosis of microcephaly , and limited causal reference on pregnant women . we made analog comparison of severe acute respiratory syndrome and chikungunya virus in terms of dna mutation and global movement to provide further research recommendation . the chance of catch - up growth may decrease the number of pervious diagnosed microcephaly.conclusions:there are some evidence available through mice models and direct isolation of zikv in affected pregnancies on kindly causal relationship but not convincible enough . we analyzed and presented the weakness or limitation of published reports with the desire to shed light to further study directions .

I Zika virus infection and its clinical manifestations Testing techniques evolving while the mechanism of microcephaly uncertain A flexible definition challenging an accurate diagnosis of microcephaly Challenges to available evidence of association between Zika virus and microcephaly Future research recommendations: Enlightenment from global movement tracking by molecular epidemiology C Financial support and sponsorship Conflicts of interest

the zika virus ( zikv ) is a mosquito - borne flavivirus with single - stranded positive rna , which is related to dengue , yellow fever , and japanese encephalitis virus . zikv was first isolated from rhesus monkeys in the zika forest of uganda in 1947 , and later , the virus was named after that place . detected zikv in blood , semen , amniotic fluid , saliva , breast milk , and cerebrospinal fluid provided further evidence of its transmission route and sent an alert to public health . it was first detected in humans in 1954 , with classic and mild clinical manifestations such as rash , conjunctivitis , fever , arthralgia , and arthritis . the disease was sporadically found in asia and africa , and then there were outbreaks in micronesia ( yap island ) , french polynesia , and brazil in 2007 , 2013 , 2015 , respectively . in early 2015 , physicians in northeast brazil first found patients with a dengue - like syndrome , and zikv was later identified as the cause . high transmissibility was found for zikv , with a range of 26.6 among the basic reproduction number , especially among those in a high migration flow . microcephaly is a rare condition defined as a smaller head size , compared to other fetuses / babies of the same age / sex / ethnicity , often with prognosis of intellectual disabilities . the possible etiology of microcephaly includes genetic cause , perinatal brain injury , postnatal brain injury , or craniosynostosis . no cause for microcephaly was found in 40% of the child victims . in november 2015 , physicians in brazil began to report that there was a surge of the number of microcephaly among newborns , which was possibly linked to zikv infection during the mothers pregnancy . a public health emergency of international concern was dispatched to explore the suspected association between zikv and microcephaly . we reviewed the published literature to search for hints of further research to clarify or verify the association . the potential association between zikv and microcephaly was reported in brazil , french polynesia , columbia , america , and slovenia [ table 1 ] . the studies were mainly conducted from 2013 to 2016 . published literature on zikv and microcephaly zikv : zika virus ; rt - pcr : reverse transcription - polymerase chain reaction ; nr : no report ; ci : confidence interval ; hbv : hepatitis b virus ; sds : standard deviations ; rr : relative risk ; ct : computed tomography . we did a comprehensive literature review with the keywords zika and/or microcephaly from inception to may 27 , 2016 , with pubmed . in this review - based viewpoint , studies were included if they met all of the following criteria : probable or confirmed infant microcephaly and probable or confirmed zikv infection among mothers or infants . we extracted the following variables : title of the study , study type , reporting place , probable infection place , study period , study description , maternal zikv identification , other maternal infections exception , fetal zikv identification , other fetus infections exception , microcephaly identification , and sample size [ table 1 ] . the characteristics of the included studies are 4 ecological studies , 19 case reports , 2 cohort studies ( 1 in brazil and 1 in columbia ) , and 1 modeling study . in total , 25 articles covered 26 studies : 16 in brazil , 5 in france , 2 in columbia , 2 in usa , and 1 in slovenia . the disease often presents as mild and nonlethal among symptomatic infections and the self - limiting disease usually lasts about 1 week , while the incubation period varies from several days to 2 weeks . such characteristics hindered the diagnosis and ecological studies in brazil made the diagnosis imprecise by mainly relying on self - reported symptoms / signs from the patients . the clinical manifestations of zikv infections are similar to dengue or chikungunya fever and are even called a dengue - like illness . specific and clear clinical manifestations of zikv are keys to identify probable patients although not unique . further monitoring should be conducted among the fetus if the patients are pregnant . however , unapparent clinical manifestations hinder the detection of zikv patients , not to mention monitoring among pregnant women . during the early stage of the zikv outbreak of yap in micronesia , the laboratory test results from the dengue igm kit mistakenly identified the dengue virus . now , the frequently used laboratory testing methods to confirm zikv include both antibody and rna detection in serum , plasma , urine , saliva , amniotic fluid specimens , cerebrospinal fluid specimens , tissues of autopsy and placenta , and conception products . a recent testing technique in the final validation may differentiate the three viruses : zika , dengue , and chikungunya in early of infection , when a physician can not confirm the infection from symptoms / signs only . although laboratory zikv testing methods are advanced , at moment , no treatment is pharmaceutically effective to reduce viral load . there are so many zikv - like symptoms which might occur in diverse diseases including zika , that even latently infected women are still potentially relevant to fetal microcephaly . scientists have realized that many other infections should be ruled out to explore the association between zikv and microcephaly . in the future , we should study and determine how often patients should be tested to timely identify infection status and have a better response to the virus . it is interesting to explore why the virus was recently reported to be related to microcephaly . zikv identified in amniotic fluids and the fetal brain showed that the viruses can get through the placental barrier . more appropriate animal models may be needed to solve the mystery between maternal zikv infection and microcephaly . the operational definition of microcephaly is a head circumference below the value of the mean 2 standard deviations or < 2% of the population of the same race , age , and sex . in brazil , the value was changed from 33 to 32 cm in all full - term newborns to estimate the number of microcephalics at birth . with no or little income , the youth of child - bearing age are in an economic - disadvantaged situation and this directly affects their nutritional status . the reproductive process study of the dutch famine in 19441945 showed that head circumference declined 2.7% during the famine , and rose 2.4% afterward . randomized controlled studies showed that adequate maternal and fetal nutrition improved prenatal head growth , the difference still existed 7 weeks after birth ( corrected value ) , but catch - up growth at 36 weeks narrowed the gap to borderline significance ( p = 0.046 ) . the economic condition may contribute to a false diagnosis of microcephaly , and postdiagnosis follow - up is necessary to make a definite diagnosis or complete correct diagnosis , considering the limitation of the operational definition in the size of head circumference . while microcephaly is often identified after 20 weeks of gestation by ultrasonography false diagnosis of microcephaly may report inexact relationship between zikv and microcephaly : exaggerated , shrink , or even reverse the links . ecological studies in brazil reported a higher prevalence of microcephaly in 2015 than previous years . a higher prevalence was also found in 15 states among the 19 states having zikv infections as reported by laboratory confirmation . nevertheless , the study can not generate causal reference because it does not rule out unknown and uncontrolled confounders . 's cohort study showed a positive association between zikv and microcephaly , 4/42 versus 0/16 among the exposure group and the nonexposure group , respectively . the limited - sample size study had a control group with a rash during pregnancy , but the cause of the rash was not clearly diagnosed or analyzed . an american case report showed zikv infections happened to pregnant women in the second and third trimester , who still delivered healthy babies . based on the nature of retrospective surveys , zikv exposure seemed to occur earlier than microcephaly diagnosis and a temporal relation was established in published articles . however , it should be taken with caution because there was either no laboratory zikv confirmation or laboratory exclusion for other infections relevant to microcephaly in the studies . in addition , there were some gaps between exposure and disease confirmation reported in the studies to verify the potential association between zikv and microcephaly [ table 2 ] . the interval between the time when the fetus was first diagnosed as microcephaly and the occurrence of maternal clinical manifestations was on average 13 weeks , with a broad range of 327 weeks . the zikv outbreak may predict a remarkable increase in the number of microcephaly cases 510 months later in brazil . as a nonspecific cause for microcephaly , we need to exclude confounders other than zikv exposure . an accurate microcephaly diagnosis will benefit the causal reference between zikv exposure and outcome of microcephaly . the cerebrospinal fluid test for zikv should be performed to verify the effectiveness of the description by the possibly infected fetus . the interval between zikv infection and diagnosis of microcephaly , literature review an ongoing cohort study in colombia will attempt to confirm zikv infection and rule out many confounders or other infections . the available etiological studies and case studies provide incomplete evidence for the confirmation of a potential association between zikv and microcephaly . a dose - response relationship was observed in three trimesters , with a higher risk in the first trimester in a model based on data from french polynesia . in future , if possible , multicenter study should be performed to explore the dose - response relationship in different trimesters of pregnancy . the estimated rna mutation rate was 0.0010.002 nt year lower than the severe acute respiratory syndrome ( sars ) mutation rate ( 0.003 nt year ) , and far higher than the human dna mutation rate . perhaps this disease can shed light on the virological study of zikv strains isolated in brazil . can zikv outbreaks in yap of micronesia , french polynesia , and countries of the americas with an asian lineage be attributed to micro - mutations of the viruses ? can the changes of the virus affect the toxicity , replication , and hosting environment ? what is the survival time in blood , amniotic fluids , brain , and other tissues ? some studies reported that the viruses had a prolonged life in amniotic fluids and fetal brain . what is the full spectrum of defects caused by congenital zikv infection ? sars research may shed light on zikv evolution at different stages in terms of nucleotide and amino acid mutation . found that china rhinolophus was the natural host of the sars coronavirus - like virus , rather than palm civets until 2013 , and this was 10 years after the first outbreak of sars . this result was consistent with the fact that few coronavirus - like viruses were separated from palm civets . the present review - based viewpoint claims that it is too early to come up with any explanation or conclusion of the zikv infection . the disease often presents as mild and nonlethal among symptomatic infections and the self - limiting disease usually lasts about 1 week , while the incubation period varies from several days to 2 weeks . such characteristics hindered the diagnosis and ecological studies in brazil made the diagnosis imprecise by mainly relying on self - reported symptoms / signs from the patients . the clinical manifestations of zikv infections are similar to dengue or chikungunya fever and are even called a dengue - like illness . specific and clear clinical manifestations of zikv are keys to identify probable patients although not unique . however , unapparent clinical manifestations hinder the detection of zikv patients , not to mention monitoring among pregnant women . during the early stage of the zikv outbreak of yap in micronesia , the laboratory test results from the dengue igm kit mistakenly identified the dengue virus . now , the frequently used laboratory testing methods to confirm zikv include both antibody and rna detection in serum , plasma , urine , saliva , amniotic fluid specimens , cerebrospinal fluid specimens , tissues of autopsy and placenta , and conception products . a recent testing technique in the final validation may differentiate the three viruses : zika , dengue , and chikungunya in early of infection , when a physician can not confirm the infection from symptoms / signs only . although laboratory zikv testing methods are advanced , at moment , no treatment is pharmaceutically effective to reduce viral load . until , microcephaly is diagnosed by ultrasonography , and women often had an inaccurate recall of their mild symptoms and signs dating back several weeks , even months . scientists have realized that many other infections should be ruled out to explore the association between zikv and microcephaly . in the future , we should study and determine how often patients should be tested to timely identify infection status and have a better response to the virus . previous case studies reported that zikv was detected in amniotic fluids and fetal brain tissues though blood / urine was negative for the zikv . zikv identified in amniotic fluids and the fetal brain showed that the viruses can get through the placental barrier . more appropriate animal models may be needed to solve the mystery between maternal zikv infection and microcephaly . the operational definition of microcephaly is a head circumference below the value of the mean 2 standard deviations or < 2% of the population of the same race , age , and sex . in brazil , the value was changed from 33 to 32 cm in all full - term newborns to estimate the number of microcephalics at birth . the reproductive process study of the dutch famine in 19441945 showed that head circumference declined 2.7% during the famine , and rose 2.4% afterward . randomized controlled studies showed that adequate maternal and fetal nutrition improved prenatal head growth , the difference still existed 7 weeks after birth ( corrected value ) , but catch - up growth at 36 weeks narrowed the gap to borderline significance ( p = 0.046 ) . the economic condition may contribute to a false diagnosis of microcephaly , and postdiagnosis follow - up is necessary to make a definite diagnosis or complete correct diagnosis , considering the limitation of the operational definition in the size of head circumference . while microcephaly is often identified after 20 weeks of gestation by ultrasonography , it 's much earlier than the possible catch - up opportunity . false diagnosis of microcephaly may report inexact relationship between zikv and microcephaly : exaggerated , shrink , or even reverse the links . ecological studies in brazil reported a higher prevalence of microcephaly in 2015 than previous years . a higher prevalence was also found in 15 states among the 19 states having zikv infections as reported by laboratory confirmation . nevertheless , the study can not generate causal reference because it does not rule out unknown and uncontrolled confounders . 's cohort study showed a positive association between zikv and microcephaly , 4/42 versus 0/16 among the exposure group and the nonexposure group , respectively . the limited - sample size study had a control group with a rash during pregnancy , but the cause of the rash was not clearly diagnosed or analyzed . an american case report showed zikv infections happened to pregnant women in the second and third trimester , who still delivered healthy babies . based on the nature of retrospective surveys , zikv exposure seemed to occur earlier than microcephaly diagnosis and a temporal relation was established in published articles . however , it should be taken with caution because there was either no laboratory zikv confirmation or laboratory exclusion for other infections relevant to microcephaly in the studies . in addition , there were some gaps between exposure and disease confirmation reported in the studies to verify the potential association between zikv and microcephaly [ table 2 ] . the interval between the time when the fetus was first diagnosed as microcephaly and the occurrence of maternal clinical manifestations was on average 13 weeks , with a broad range of 327 weeks . the zikv outbreak may predict a remarkable increase in the number of microcephaly cases 510 months later in brazil . as a nonspecific cause for microcephaly , we need to exclude confounders other than zikv exposure . an accurate microcephaly diagnosis will benefit the causal reference between zikv exposure and outcome of microcephaly . the cerebrospinal fluid test for zikv should be performed to verify the effectiveness of the description by the possibly infected fetus . the interval between zikv infection and diagnosis of microcephaly , literature review an ongoing cohort study in colombia will attempt to confirm zikv infection and rule out many confounders or other infections . the available etiological studies and case studies provide incomplete evidence for the confirmation of a potential association between zikv and microcephaly . a dose - response relationship was observed in three trimesters , with a higher risk in the first trimester in a model based on data from french polynesia . in future , if possible , multicenter study should be performed to explore the dose - response relationship in different trimesters of pregnancy . the estimated rna mutation rate was 0.0010.002 nt year lower than the severe acute respiratory syndrome ( sars ) mutation rate ( 0.003 nt year ) , and far higher than the human dna mutation rate . perhaps this disease can shed light on the virological study of zikv strains isolated in brazil . can zikv outbreaks in yap of micronesia , french polynesia , and countries of the americas with an asian lineage be attributed to micro - mutations of the viruses ? can the changes of the virus affect the toxicity , replication , and hosting environment ? what is the survival time in blood , amniotic fluids , brain , and other tissues ? some studies reported that the viruses had a prolonged life in amniotic fluids and fetal brain . what is the full spectrum of defects caused by congenital zikv infection ? sars research may shed light on zikv evolution at different stages in terms of nucleotide and amino acid mutation . found that china rhinolophus was the natural host of the sars coronavirus - like virus , rather than palm civets until 2013 , and this was 10 years after the first outbreak of sars . this result was consistent with the fact that few coronavirus - like viruses were separated from palm civets . the present review - based viewpoint claims that it is too early to come up with any explanation or conclusion of the zikv infection . it is a prerequisite to explore the association of zikv and microcephaly from the perspective of the global health . zikv - like clinical manifestations can corroborate the diagnosis , but maternal and fetal laboratory confirmation , especially autopsy or detection of miscarriage products , could provide more objective evidence . microcephaly diagnosis relies on ultrasonography during pregnancy , physical birth examination , and/or autopsy of still - birth or miscarriage products , and postdelivery follow - up . bioinformatics techniques and findings on similar damages of central nervous system diseases might shed light on the choice of an appropriate animal model , which will be better than mice .

mitochondrial dysfunction is implicated in the etiology of various diseases , such as neurodegenerative diseases , diabetes , cardiovascular disease , various forms of hepatic disorders , skeletal muscle disorders , sepsis , and psychiatric disorders [ 110 ] . abnormalities in mitochondrial functions such as defects in the electron transport chain ( etc)/oxidative phosphorylation ( oxphos ) system , krebs 's cycle enzymes , and atp production have all been suggested as the primary causative factors in the pathogenesis of neurodegenerative disorders and sepsis . enhanced production of reactive oxygen species ( ros ) and possibly accumulation of mitochondrial ( mt ) dna mutations in postmitotic cells are considered to be contributory factors to age - related degeneration . mitochondria not only generate ros / reactive nitrogen species ( rns ) but are also the main target of their actions . as a result of this action , damage occurs in the mitochondrial respiratory chain , thus producing further increases in free radical generation , ultimately self - inducing a vicious cycle . during the last decade , a number of studies have demonstrated that melatonin plays an effective role in regulating mitochondrial homeostasis . in addition to being a free radical scavenger , melatonin reduces nitric oxide ( no ) generation within mitochondria . it maintains the electron flow , efficiency of oxidative phosphorylation , atp production and bioenergetic function of the cell by regulating respiratory complex activities , ca influx , and mitochondrial permeability transition pore opening [ 1418 ] . in this article , the several mechanisms through which melatonin exerts neuroprotective actions in neurodegenerative disorders such as parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , and huntington 's disease ( hd ) and in a number of mitochondrial dysfunction related conditions such as aging , ischemia / reperfusion ( i / r ) , or septic shock , are reviewed . mitochondria contain multiple copies of a circular genome ( chromosome ) known as mtdna as it has been characterized in humans . although the majority of mitochondrial proteins essential for normal bioenergetic function are encoded by nuclear dna , some proteins needed for etc / oxphos are encoded by mtdna . human mitochondrial genome encodes for 13 peptides of subunits of complexes i , iii , and iv , and atp synthase complex , 22 transfer rnas and 2 ribosomal nucleic acids , while nuclear dna encodes for at least 1000 mitochondrial proteins . the primary function of mitochondria is to generate atp within the cell through the etc resulting in oxphos . the etc , which is present in the inner mitochondrial membrane , comprises a series of electron carriers grouped into four enzyme complexes , namely , complex i ( nadh ubiquinone reductase ) , complex ii ( succinate ubiquinone reductase ) , complex iii ( ubiquinol cytochrome - c - reductase ) , and complex iv ( cytochrome c oxidase ) . the main function of the etc is to convert redox energy into an electrochemical gradient of protons that subsequently causes atp formation from adp and phosphate by atp synthase . the end product of the respiratory chain is water that is generated in a four - electron reduction of molecular oxygen ( o2 ) by complex iv . during this process ( electron leakage especially at complex i and iii ) , a small percentage of o2 is converted into ros , such as superoxide anion radical ( o2 ) and its secondary products hydrogen peroxide ( h2o2 ) and reactive hydroxyl radical ( oh ) [ 23 , 24 ] . under normal conditions , the iron - sulfur cluster n2 of complex i appears to be the primary source of free radicals in the brain [ 23 , 25 ] . mitochondrial no synthase ( mtnos ) localized in the inner mitochondrial membrane is responsible for generating no radical ( no ) from l - arginine . localization of mtnos refers only to myristoylated nnos splice variant alpha , which is also relevant because of its interaction with complex iv . high rates of no synthesis , which typically occur in the calcium - dependent excited state of neurons , can contribute to oxidative and nitrosative stress . the availability of no determines the rates at which the adduct peroxynitrite ( onoo ) and the decomposition products are generated . the free radical no is produced by several forms of nos . in the mitochondria , two nos isoforms , namely constitutive and inducible have been reported ( c - mtnos and i - mtnos ) . since no is an uncharged gaseous compound , it crosses membranes with ease and can enter mitochondria regardless of their neuronal , glial , or vascular origin . no strongly interferes with components of the respiratory chain , in particular cytochrome c oxidase [ 2729 ] . other nitrosation processes , like transnitrosation or reversible nitrosation and nitration as well as irreversible protein and lipid oxidation , can occur . upon entering neuronal mitochondria , no , in combination with onoo ( formed there by combination with o2 from electron leakage ) , not only interferes with respiratory chain complexes but , when it reaches elevated levels , can trigger free radical - mediated chain reactions that in turn destroy protein , lipid , and dna molecules [ 3032 ] . as it has been stated above , damage to the mitochondrial respiratory chain can cause a breakdown of the proton potential , apoptosis , or lead to further generation of free radicals , thus maintaining a vicious cycle that ultimately results in cell death [ 23 , 33 ] . in the mitochondrial - mediated cell death pathway , a nonspecific increase in the permeability of the inner mitochondrial this is known as mitochondrial permeability transition ( mtpt ) , a process associated with the opening of channels in the inner mitochondrial membrane , which in turn causes a flux of molecules of < 1500 daltons . the inner mitochondrial membrane possesses a uniporter to transport ca into the matrix . with ca overload , there is complete uniporter inhibition , mitochondrial swelling , loss of respiratory control , and a release of matrix calcium caused by mtpt pore opening [ 35 , 36 ] . under these conditions , atp is hydrolyzed by mitochondria , the mitochondria undergo swelling , and mitochondrial - mediated apoptosis occurs . a number of mechanisms take part in the control of ros / rns production . among these is the action of the enzyme superoxide dismutase ( sod ) , which occurs in the inner side of the inner mitochondrial membrane ( mn - sod ) , that remove o2 . oh generated from h2o2 in the presence of reduced transition metals are scavenged by the enzyme glutathione peroxidase ( gpx ) , during the process of metabolism of reduced glutathione ( gsh ) to its disulfide ( gssg ) , which in turn is reduced back to gsh by the enzyme glutathione reductase ( grd ) . these enzymes form part of the endogenous antioxidant defense system and suppress ros levels within the cell as well as within the mitochondria . antioxidants such as ascorbate , ubiquinone , or -tocopherol can participate in the mitochondrial antioxidative defense system , but none of them can convert o2 to o2 . it is gsh that participates in scavenging o2 , as well as in several redox reactions and maintains the mttp pore closed . the redox cycling in the mitochondria is very active and serves to prevent significant loss of gsh . this is important because the mitochondria contain gpx and grd activities and depend only on gsh uptake from the cytoplasm to keep adequate gsh levels . since melatonin promotes de novo synthesis of gsh by stimulating the activity of the enzyme -glutamyl - cysteine synthetase and also through its effects on gene expression of gpx , grd , sod , and cat [ 4145 ] helping in the recycling of gsh and in maintaining high gsh / gssg ratio , the role it plays in mitochondrial physiology is important . the synthesis of melatonin that occurs in a number of other tissues and cells does not contribute significantly to the circulating melatonin levels but rather exerts an autocrine or paracrine role . within this category , melatonin synthesis by lymphocytes , skin , the gastrointestinal tract , thymus , serotonin is then acetylated to form n - acetylserotonin through the action of arylalkylamine n - acetyltransferase , one of the key enzymes in melatonin synthesis . n - acetylserotonin is then converted to melatonin by hydroxyindole - o - methyltransferase ( hiomt ) , which has been identified as a rate - limiting enzyme in the biosynthesis of pineal melatonin [ 55 , 56 ] . once formed , melatonin is not stored within the pineal gland but diffuses into the capillary blood and cerebrospinal fluid . in a recent study conducted in humans , csf melatonin levels were found to be higher in the third ventricle compared to the lateral one , thus indicating that melatonin enters the csf through the pineal recess , even during daytime . the brain has much higher concentrations of melatonin than any other tissue in the body . in the circulation circulating melatonin is metabolized mainly in the liver where it is first hydroxylated by cytochrome p450 mono - oxygenases ( isoenzymes cyp1b1 , cyp1a2 , cyp1a1 ) and , thereafter , conjugated with sulphate to be excreted as 6-sulfatoxymelatonin . under certain circumstances these metabolites of melatonin which are formed in the brain , namely , n -acetyl - n -formyl-5-methoxy kynuramine ( afmk ) and n -acetyl-5-methoxykynuramine ( amk ) , also share the antioxidant and anti - inflammatory properties of melatonin [ 64 , 65 ] . melatonin is involved in the control of various physiological functions of the body such as seasonal control of reproductive processes [ 66 , 67 ] , sleep regulation , immune mechanisms [ 69 , 70 ] , and regulation of circadian [ 71 , 72 ] and sleep - wake rhythms [ 73 , 74 ] . in addition to the above - mentioned physiological actions , melatonin in pharmacological doses inhibits tumor growth and may have a potential therapeutic value in treating breast cancer , prostate cancer , melanoma , and cancer of gi tract [ 75 , 76 ] . as the prototype of the chronobiotic class of drugs [ 74 , 7880 ] , melatonin regulates the phase and amplitude of circadian rhythmicity by interaction with mt1 and mt2 receptors expressed in the hypothalamic suprachiasmatic nuclei ( scn ) and other brain areas . melatonin exerts many of its actions via membrane receptors , namely , mt1 and mt2 receptors that are expressed both singly and together in various tissues of the body [ 8183 ] . a third melatonin binding site that was isolated and purified from hamster kidney has been characterized as quinone reductase type 2 . this enzyme belongs to a group of reductases that participate in the protection against oxidative stress by preventing electron transfer reactions of quinones . the melatonin mt1 receptor is coupled to different g proteins that mediate adenylyl cyclase inhibition and phospholipase c activation . the mt2 receptor is coupled to a number of signal transduction mechanisms among them phosphoinositide production , inhibition of adenylyl cyclase , and inhibition of guanylyl cyclase . melatonin gets access freely to all compartments of the cell , and can be especially concentrated in the nucleus and mitochondria [ 15 , 47 , 88 ] . the discovery that melatonin was a remarkably potent scavenger of the particularly reactive , mutagenic , and carcinogenic oh was the finding that initiated numerous studies on melatonin 's role as a protector against free radicals . melatonin was shown to be much more specific than its structural analogs in undergoing reactions which lead to the termination of the radical reaction chain and in avoiding pro - oxidant , c- or o - centered intermediates [ 8991 ] . moreover , melatonin scavenged numerous different free radical species and other oxidants , among which the carbonate radical is important because of its presumed role in mitochondrial damage . although direct radical scavenging has been effective under numerous experimental conditions at clearly supraphysiological melatonin concentrations , its relevance at physiological levels has been questioned for reasons of stoichiometry . even though a single melatonin molecule may generate products in a scavenger cascade which may collectively eliminate up to ten free radicals , such findings from chemical systems a possible indirect action as mediated by upregulation of antioxidant enzymes by melatonin was proposed ( reviewed in [ 96 , 97 ] ) . an alternate concept has been put forth to explain the protective effect at the level of radical generation rather than detoxification of already formed radicals [ 98100 ] . if melatonin is capable of decreasing the processes leading to enhanced radical formation , this might be achieved by low , physiological , concentrations of the methoxyindole . apart from oxidants released by leukocytes , the isoforms of nad(p)h oxidases ( nox ) and mitochondria should be mentioned as main sources of free radicals in the cell . a recent study showed that melatonin inhibits free radical formation in microglia exposed to amyloid-142 by preventing the phosphorylation of the p47 nox subunit via the pi3k / akt pathway thus giving support to the hypothesis that melatonin has a protective effect at the level of radical generation . melatonin 's ability to influence mitochondrial function has been tested both in vivo and in vitro . in initial in vivo studies conducted on rats , etc complexes from the mitochondria of brain and liver tissues melatonin was found to increase the activity of c - i and c - iv of mitochondrial etc in a time - dependent manner , c - ii and c - iii not being affected . ruthenium red was found to impair mitochondrial metabolism by reducing etc and atp synthesis through its cellular oxidative stress action . inhibition of both c - i and c - iv of the etc were noted . injections of melatonin were found to counteract the inhibitory effect of ruthenium red on c - i , c - iv , and gpx enzyme . in an in vitro study , the effect of melatonin on t - butyl hydroperoxide- ( t - bhp- ) induced mitochondrial oxidative stress was evaluated . t - bhp depletes mitochondrial gsh and inhibits gpx and grd activities . in mitochondrial preparations , 100 nm melatonin was found to prevent the oxidation of gsh to gssg induced by t - bhp and also restored the normal activities of both gpx and grd . melatonin increased c - i and c - iv in a dose - dependent manner , the effect being significant at 1 nm . melatonin also counteracted cyanide - induced inhibition of c - iv showing thereby that melatonin can increase the activity of etc coupled to oxphos and increase atp synthesis in normal mitochondria as well as in mitochondria depleted of atp by cyanide . the effects of melatonin in regulating complexes i and iv presumably do not reflect its antioxidant role but indicates an interaction with etc complexes by donating and accepting electrons , thereby increasing electron flow , an effect not shared by other antioxidants . the major consequence of melatonin 's action on mitochondria may be avoidance of damage and dysfunction thus contributing to increase atp production [ 16 , 107 ] . melatonin increases the efficiency of etc thereby limiting electron leakage and free radical generation , and consequently promoting protein synthesis [ 17 , 47 ] . the possible mechanism by which melatonin controls mitochondrial respiration in the liver was examined in two groups of rats . in one group , melatonin ( 16 to 50 g / ml ) or vehicle was administered for a period of 45 days . in another study , rats received melatonin in drinking water ( 50 g / ml ) for 45 days or the same amount for 30 days , followed by a withdrawal period of 15 days . at sacrifice , the liver mitochondrial fraction was prepared and oxygen consumption was measured in the presence of excess concentration dl-3 -hydroxybutyrate or l - succinate . melatonin treatment decreased krebs 's cycle substrate - induced respiration significantly at both examined doses . the stimulation of mitochondrial respiration , caused by excess concentration of substrate , recovered after melatonin withdrawal . this study shows that melatonin can protect mitochondria from oxidative damage resulting from overstimulation of cellular respiration caused by excess krebs ' cycle substrate . a similar study on melatonin 's mechanism of action on mitochondrial respiration was carried out by another group of investigators . in this study , mitochondria from mouse liver cells was incubated in vitro with melatonin at concentrations ranging from 1 nm to 1 mm . melatonin decreased oxygen consumption , inhibited the increase in oxygen flux in the presence of excess of adp , reduced membrane potential and inhibited the production of o2 and h2o2 . melatonin was also able to maintain the efficiency of oxidative phosphorylation and atp synthesis by increasing the activity of the respiratory complexes i , iii , and iv . these effects were attributed to the intramitochondrial presence of melatonin , thus showing melatonin 's participation in the physiological regulation of mitochondrial homeostasis . melatonin 's action in preventing the opening of the mtpt pore given by oxidative stress caused by t - bhp was shown in another study on primary skeletal muscle cultures . using isolated mitochondria , melatonin ( 1100 m ) fully prevented myotube death induced by t - bhp . melatonin desensitized the mtpt pore to ca and prevented t - bhp - induced mitochondrial swelling and gsh oxidation . the inhibition of the mtpt pore opening by melatonin was suggested as an explanation for the protective action of melatonin against oxidative stress in myotubes . recently , the role of melatonin on cardiolipin and mitochondrial bioenergetics was explored [ 110 , 111 ] . cardiolipin , a phospholipid located at the level of inner mitochondrial membrane , is required for several mitochondrial bioenergetic processes as well as in mitochondrial - dependent steps of apoptosis . alterations in cardiolipin structure , content , and acyl chain composition have been associated with mitochondrial dysfunction in various tissues under a variety of pathophysiological conditions . melatonin was reported to protect the mitochondria from oxidative damage by preventing cardiolipin oxidation and this may explain , at least in part , the beneficial effect of this molecule in mitochondrial physiology [ 110 , 111 ] . the enhanced production of ros and accumulation of mtdna mutations in mitochondria may be contributory factors to human aging . many studies have established that the respiratory function of mitochondria declines with age [ 113115 ] . the increased production of free radicals such as o2 and h2o2 in mitochondria along with advancing age has been demonstrated [ 116 , 117 ] . accumulation of mtdna mutations can cause defective respiratory function resulting in enhanced production of ros . many of these mtdna mutations begin after adults reach their the mid thirties and accumulate with age in postmitotic tissues . overproliferation of abnormal mitochondria has been shown to occur in the muscle of aged individuals and in patients with mitochondrial myopathies [ 115 , 118 ] . the presence of these defective mitochondria is one of the factors involved in the decline in respiratory function during the aging process . enhanced activation of the mtpt pore in the brain and liver of aging mice has also been demonstrated . this in turn causes the release of proapoptotic factors from the intermembrane space of mitochondria . hence , an increased mitochondrial ros production , oxidative stress , respiratory functional decline , and susceptibility to apoptosis constitute central events in the aging process . the mechanism of the aging process can be studied in experimental models like the senescence accelerated mouse ( samp8 ) and senescence resistant mouse ( samr1 ) . the accelerated aging seen in this mouse strain is due to oxidative stress , which occurs with greater intensity as compared to the samr1 . at an age of 12 months , reductions in the activities of respiratory complexes i and iv have been demonstrated in liver mitochondria of samp8 mice , but not in samr1 mice . greater concentrations of lipid peroxidation products in the liver and brain homogenates were found in samp8 mice as compared to samr1 mice . in contrast to this , the concentration of the antioxidant enzyme gpx from samp8 mice at 12 months of age was found to be significantly lower than in samr1 mice . these studies support the conclusion that excess free radical generation coupled with less effective defense against the oxidative stress is responsible for alteration of mitochondrial function seen in samp 8 mice [ 122124 ] . since melatonin can readily reach the mitochondria due to its high lipophilicity , it seems feasible that , upon its entry into the cell , it could become concentrated at a superficial position in lipid layers near the polar heads of membrane phospholipids , a key place to function as a free radical scavenger . the effect of melatonin on age - dependent changes in the redox status of mitochondria in the heart and diaphragm was thus evaluated . melatonin , administered in the drinking water at a dose of 10 mg / kg for 9 months , was shown ( i ) to counteract the age - dependent increase in lipoperoxidation level , ( ii ) to increase gsh content in muscle mitochondria of both samp8 and samr1 mice , ( iii ) to counteract the reduction of gsh / gssg ratio in diaphragmatic mitochondria of samr1 and samp8 mice , ( iv ) to increase the activity of the antioxidant enzymes gpx and grd in the mitochondria of samp8 mice with no effect on samr 1 mice , and ( v ) to increase the activity of grd in samr1 mice . therefore , long - term melatonin administration prevented the age - dependent mitochondrial stress in both senescence - accelerated and senescence - resistant mice . as a continuation of the above - mentioned study , the effect of melatonin at earlier stages of the life span was evaluated at the 5th and 10th months of age in samp8 and samr1 mice . mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation , gsh and gssg , and the activities of gpx and grd in diaphragmatic mitochondria . age did not affect diaphragmatic mitochondrial levels of lipid peroxidation in samr1 mice but increased them in samp8 animals . when melatonin was administered in the drinking water at a daily dose of 10 mg / kg , the level of lipid peroxidation in 10-month - old samp8 mice was reduced to that found at 5 months of age . the decrease of gpx seen with age in both strains of mice was counteracted by melatonin administration with a higher effect in samp8 mice . as far as grd , although age caused significant reductions in both strains of mice , treatment with melatonin partially restored grd activity in samr1 mice only . the chronic administration of melatonin significantly increased complex ii and complex iii activity in samr1 and samp 8 mice and complex iv activity in samp 8 mice . measurement of atp levels and atp / adp ratio showed that treatment with melatonin counteracted the reduction in atp levels and atp / adp ratio found in samp8 mice . as far as longevity , melatonin treatment increased the half - life of samp8 mice from 16 to 22 months while longevity increased from 23 to 27 months . melatonin 's beneficial effects on longevity were significantly higher in samp8 mice than in samr1 mice . the study thus showed that melatonin administration counteracted age - dependent oxidative damage and mitochondrial dysfunction in senescence accelerated mice by improving mitochondrial function as reflected by the increase in atp production and a prolonged longevity . another study using rat brain mitochondria was designed to evaluate the beneficial effects of melatonin on age - associated reductions in mitochondrial bioenergetic function . mitochondria from control and aged rats treated or not with melatonin were obtained , and various bioenergetic parameters such as complex i activity , rates of state 3 respiration , mitochondrial h2o2 production , and membrane potential were evaluated . melatonin was found to prevent the significant age - related changes that occurred in all of these parameters in untreated animals . the ability to prevent complex i dysfunction and cardiolipin peroxidation was melatonin 's principal mechanism of action for achieving its effects . age - associated impairments in mitochondrial oxphos found in the brain of samp8 mice did not exhibit any major gender differences . however , a higher reduction in the gsh / gssg ratio at 10 months of age in female than in male samp8 mice has been reported in one study . chronic melatonin treatment completely prevented age - dependent oxidative stress as assessed by the recovery of the gsh / gssg in mitochondria of brain samples of both male and female mice . the ability of melatonin to prevent gsh loss with age probably reflects its influence on the activities of the gsh redox cycle enzymes . this is not due to reduction of brain mitochondria with aging but has been demonstrated to be due to diminished activities of respiratory complexes i , ii , and iii . mitochondrial dysfunction with aging is not an irreversible process as shown by studies using melatonin to prevent age - dependent declines in bioenergetic impairment of brain mitochondria in mice . i / r lesions are seen in many clinical conditions and are triggered by multiple factors including overproduction of ros [ 131133 ] . for example , ros produced at the level of complex i and iii of the respiratory chain are responsible for injury seen in cardiac i / r [ 134 , 135 ] as well as in stroke . the available evidence indicates the opening of the mtpt pore is responsible for the cardiomyocyte death occurring during i / r . while these pores remain closed during the ischemic period , at reperfusion the influx of ca into the mitochondria and an associated burst of ros production caused the opening of mtpt channels . this leads to mitochondrial depolarization , swelling , and rupture of the external mitochondrial membrane , with uncoupling of the respiratory chain and efflux of cytochrome c and other proapoptotic factors , all of which lead to either cell death by either apoptosis or necrosis . melatonin has also been shown to be effective in protecting the cardiac musculature against i / r [ 137139 ] . melatonin 's protective effect during i / r has been attributed to its action in inhibiting the mtpt pore , and in preserving the content and integrity of cardiolipin molecules . the fact that melatonin treatment inhibits both mtpt pore opening and cardiolipin peroxidation following i / r suggests a possible link between these two processes . it has been suggested that that increased levels of peroxidized cardiolipin together with increased ca overload can contribute to the mtpt pore opening during reperfusion . it must be noted that a significant cytoprotective effect of melatonin was described at a very early phase of a myocardial infarction , when i / r and thus oxidative damage were minimal , indicating that not all cardiac protective effects of melatonin are attributable to its antioxidant activity . septic shock is a lethal condition caused by a complex chain of pathogen - induced events involving immune cells , the epithelium , the endothelium , and the neuroendocrine system . the lethal effects of septic shock are associated with the production and release of numerous proinflammatory mediators as well as no and ros , thus inducing massive apoptosis . since many years ago , research interest was focused on the hypothesis that mitochondrial dysfunction plays a pivotal role in septic shock . this hypothesis was indeed confirmed by the finding of decreased respiratory complex i activity and low levels of atp levels in skeletal muscle biopsies obtained from critically ill patients with septic shock . increased no production and decreased levels of gsh were also found in septic shock patients . the protective effect of melatonin on the lethal effects of bacterial lipopolysaccharide ( lps ) on respiratory complex activities i and iv and a mitochondrial subform of inos ( mt inos ) activity was examined in liver and lung mitochondria of rats . lps administered at a dose of 10 mg / kg i.v . was found to increase mt inos activity and no , an effect that was greater in old rats than in young ones . melatonin administration ( 60 mg / kg , i.p . ) prevented lps toxicity by decreasing mt inos activity and no production . it also counteracted lps - induced inhibition of the activity of respiratory complexes i and iv . it is interesting to note that the effectiveness of melatonin to prevent the mitochondrial failure that occurs during endotoxemia were more pronounced in older animals than in young ones . using a long - term ( 3-day ) rat model of sepsis , the model comprises a long - term , fluid - resuscitated , fecal peritonitis model utilizing male wistar rats that closely replicates human physiological , biochemical , and histological findings with a 40% mortality . compared to sham - operated controls severely septic rats had lower ( 2022% ) hepatic and muscle complex i activities . moderate increases in nitrite / nitrate production were seen in both muscle and liver peaking at 2448 h and returning to sham - operated levels at 72 h. a fall in gsh was associated with lower complex i and increased no production was also demonstrated . a number of animal model studies and a few clinical observations have now shown that melatonin is beneficial for treating septic shock ( see , for a recent review , ) . to examine the effect of melatonin on changes in mt inos in septic skeletal muscles wild - type ( inos ) and inos knockout ( inos ) mice after sepsis , increases in mt inos and no levels , and decreases in electron chain activity were noted in inos mice but not in inos mice . in addition , an increase in oxidative stress was also found as indicated by an increase in lipid peroxidation products as well as a reduction in gsh levels and in the activities of gpx and grd . melatonin treatment counteracted the changes in mt inos activities and oxidative stress , and , further , restored the mitochondrial respiratory chain in inos mice . this study confirmed that mtnos is responsible for the mitochondrial dysfunction seen during sepsis and thus supported the conclusion that melatonin has the ability to protect against mt inos - mediated mitochondrial failure . a similar study performed in mitochondria isolated from the diaphragm of septic mice indicated that melatonin administration to inos mice counteracted mt inos induction and respiratory chain failure , and , finally , normalized the redox state after sepsis . considering the effects of melatonin and its virtual absence of toxicity , the use of melatonin along with conventional therapy to preserve mitochondrial bioenergetics as well as to limit inflammatory response and oxidative damage should be taken into account as a treatment option . pd is a neurodegenerative disorder with a multifactorial etiology , mainly characterized by the death of dopaminergic neurons in the pars compacta of substantia nigra and by the formation of lewy bodies . the initiating factor in pd is increased release of free radicals and enhanced signs of oxidative stress as demonstrated in brains of pd patients [ 150152 ] . although the molecular mechanisms responsible for the pathogenesis of ad are still under intense investigation , reduced complex i activity in the substantia nigra and loss of gsh have been reported in pd patients . the selective inhibition of complex i in the etc compromises energy availability and leads to apoptosis and death of the dopaminergic cells of substantia nigra . a commonly accepted model of pd is that achieved by the systemic or intracerebral administration of neurotoxins like 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine ( mptp ) . the loss of dopamine neurons occurring in these animal models causes severe sensory and motor impairment which in turn gives rise to tremor , rigidity and akinesia similar to those seen in pd patients [ 155 , 156 ] . the active glial metabolite of mptp , 1-methyl-4-phenylpyridinium ( mpp ) , is taken up into the dopaminergic neurons through the dopamine transporter , and then accumulates in the mitochondria of substantia nigra pars compacta . by binding to complex i , mpp increases the production of ros and enhances oxidative stress causing reduction of atp and death of cells in the substantia nigra . in the striatum , the damage caused by mpp is attributed to increased generation of o2 that reacts with no to generate the highly toxic onoo . this impairs mitochondrial function as a result of irreversible inhibition of all etc complexes leading to the death of neuronal cells . a neuroprotective effect of melatonin in isolated rat striatal synaptosomes and liver mitochondria treated with mpp has been demonstrated . melatonin prevented the inhibition of mitochondrial respiration by limiting the interaction of mpp with complex i of etc . although the role of ros generation has been demonstrated in the etiology of pd , the participation of mtnos in the mitochondrial dysfunction and nigrostriatal degeneration has only recently been examined . in a study conducted in adult male mice , mptp was administered at a dose of 15 mg / kg in four separate doses . animals also received melatonin or its metabolite amk ( 20 mg / kg ) 1 h prior to mptp injection . the administration of melatonin or amk concomitantly with mptp significantly reduced the inos activity stimulated by mptp . in the mitochondria , two nos isoforms , namely , constitutive and inducible , mptp administration significantly increased the activity of mt inos without affecting mt constitutive nos activity . interestingly , mptp administration induced i - mtnos activity in the mitochondria of substantia nigra whereas i - mtnos was only slightly induced by mptp in striatal mitochondria . treatment with either melatonin or its brain metabolite amk effectively counteracted i - mtnos induction , oxidative stress , and mitochondrial dysfunction induced by mptp . the nitrosative / oxidative stress reduction seen after therapeutic intervention with melatonin or amk in mptp treated mice was attributed to an effect in preventing damage to mitochondria . as already mentioned , mitochondria take up melatonin in a concentration- and time - dependent manner . a small number of controlled trials indicate that melatonin is useful to treat disturbed sleep in pd [ 162 , 163 ] , particularly rapid eye movement - associated sleep behavior disorder [ 164169 ] . whether melatonin or the recently introduced melatonergic agents ( ramelteon , agomelatine ) have the potential for treating insomnia in pd patients and , more generally , for arresting the progression of pd merits further investigation . several recent studies have confirmed the involvement of mitochondrial ros production and abnormal mitochondrial function in the pathophysiology of ad [ 170177 ] . ad is characterized by extracellular senile plaques of aggregated -amyloid ( a ) and intracellular neurofibrillary tangles that contain hyperphosphorylated tau protein . the resulting clinical effect is a progressive loss of memory and deterioration of cognition . there is substantial evidence to prove that mitochondrial toxicity is linked to the progressive accumulation of mitochondrial a . in the early phase of ad , inhibitors of and -secretase can be therapeutically effective to halt ad disease progression by inhibition of the protein misfolding of a into neurotoxic oligomeric aggregates . an increased expression of mitochondrial antioxidant enzyme sod-2 has been shown to prevent memory deficits and amyloid plaque deposition associated with ad . although a hypothetical occurrence of mutations in mtdna could cause increased oxidative stress and energy failure , no causative mutations in mtdna have been detected in ad so far . several actions of melatonin have been described which antagonize the deleterious effects of a. the effects of melatonin can be grouped as ( i ) antioxidant , including influences on mitochondrial metabolism ; ( ii ) antifibrillogenic , blocking a synthesis ; ( iii ) cytoskeletal , including suppression of tau protein hyperphosphorylation ( for a recent review see ) . the antifibrillogenic effects of melatonin were observed not only in vitro but also in vivo in transgenic mouse models [ 182184 ] . one such pathway is the bcl-2 pathway , which stabilizes mitochondrial function by antiapoptotic bcl-2 family modulators . melatonin inhibited free radical formation in microglia exposed to amyloid-142 by preventing the phosphorylation of the p47 nox subunit via the pi3k / akt pathway . in view of the consequences of excitation - dependent calcium overload on mitochondrial membrane potential and mtpt pore sensitivity towards excitotoxins like a , the actions of melatonin at the level of this important cellular compartment deserve particular attention . modulation of mitochondrial ca handling has been suggested as the potential pharmacological target for ad . in a recent study , a possible melatonin prevention of damage induced by a was evaluated in young and senescent hippocampal neurons . rat hippocampal neurons were incubated with a2535 and cell viability , mitochondrial membrane potential , atp , and the activity of the respiratory chain complexes were measured . cells exposed to a2535 showed decreased mitochondrial membrane potential , inhibited activity of respiratory chain complexes , and a depletion of atp levels . molecular studies undertaken with mitochondrial preparations suggest that melatonin has a therapeutic value in treating ad through its antiapoptotic activities . as outlined , melatonin acts at different levels relevant to the development and manifestation of ad . the antioxidant , mitochondrial , and antiamyloidogenic effects may be seen as a possibility of interfering with the onset of the disease . mild cognitive impairment ( mci ) is an etiologically heterogeneous syndrome characterized by cognitive impairment shown by objective measures adjusted for age and education in advance of dementia . a small number of controlled trials indicate that melatonin is useful to treat mci and to prevent progression to ad [ 181 , 191194 ] . a huntington 's chorea animal model was developed by using 3-nitropropionic acid , an inhibitor of mitochondrial complex ii . in this model , that replicates the neurochemical , histological , and clinical features of hd current evidence from genetic models of hd including mutation of the huntingtin gene ( mhtt ) , supports the mitochondrial dysfunction as major cause of the disease , with respiratory chain impairment relegated to a late secondary event . upstream events include defective mitochondrial calcium handling and impaired atp production . also , transcription abnormalities affecting mitochondria composition , reduced mitochondria trafficking to synapses , and direct interference with mitochondrial structures enriched in striatal neurons , are possible mechanisms by which mhtt amplifies striatal vulnerability . evidence is lacking on whether melatonin 's action on mitochondria could affect evolution in the genetic model of hd . at least on the accumulation of insoluble protein aggregates in intra- and perinuclear inclusions in hd melatonin had little or no inhibitory effect on huntingtin aggregation . mitochondrial dysfunction is implicated as the major causative factor in a variety of conditions such as the aging process , i / r , and septic shock . in addition , abnormal mitochondrial function , decreased respiratory enzyme complex activities , increased electron leakage , opening of the mtpt pore , and increased ca entry have all been shown to play a role in the pathophysiology of neurodegenerative disorders such as pd , ad , and hd . in addition to aging as a factor for low melatonin levels , it is well documented that there is a huge interindividual variation in circulating levels of melatonin which is stable within individuals and , which has been hypothesized to be genetic in origin [ 198201 ] . there is now evidence that there are polymorphisms in the gene for hiomt , the rate limiting enzyme in melatonin synthesis , and that the hiomt transcript level depends significantly on the genotype distributions . thus , there may be a genetically determined low melatonin syndrome that causes a predisposition to a variety of diseases . among the number of substances involved in maintaining mitochondrial bioenergetics a number of in vivo and in vitro studies in animals ( table 1 ) indicate that melatonin may emerge as a major therapeutic candidate to preserve the bioenergetic function of mitochondria . double - blind placebo controlled studies are needed to assess to what extent melatonin has therapeutic value in the treatment of the several disorders associated with mitochondrial dysfunction . s. r. pandi - perumal is a stockholder and the president and chief executive officer of somnogen inc . , a new york corporation . he declared no competing interests that might be perceived to influence the content of this paper . all remaining authors declare that they have no proprietary , financial , professional , nor any other personal interest of any kind in any product or services and/or company that could be construed or considered to be a potential conflict of interest that might have influenced the views expressed in this paper .

mitochondrial dysfunction is considered one of the major causative factors in the aging process , ischemia / reperfusion ( i / r ) , septic shock , and neurodegenerative disorders like parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , and huntington 's disease ( hd ) . increased free radical generation , enhanced mitochondrial inducible nitric oxide ( no ) synthase activity , enhanced no production , decreased respiratory complex activity , impaired electron transport system , and opening of mitochondrial permeability transition pore all have been suggested as factors responsible for impaired mitochondrial function . melatonin , the major hormone of the pineal gland , also acts as an antioxidant and as a regulator of mitochondrial bioenergetic function . both in vitro and in vivo , melatonin was effective for preventing oxidative stress / nitrosative stress - induced mitochondrial dysfunction seen in experimental models of pd , ad , and hd . in addition , melatonin is known to retard aging and to inhibit the lethal effects of septic shock or i / r lesions by maintaining respiratory complex activities , electron transport chain , and atp production in mitochondria . melatonin is selectively taken up by mitochondrial membranes , a function not shared by other antioxidants . melatonin has thus emerged as a major potential therapeutic tool for treating neurodegenerative disorders such as pd or ad , and for preventing the lethal effects of septic shock or i / r .

1. Introduction 2. Mitochondrial Function and Free Radical Generation 3. Melatonin: Biosynthesis, Metabolism, and Receptors 4. Melatonin's Free Radical Scavenging and Antioxidant Actions 5. Melatonin and Mitochondrial Function 6. Conclusions Conflict of Interests

mitochondrial dysfunction is implicated in the etiology of various diseases , such as neurodegenerative diseases , diabetes , cardiovascular disease , various forms of hepatic disorders , skeletal muscle disorders , sepsis , and psychiatric disorders [ 110 ] . abnormalities in mitochondrial functions such as defects in the electron transport chain ( etc)/oxidative phosphorylation ( oxphos ) system , krebs 's cycle enzymes , and atp production have all been suggested as the primary causative factors in the pathogenesis of neurodegenerative disorders and sepsis . as a result of this action , damage occurs in the mitochondrial respiratory chain , thus producing further increases in free radical generation , ultimately self - inducing a vicious cycle . in addition to being a free radical scavenger , melatonin reduces nitric oxide ( no ) generation within mitochondria . it maintains the electron flow , efficiency of oxidative phosphorylation , atp production and bioenergetic function of the cell by regulating respiratory complex activities , ca influx , and mitochondrial permeability transition pore opening [ 1418 ] . in this article , the several mechanisms through which melatonin exerts neuroprotective actions in neurodegenerative disorders such as parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , and huntington 's disease ( hd ) and in a number of mitochondrial dysfunction related conditions such as aging , ischemia / reperfusion ( i / r ) , or septic shock , are reviewed . the etc , which is present in the inner mitochondrial membrane , comprises a series of electron carriers grouped into four enzyme complexes , namely , complex i ( nadh ubiquinone reductase ) , complex ii ( succinate ubiquinone reductase ) , complex iii ( ubiquinol cytochrome - c - reductase ) , and complex iv ( cytochrome c oxidase ) . during this process ( electron leakage especially at complex i and iii ) , a small percentage of o2 is converted into ros , such as superoxide anion radical ( o2 ) and its secondary products hydrogen peroxide ( h2o2 ) and reactive hydroxyl radical ( oh ) [ 23 , 24 ] . mitochondrial no synthase ( mtnos ) localized in the inner mitochondrial membrane is responsible for generating no radical ( no ) from l - arginine . high rates of no synthesis , which typically occur in the calcium - dependent excited state of neurons , can contribute to oxidative and nitrosative stress . upon entering neuronal mitochondria , no , in combination with onoo ( formed there by combination with o2 from electron leakage ) , not only interferes with respiratory chain complexes but , when it reaches elevated levels , can trigger free radical - mediated chain reactions that in turn destroy protein , lipid , and dna molecules [ 3032 ] . in the mitochondrial - mediated cell death pathway , a nonspecific increase in the permeability of the inner mitochondrial this is known as mitochondrial permeability transition ( mtpt ) , a process associated with the opening of channels in the inner mitochondrial membrane , which in turn causes a flux of molecules of < 1500 daltons . among these is the action of the enzyme superoxide dismutase ( sod ) , which occurs in the inner side of the inner mitochondrial membrane ( mn - sod ) , that remove o2 . antioxidants such as ascorbate , ubiquinone , or -tocopherol can participate in the mitochondrial antioxidative defense system , but none of them can convert o2 to o2 . since melatonin promotes de novo synthesis of gsh by stimulating the activity of the enzyme -glutamyl - cysteine synthetase and also through its effects on gene expression of gpx , grd , sod , and cat [ 4145 ] helping in the recycling of gsh and in maintaining high gsh / gssg ratio , the role it plays in mitochondrial physiology is important . within this category , melatonin synthesis by lymphocytes , skin , the gastrointestinal tract , thymus , serotonin is then acetylated to form n - acetylserotonin through the action of arylalkylamine n - acetyltransferase , one of the key enzymes in melatonin synthesis . n - acetylserotonin is then converted to melatonin by hydroxyindole - o - methyltransferase ( hiomt ) , which has been identified as a rate - limiting enzyme in the biosynthesis of pineal melatonin [ 55 , 56 ] . once formed , melatonin is not stored within the pineal gland but diffuses into the capillary blood and cerebrospinal fluid . under certain circumstances these metabolites of melatonin which are formed in the brain , namely , n -acetyl - n -formyl-5-methoxy kynuramine ( afmk ) and n -acetyl-5-methoxykynuramine ( amk ) , also share the antioxidant and anti - inflammatory properties of melatonin [ 64 , 65 ] . melatonin is involved in the control of various physiological functions of the body such as seasonal control of reproductive processes [ 66 , 67 ] , sleep regulation , immune mechanisms [ 69 , 70 ] , and regulation of circadian [ 71 , 72 ] and sleep - wake rhythms [ 73 , 74 ] . in addition to the above - mentioned physiological actions , melatonin in pharmacological doses inhibits tumor growth and may have a potential therapeutic value in treating breast cancer , prostate cancer , melanoma , and cancer of gi tract [ 75 , 76 ] . as the prototype of the chronobiotic class of drugs [ 74 , 7880 ] , melatonin regulates the phase and amplitude of circadian rhythmicity by interaction with mt1 and mt2 receptors expressed in the hypothalamic suprachiasmatic nuclei ( scn ) and other brain areas . the mt2 receptor is coupled to a number of signal transduction mechanisms among them phosphoinositide production , inhibition of adenylyl cyclase , and inhibition of guanylyl cyclase . melatonin gets access freely to all compartments of the cell , and can be especially concentrated in the nucleus and mitochondria [ 15 , 47 , 88 ] . the discovery that melatonin was a remarkably potent scavenger of the particularly reactive , mutagenic , and carcinogenic oh was the finding that initiated numerous studies on melatonin 's role as a protector against free radicals . melatonin was shown to be much more specific than its structural analogs in undergoing reactions which lead to the termination of the radical reaction chain and in avoiding pro - oxidant , c- or o - centered intermediates [ 8991 ] . moreover , melatonin scavenged numerous different free radical species and other oxidants , among which the carbonate radical is important because of its presumed role in mitochondrial damage . a recent study showed that melatonin inhibits free radical formation in microglia exposed to amyloid-142 by preventing the phosphorylation of the p47 nox subunit via the pi3k / akt pathway thus giving support to the hypothesis that melatonin has a protective effect at the level of radical generation . melatonin 's ability to influence mitochondrial function has been tested both in vivo and in vitro . in initial in vivo studies conducted on rats , etc complexes from the mitochondria of brain and liver tissues melatonin was found to increase the activity of c - i and c - iv of mitochondrial etc in a time - dependent manner , c - ii and c - iii not being affected . in an in vitro study , the effect of melatonin on t - butyl hydroperoxide- ( t - bhp- ) induced mitochondrial oxidative stress was evaluated . melatonin also counteracted cyanide - induced inhibition of c - iv showing thereby that melatonin can increase the activity of etc coupled to oxphos and increase atp synthesis in normal mitochondria as well as in mitochondria depleted of atp by cyanide . the effects of melatonin in regulating complexes i and iv presumably do not reflect its antioxidant role but indicates an interaction with etc complexes by donating and accepting electrons , thereby increasing electron flow , an effect not shared by other antioxidants . melatonin increases the efficiency of etc thereby limiting electron leakage and free radical generation , and consequently promoting protein synthesis [ 17 , 47 ] . melatonin was also able to maintain the efficiency of oxidative phosphorylation and atp synthesis by increasing the activity of the respiratory complexes i , iii , and iv . these effects were attributed to the intramitochondrial presence of melatonin , thus showing melatonin 's participation in the physiological regulation of mitochondrial homeostasis . melatonin 's action in preventing the opening of the mtpt pore given by oxidative stress caused by t - bhp was shown in another study on primary skeletal muscle cultures . the inhibition of the mtpt pore opening by melatonin was suggested as an explanation for the protective action of melatonin against oxidative stress in myotubes . alterations in cardiolipin structure , content , and acyl chain composition have been associated with mitochondrial dysfunction in various tissues under a variety of pathophysiological conditions . the presence of these defective mitochondria is one of the factors involved in the decline in respiratory function during the aging process . enhanced activation of the mtpt pore in the brain and liver of aging mice has also been demonstrated . hence , an increased mitochondrial ros production , oxidative stress , respiratory functional decline , and susceptibility to apoptosis constitute central events in the aging process . the mechanism of the aging process can be studied in experimental models like the senescence accelerated mouse ( samp8 ) and senescence resistant mouse ( samr1 ) . the accelerated aging seen in this mouse strain is due to oxidative stress , which occurs with greater intensity as compared to the samr1 . at an age of 12 months , reductions in the activities of respiratory complexes i and iv have been demonstrated in liver mitochondria of samp8 mice , but not in samr1 mice . these studies support the conclusion that excess free radical generation coupled with less effective defense against the oxidative stress is responsible for alteration of mitochondrial function seen in samp 8 mice [ 122124 ] . since melatonin can readily reach the mitochondria due to its high lipophilicity , it seems feasible that , upon its entry into the cell , it could become concentrated at a superficial position in lipid layers near the polar heads of membrane phospholipids , a key place to function as a free radical scavenger . melatonin , administered in the drinking water at a dose of 10 mg / kg for 9 months , was shown ( i ) to counteract the age - dependent increase in lipoperoxidation level , ( ii ) to increase gsh content in muscle mitochondria of both samp8 and samr1 mice , ( iii ) to counteract the reduction of gsh / gssg ratio in diaphragmatic mitochondria of samr1 and samp8 mice , ( iv ) to increase the activity of the antioxidant enzymes gpx and grd in the mitochondria of samp8 mice with no effect on samr 1 mice , and ( v ) to increase the activity of grd in samr1 mice . as a continuation of the above - mentioned study , the effect of melatonin at earlier stages of the life span was evaluated at the 5th and 10th months of age in samp8 and samr1 mice . when melatonin was administered in the drinking water at a daily dose of 10 mg / kg , the level of lipid peroxidation in 10-month - old samp8 mice was reduced to that found at 5 months of age . the study thus showed that melatonin administration counteracted age - dependent oxidative damage and mitochondrial dysfunction in senescence accelerated mice by improving mitochondrial function as reflected by the increase in atp production and a prolonged longevity . another study using rat brain mitochondria was designed to evaluate the beneficial effects of melatonin on age - associated reductions in mitochondrial bioenergetic function . mitochondria from control and aged rats treated or not with melatonin were obtained , and various bioenergetic parameters such as complex i activity , rates of state 3 respiration , mitochondrial h2o2 production , and membrane potential were evaluated . chronic melatonin treatment completely prevented age - dependent oxidative stress as assessed by the recovery of the gsh / gssg in mitochondria of brain samples of both male and female mice . i / r lesions are seen in many clinical conditions and are triggered by multiple factors including overproduction of ros [ 131133 ] . for example , ros produced at the level of complex i and iii of the respiratory chain are responsible for injury seen in cardiac i / r [ 134 , 135 ] as well as in stroke . the available evidence indicates the opening of the mtpt pore is responsible for the cardiomyocyte death occurring during i / r . melatonin has also been shown to be effective in protecting the cardiac musculature against i / r [ 137139 ] . melatonin 's protective effect during i / r has been attributed to its action in inhibiting the mtpt pore , and in preserving the content and integrity of cardiolipin molecules . the fact that melatonin treatment inhibits both mtpt pore opening and cardiolipin peroxidation following i / r suggests a possible link between these two processes . it must be noted that a significant cytoprotective effect of melatonin was described at a very early phase of a myocardial infarction , when i / r and thus oxidative damage were minimal , indicating that not all cardiac protective effects of melatonin are attributable to its antioxidant activity . septic shock is a lethal condition caused by a complex chain of pathogen - induced events involving immune cells , the epithelium , the endothelium , and the neuroendocrine system . the lethal effects of septic shock are associated with the production and release of numerous proinflammatory mediators as well as no and ros , thus inducing massive apoptosis . since many years ago , research interest was focused on the hypothesis that mitochondrial dysfunction plays a pivotal role in septic shock . this hypothesis was indeed confirmed by the finding of decreased respiratory complex i activity and low levels of atp levels in skeletal muscle biopsies obtained from critically ill patients with septic shock . the protective effect of melatonin on the lethal effects of bacterial lipopolysaccharide ( lps ) on respiratory complex activities i and iv and a mitochondrial subform of inos ( mt inos ) activity was examined in liver and lung mitochondria of rats . using a long - term ( 3-day ) rat model of sepsis , the model comprises a long - term , fluid - resuscitated , fecal peritonitis model utilizing male wistar rats that closely replicates human physiological , biochemical , and histological findings with a 40% mortality . moderate increases in nitrite / nitrate production were seen in both muscle and liver peaking at 2448 h and returning to sham - operated levels at 72 h. a fall in gsh was associated with lower complex i and increased no production was also demonstrated . a number of animal model studies and a few clinical observations have now shown that melatonin is beneficial for treating septic shock ( see , for a recent review , ) . in addition , an increase in oxidative stress was also found as indicated by an increase in lipid peroxidation products as well as a reduction in gsh levels and in the activities of gpx and grd . this study confirmed that mtnos is responsible for the mitochondrial dysfunction seen during sepsis and thus supported the conclusion that melatonin has the ability to protect against mt inos - mediated mitochondrial failure . a similar study performed in mitochondria isolated from the diaphragm of septic mice indicated that melatonin administration to inos mice counteracted mt inos induction and respiratory chain failure , and , finally , normalized the redox state after sepsis . considering the effects of melatonin and its virtual absence of toxicity , the use of melatonin along with conventional therapy to preserve mitochondrial bioenergetics as well as to limit inflammatory response and oxidative damage should be taken into account as a treatment option . although the molecular mechanisms responsible for the pathogenesis of ad are still under intense investigation , reduced complex i activity in the substantia nigra and loss of gsh have been reported in pd patients . the active glial metabolite of mptp , 1-methyl-4-phenylpyridinium ( mpp ) , is taken up into the dopaminergic neurons through the dopamine transporter , and then accumulates in the mitochondria of substantia nigra pars compacta . in the striatum , the damage caused by mpp is attributed to increased generation of o2 that reacts with no to generate the highly toxic onoo . although the role of ros generation has been demonstrated in the etiology of pd , the participation of mtnos in the mitochondrial dysfunction and nigrostriatal degeneration has only recently been examined . treatment with either melatonin or its brain metabolite amk effectively counteracted i - mtnos induction , oxidative stress , and mitochondrial dysfunction induced by mptp . whether melatonin or the recently introduced melatonergic agents ( ramelteon , agomelatine ) have the potential for treating insomnia in pd patients and , more generally , for arresting the progression of pd merits further investigation . several recent studies have confirmed the involvement of mitochondrial ros production and abnormal mitochondrial function in the pathophysiology of ad [ 170177 ] . in the early phase of ad , inhibitors of and -secretase can be therapeutically effective to halt ad disease progression by inhibition of the protein misfolding of a into neurotoxic oligomeric aggregates . several actions of melatonin have been described which antagonize the deleterious effects of a. the effects of melatonin can be grouped as ( i ) antioxidant , including influences on mitochondrial metabolism ; ( ii ) antifibrillogenic , blocking a synthesis ; ( iii ) cytoskeletal , including suppression of tau protein hyperphosphorylation ( for a recent review see ) . the antifibrillogenic effects of melatonin were observed not only in vitro but also in vivo in transgenic mouse models [ 182184 ] . melatonin inhibited free radical formation in microglia exposed to amyloid-142 by preventing the phosphorylation of the p47 nox subunit via the pi3k / akt pathway . modulation of mitochondrial ca handling has been suggested as the potential pharmacological target for ad . the antioxidant , mitochondrial , and antiamyloidogenic effects may be seen as a possibility of interfering with the onset of the disease . in this model , that replicates the neurochemical , histological , and clinical features of hd current evidence from genetic models of hd including mutation of the huntingtin gene ( mhtt ) , supports the mitochondrial dysfunction as major cause of the disease , with respiratory chain impairment relegated to a late secondary event . mitochondrial dysfunction is implicated as the major causative factor in a variety of conditions such as the aging process , i / r , and septic shock . in addition , abnormal mitochondrial function , decreased respiratory enzyme complex activities , increased electron leakage , opening of the mtpt pore , and increased ca entry have all been shown to play a role in the pathophysiology of neurodegenerative disorders such as pd , ad , and hd . there is now evidence that there are polymorphisms in the gene for hiomt , the rate limiting enzyme in melatonin synthesis , and that the hiomt transcript level depends significantly on the genotype distributions . among the number of substances involved in maintaining mitochondrial bioenergetics a number of in vivo and in vitro studies in animals ( table 1 ) indicate that melatonin may emerge as a major therapeutic candidate to preserve the bioenergetic function of mitochondria . double - blind placebo controlled studies are needed to assess to what extent melatonin has therapeutic value in the treatment of the several disorders associated with mitochondrial dysfunction .

given the frequency of reported white matter abnormalities in psychotic disorders , recent hypotheses regarding the etiology of schizophrenia have begun to focus more closely on the possibility of abnormal myelination of late developing frontal white matter tracts ( whitford et al . , 2012 ) . until recently , our ability to explore abnormal myelination and subsequent frontal mis- or disconnection as an underlying central mechanism in the emergence of schizophrenia has been hampered by technological limitations . the advent of new magnetic resonance imaging ( mri ) modalities has made it possible to explore little characterized facets of underlying structural deficits in psychotic disorders and to more fully explore the hypothesis of disconnectivity in schizophrenia and schizophrenia spectrum disorders . disconnectivity or aberrant connectivity may be a common feature of the psychopathologies associated with schizophrenia including decreased information processing speed , difficulties with memory , poor attention and passive withdrawal from normal activities ( girard et al . white matter deficits and white matter disarray ( i.e. axonal disorganization ) have been well established based on in vivo imaging of older , more chronic schizophrenia patients ; however , the evidence for similar deficits early in the illness ( i.e. at first episode ) is more equivocal , with some reports of reduced white matter volumes or altered white matter integrity ( chan et al . , 2010 ; hao et al . , some fronto - medial regions ( particularly the superior frontal gyri and the entorhinal cortex ) appear to have both reduced volume and abnormal tissue integrity in first - episode psychosis patients , based on correlations between findings from standard structural imaging and magnetization transfer imaging ( price et al . , 2010 ) . a number of diffusion tensor studies have reported decreased fractional anisotropy in schizophrenia , particularly in the frontal and temporal white matter , providing corollary evidence of white matter abnormalities as a feature of this illness ( debette and markus , 2010 ; lee et al . , 2013 ) . in contrast , other diffusion tensor imaging studies of white matter alterations in first - episode schizophrenia have failed to demonstrate differences between first - episode patients and healthy age and gender matched volunteers ( kong et al . , 2011 ) . it is postulated that the observed alterations in fa are a result of alterations in the myelin and that a subtle form of axonopathy underlies reduction in anisotropy with relative sparing of the normal white matter histology ( highley et al . , 2002 ) . numerous white matter tracts have been implicated in schizophrenia , including aberrant interhemispheric connectivity through the corpus callosum ( crow et al . , 2007 ) , corticothalamocerebellar circuits ( andreasen , 1999 ) , and fronto - temporal pathways ( friston and frith , 1995 ) . the frontal white matter tracts coursing through the anterior limb of the internal capsule ( alic ) are of particular interest as they contain corticothalamic projections that subserve sensory processing , sensory gating and cognitive processes such as memory , attention , and psychomotor control ( buchsbaum et al . , 2006 ) . imaging investigations using diffusion as a tool for exploration have suggested that white matter integrity and/or quantity in patients with schizophrenia are reduced , particularly in frontal white matter regions ( du et al . , 2012 ; garver et al . , 2008 ) . whether the observed changes in frontal white matter integrity are clearly associated with myelin deficits in schizophrenia has not yet been established . moreover , any potential deficits in myelin need to be interpreted in the context of normal progression of myelination in the developing human brain over the lifespan . both early and more recent studies have shown that myelination continues well in the 3rd decade of life ( benes et al . , 1994 ; yakelov and lecours , 1967 ) . aberrant developmental trajectories that could contribute to the emergence of schizophrenia may result in differential relationships between myelination and age in patients compared to the normal population . we are able to apply a newer , highly specific magnetic resonance ( mr ) methodology developed to assess the myelin - associated water fraction ( mwf ) in white matter ( mackay et al . this novel technique exploits the rate of signal decay from water within specific components of tissue ; as such mwf imaging is well suited to differentiating the specific t2 water signal associated with myelin only , as opposed to water signal from extracellular , neuronal or cerebral spinal fluid compartments ( kolind et al . , 2009 ) . myelin water imaging was validated as a measure of myelin content in fixed brain samples ( laule et al . , 2006 ) , and has been used to follow myelination changes in multiple sclerosis ( laule et al . , 2003 ) and phenylketonuria ( sirrs et al . , 2007 ) . in a previous study , mwf was successfully used to obtain index measures of frontal white matter integrity in chronic schizophrenia patients and healthy normal volunteers ( flynn et al . both age and education were found to have significant positive associations with measures of myelin integrity ( flynn et al . these relationships were not seen in chronic schizophrenia patients ( flynn et al . , 2003 ) . both chronicity of illness and long - term exposure to medications may contribute to these observations , as it has been posited that some antipsychotic medications may act to at least partially restore myelin integrity ( garver et al . it is predicted that 1 ) first - episode schizophrenia patients will have reduced frontal mwf and 2 ) positive relationships between age and years of education with mwf will be present in healthy volunteers , but not in fep subjects . mwf is not expected to be associated with local white matter volume , as the mwf signal is not dependent upon white matter volume . ethics approval for this study was granted by the university of british columbia 's research ethics board in accordance with the canadian federal tri - council policy statement . all subjects provided full written consent . first - episode psychosis ( fep ) subjects were recruited as part of a longitudinal study of the interactions of environment , genetics and neurodevelopment in schizophrenia ( the net - epi study : pi wg honer ) . for the current analysis of white matter integrity , a sample of 58 fep subjects and 44 age and gender - matched healthy volunteers were included . entry criteria for the current study included a current episode of psychosis , ability to give written and verbal consent in english , normal vision with or without correction , minimum iq of 80 , age 1440 , and ability to tolerate mr scanning . exclusion criteria for psychosis subjects included any prior exposure to antipsychotic medications , a history of meningitis or other cns infections , history of head injury leading to loss of consciousness for more than 5 min , dsm - iv drug dependence , severe claustrophobia , and presence of metal surgical implants or metal dental devices . healthy age and gender - matched volunteers who had no history of psychosis or any other major dsm - iv disorder were recruited by poster advertisement within the local community . all subjects had no more than 10 weeks of lifetime exposure to antipsychotic medications ( see table 1 for mean weeks treatment and average daily doses ) . at time of baseline scans 24 subjects were antipsychotic naive , 19 were treated with risperidone , 12 were receiving olanzapine and 1 was receiving quetiapine . diagnoses were made using dsm - iv criteria and included review of a structured clinical interview and longitudinal clinical notes ( wgh ) . demographic and clinical data ( age , gender , ethnicity , iq , years of education , diagnosis , symptom severity ) were assessed at intake ( see table 1 ) . all subjects were given a full diagnostic interview by a clinician ( gwm or lck ) at intake ( baseline ) . panss scores were available for 57 of 58 subjects , as one subject was unavailable for symptom assessment at study entry . additionally , one fep subject did not complete iq assessments at time of entry . intelligence ( iq ) was assessed by the kaufman brief intelligence test ( kbit ) ( kaufman and kaufman , 1990 ) premorbid iq was assessed with the national american adult reading test ( naart ) ( blair and spreen , 1989 ) . all images were inspected for motion artifact and susceptibility artifact ( djl , ws ) to determine suitability for inclusion in the study . optimized for assessment of the mwf in white matter were acquired on a 1.5 t ge , signa echospeed scanner ( software version 5.7 ) . after a sagittal localizer ( tr = 350 ms , te = 14 ms , 15 slices ) , single - slice myelin water imaging data were acquired using a 48-echo cpmg sequence , consisting of a 90 slice selective pulse followed by 48 rectangular composite 180 pulses flanked by slice - selective crusher gradient pulses for elimination of signal from outside the selected slice ( tr = 3800 ms , echo spacing = 10 ms for the first 32 echoes and 50 ms for the next 16 echoes , bw = 31 khz , fov = 22 cm , thickness = 10 mm , matrix 256 128 , averages = 4 ) . for the 20 central lines of k - space , the repetition time was 3.8 s and was ramped linearly to 2.12 s at the extremities of k - space . the reduction in repetition time had a negligible effect on the estimated t2 distributions but substantially decreased the acquisition time ( laule et al . , 2007 ) . the myelin water image slice was positioned transversely to the slice parallel to the anterior commissure posterior commissure line to optimize simultaneous visualization of frontal white matter , basal ganglia structures , thalamic nuclei and posterior white matter . a 3d spgr sequence ( graphic prescription , minimum tr , te = 5 ms , fov = 22 cm , matrix 256 256 , 124 continuous 1.5 mm thick slices , flip angle = 45 ) was acquired for volumetric assessments . parallel t1-weighted fspgr ir 3-d structural images were acquired with the following parameters : tr = 11.5 ms , te = 5 ms , fa 250 , b value = 1000 , fov 24 cm , nex = 1 , acquisition and reconstruction matrices = 256 192 and 256 256 respectively , voxel dimensions = 0.9375 mm 0.9375 mm 2 mm , and interslice thickness = 1 mm . dti was performed with the following parameters ; tr = 13,000 ms , te = 72.8 ms , nex = 2 , voxel dimensions = 1.25 mm 1.25 mm 2.5 mm , fov = 32 32 cm , and acquisition and reconstruction matrices = 128 128 and 256 256 respectively . ( genu , minor forceps , anterior internal capsule , posterior internal capsule , splenium and major forceps ) were manually selected by a trained rater ( ey ) and averaged over 3 trials ( see fig . 1 ) . t2 relaxation decay curves from these regions were decomposed into an unspecified number of exponentials by using a non - negative least squares algorithm ( whittall et al . , 1997 ) and myelin water fraction was defined as the signal with t2 below 50 ms divided by the total signal in the t2 distribution ( mackay et al . , 1994 ) . all rois were analyzed using a pixel - by - pixel analysis based on mwf maps ( mackay et al . , 1994 ; meyers et al . , 2009 ) whereby the average of the pixels within each roi was ascertained to obtain an mwf value for each roi . raw scanner dicom ( digital imaging & communications in medicine ) images were converted to nifti ( neuroimaging informatics technology initiative ) file format using the dcm2nii tool from mricron ( http://www.sph.sc.edu/comd/rorden/mricron/ ) prior to processing . fsl 4.1 software ( i.e. the fsl bet brain extraction tool ) was used to perform skull - stripping and then to register extracted whole - brain images to standard mni152 space . subsequently , using the fast v4.1 tool in fsl , all brain images were parcellated into gray matter , white matter and cerebrospinal fluid ( csf ) . individual lobar volumes were calculated on the basis of lobar masks from the standard mni whole brain template . preliminary comparisons included an independent t - test of age between groups , a 1-way anova to explore diagnosis by age interactions on years of education completed and chi - square analysis of gender distribution between groups . medication dosages were converted from raw lifetime doses to chlorpromazine equivalents in accordance with standard pharmacokinetic d2 occupancy rates to investigate potential relationships between medication dose and mwf ( virani et al . , 2012 ) . left and right mwf values of the major white matter regions were summed to obtain pooled measures , as preliminary comparisons by repeated measures t - tests of left and right sides did not reveal hemispheric differences for any regions of interest ( all p - values > 0.10 ) . relationships between age , education , iq , panss score and frontal white matter ( i.e. minor forceps ) mwf measures were examined with pearson 's linear correlations within each group . omnibus ancova models were use to compare mwf scores between groups with age entered as a co - variate and gender entered as an independent factor , as both age and gender are associated with alterations in myelination ( de bellis et al . , 2001 ) . the ancova model bonferroni correction level for comparisons of mwfs across regions was set to .025 as our a priori specifically hypothesized differences between groups in the frontal white matter ( minor forceps ) , with the investigation of other regions being more exploratory in nature . omnibus group comparisons of frontal white matter volumes included total age , gender and total brain volume as covariates . linear pearson 's correlation and univariate regression models were used to explore relationships between white matter volume and frontal mwf scores on the left and right sides in both groups . standard bonferroni correction was applied for multiple comparisons ( significance set at the p = 0.008 level ) . healthy volunteers and fep subjects were similar in age , t(df 100 ) = 1.23 , p = 0.11 , and had similar levels of educational attainment , f(df 100 ) = 0.01 , p = 0.90 ( note : education data were not available for one fep subject ) . gender distribution across groups was significantly different , with females making up a greater proportion of the healthy volunteer group compared to the fep group , (df 1 ) = 6.82 , p = 0.01 . no within group differences in mwf scores were seen between male and female fep subjects or healthy volunteers ( all p - values > .01 ) . healthy volunteers also had greater crystallized iq as assessed by the naart ; f(1,99 ) = 22.23 , p 0.0001 . no relationships between mean baseline medication dose ( chlorpromazine equivalents per day ) or total lifetime chlorpromazine dose and mwf in any of the six regions of interest were observed in the fep sample ( all p - values > 0.05 ) . analysis of covariance did not reveal differences in mwf scores for any region of interest between fep subjects and healthy volunteers ( all p - values > 0.10 ) . in healthy volunteers , significant positive relationships were found between frontal white matter mwf and age ( r - value .48 , p = .0005 ) , naart iq ( r - value .43 , p = .0045 ) and years of completed education ( r - value .52 , p = 0.0003 ) ( see fig . 2 ) . these relationships remained significant after bonferroni correction ( p - level .008 ) . healthy volunteers also had positive relationships between age and mwf in the anterior and posterior internal capsules , the genu , and the splenium ( all p - values < 0.008 ) , but not with years of completed education or naart iq ( all p - values > .008 ) . in fep subjects , correlation of frontal white mwf and age ( r - value .34 , p = 0.009 ) did not remain significant after bonferroni correction , nor did the correlation of frontal mwf and years of education completed ( r - value .33 , p = 0.01 ) ( see fig . 2 , a c ) . however , the relationship of frontal mwf and naart iq remained significant in the fep group ( r - value .38 , p = .004 ) . additionally , a significant correlation between age and mwf of the splenium in fep subjects was observed ( r - value .35 , p = 0.0006 ) . in fep subjects , no significant relationships between age , naart iq and years of completed education were observed for all other regions of interest . no between - group differences in frontal lobe white matter volume were observed for either the left or right side ( all p - values > 0.50 ) , nor were there left versus right side difference in frontal white volumes within either group ( all p - values > 0.40 ) . neither left nor right frontal mwf was associated with ipsilateral frontal white matter volumes in either patients or healthy volunteers ( all p - values > 0.10 ) . no relationships between naart iq or age and frontal white matter volumes were observed for either left or right side in fep subjects or healthy volunteers ( all p - values > 0.30 ) . in fep subjects , positive relationships between frontal white volume and education were observed on the left ( r = .34 , f(1,55 ) = 7.40 , p = 0.009 ) and the right ( r = .28 , f(1,55 ) = 4.6 , p = 0.037 ) , however these relationships did not remain significant after bonferroni correction at the p 0.008 level . similarly , in healthy volunteers , no significant relationships of white matter volume and education on either the left or right were observed ( all p - values > 0.15 ) . in the current study of first - episode schizophrenia patients , we did not observe any significant reduction in normal mwf in the frontal white matter using t2 myelin water imaging . this result contrasts our previous findings in chronic patients ( flynn et al . , 2003 ) ; rather , they are consistent with previous data presented by hirayasu et al . and ho and colleagues indicating that volumetric frontal white matter reductions are not seen at the first episode , but are progressive over the first 35 years of illness ( hirayasu et al . , 2001 ; ho et al the failure to detect significant frontal white matter mwf abnormality in the present fe sample is congruent with white matter that has not yet undergone measurable physical change . the lack of reductions in frontal white matter volumes in this first - episode cohort provides empirical support for our expectation that 1 ) mwf indices are unrelated to underlying white matter volume and 2 ) in the early stages of illness , frontal white matter volumes are preserved in schizophrenia patients . this observation is of interest as frontal areas are thought to have greater vulnerability in schizophrenia ( iwashiro et al . , 2012 ) ( yang et al . , 2012 ) and have been suggested to be affected at very early stages in the disease ( kanahara et al . , 2013 ) . the current data suggest a lack of significant reduction in frontal white matter mwf early in the illness , and support the hypothesis of progressive change in later schizophrenia . interestingly , yao et al . recently observed decreased posterior internal capsule and frontal white matter volume in untreated first episode patients . these anatomical alterations were related to the clinical symptoms but not the untreated illness duration , suggesting that these deficits are related to aberrations in the myelination process ( hoshi et al . , 2006 ) . the myelin - associated fraction index offers a distinct assessment of a physical property of myelin that is independent of white matter volume in our cohort . in our study , associations between mwf and age in the anterior and posterior internal capsules , the genu , and the splenium in healthy volunteers were observed . the present results also suggest attenuated associations between years of education and iq in first - episode patients , particularly in the frontal white matter . these results are consistent with our earlier report in a cohort of chronic schizophrenia patients ( flynn et al . , 2003 ) . flynn and colleagues reported significant relationships between age and frontal white mwf ( r = .47 , p = .01 ) and years of education and frontal white mwf ( r = .51 , p = 0.006 ) in healthy controls , but not in chronic schizophrenia patients . such alterations in the relationships between frontal mwf and age , iq and education suggest that subtle myelin - associated abnormalities already exist during the first - episode . processes that are expected to drive normal myelination , such as maturation and learning , do not appear to confer the same results in persons with schizophrenia . with respect to age , associations between the anterior and posterior internal capsules , and the genu were observed in healthy volunteers . these findings suggest that aberrant myelination patterns in schizophrenia may result in the emergence of altered white - matter maturation early in illness . it has been suggested that myelin , an under - appreciated , yet vital component of human brain structure and function , is a possible unifying agent involved in the emergence of many neuropsychiatric disorders ( bartzokis , 2004 ) . myelination of the human brain is a continuous process in the first 3540 years of life , undergoing periods of pruning and rapid expansion as part of the normal maturation process ( miller et al . , 2012 ) . genetically mediated alterations , developmental interruptions or externally driven insults may result in perturbations of normal myelination , resulting in different psychiatric sequelae based on the timing of the perturbations . the natural sequencing of functional neurodevelopment would suggest that normally , mid- to late adolescence is a particularly critical phase of expansion in cognitive processing capacity and this increased capacity is supported by molecular and cellular maturation of the frontal circuitry ( catts et al . , 2013 ) . striatal myelination , that occurs in parallel with the pubertal to post - pubertal maturational stages ( asato et al . , 2010 ) . this timeframe coincides with the typical age at onset for schizophrenia , suggesting that abnormal myelination in the context of post - natal neurodevelopment may be involved in the emergence of psychosis . given the accumulating evidence for widespread subtle abnormalities of white matter at illness onset , the role of white matter pathophysiology may be important for the emergence of schizophrenia ( lee et al . , 2013 ) . these neuroglial cells form myelin sheaths around axons ( uranova et al . , 2011 ) . additionally , genetic studies of risk - variants of the myelin - associated glycoprotein ( mag ) gene have reported that specific genotypes may predispose individuals to alterations of brain morphology , surprisingly in the temporal and parietal gray matter ( felsky et al . , 2012 ) . whether some regions are more likely to be affected early in the course of illness , has not yet been established , although frontal areas appear to have greater vulnerability ( yang et al . . recent work using phosphorus spectroscopy found reduced brain phospholipid content in people with schizophrenia , which could be interpreted as reduced myelin content considering the high phospholipid content in the myelin bilayer ( vostrikov , 2007 ) . in addition , the efficacy of antipsychotic medication may be partially mediated by its salutary effect on myelin and oligodendrocyte function . this finding provides additional support for a generalized underlying pathophysiology of myelin in psychosis ( ren et al . , 2013 ) . the current application of myelin water imaging in a cohort of minimally treated patients , provides further evidence of abnormalities in frontal myelination patterns with respect to normal aging , iq and educational attainment . the multiple lines of evidence suggest that aberrant myelination in schizophrenia plays a central role in the emergence of schizophrenia , and in particular , frontal myelin disruptions or deficits may be a key component of the illness .

myelin water imaging provides a novel strategy to assess myelin integrity and corresponding clinical relationships in psychosis , of particular relevance in frontal white matter regions . in the current study , t2 myelin water imaging was used to assess the myelin water fraction ( mwf ) signal from frontal areas in a sample of 58 individuals experiencing first - episode psychosis ( fep ) and 44 healthy volunteers . no differences in frontal mwf were observed between fep subjects and healthy volunteers ; however , differences in normal patterns of associations between frontal mwf and age , education and iq were seen . significant positive relationships between frontal mwf and age , north american adult reading test ( naart ) iq , and years of completed education were observed in healthy volunteers . in contrast , only the relationship between frontal mwf and naart iq was significant after bonferroni correction in the fep group . additionally , significant positive relationships between age and mwf in the anterior and posterior internal capsules , the genu , and the splenium were observed in healthy volunteers . in fep subjects , only the relationship between age and mwf in the splenium was statistically significant . frontal mwf was not associated with local white matter volume . altered patterns of association between age , years of education , and mwf in fep suggest that subtle disturbances in myelination may be present early in the course of psychosis .

Introduction Methods Discussion

given the frequency of reported white matter abnormalities in psychotic disorders , recent hypotheses regarding the etiology of schizophrenia have begun to focus more closely on the possibility of abnormal myelination of late developing frontal white matter tracts ( whitford et al . until recently , our ability to explore abnormal myelination and subsequent frontal mis- or disconnection as an underlying central mechanism in the emergence of schizophrenia has been hampered by technological limitations . disconnectivity or aberrant connectivity may be a common feature of the psychopathologies associated with schizophrenia including decreased information processing speed , difficulties with memory , poor attention and passive withdrawal from normal activities ( girard et al . axonal disorganization ) have been well established based on in vivo imaging of older , more chronic schizophrenia patients ; however , the evidence for similar deficits early in the illness ( i.e. at first episode ) is more equivocal , with some reports of reduced white matter volumes or altered white matter integrity ( chan et al . , some fronto - medial regions ( particularly the superior frontal gyri and the entorhinal cortex ) appear to have both reduced volume and abnormal tissue integrity in first - episode psychosis patients , based on correlations between findings from standard structural imaging and magnetization transfer imaging ( price et al . a number of diffusion tensor studies have reported decreased fractional anisotropy in schizophrenia , particularly in the frontal and temporal white matter , providing corollary evidence of white matter abnormalities as a feature of this illness ( debette and markus , 2010 ; lee et al . in contrast , other diffusion tensor imaging studies of white matter alterations in first - episode schizophrenia have failed to demonstrate differences between first - episode patients and healthy age and gender matched volunteers ( kong et al . it is postulated that the observed alterations in fa are a result of alterations in the myelin and that a subtle form of axonopathy underlies reduction in anisotropy with relative sparing of the normal white matter histology ( highley et al . numerous white matter tracts have been implicated in schizophrenia , including aberrant interhemispheric connectivity through the corpus callosum ( crow et al . , 2007 ) , corticothalamocerebellar circuits ( andreasen , 1999 ) , and fronto - temporal pathways ( friston and frith , 1995 ) . the frontal white matter tracts coursing through the anterior limb of the internal capsule ( alic ) are of particular interest as they contain corticothalamic projections that subserve sensory processing , sensory gating and cognitive processes such as memory , attention , and psychomotor control ( buchsbaum et al . imaging investigations using diffusion as a tool for exploration have suggested that white matter integrity and/or quantity in patients with schizophrenia are reduced , particularly in frontal white matter regions ( du et al . whether the observed changes in frontal white matter integrity are clearly associated with myelin deficits in schizophrenia has not yet been established . aberrant developmental trajectories that could contribute to the emergence of schizophrenia may result in differential relationships between myelination and age in patients compared to the normal population . we are able to apply a newer , highly specific magnetic resonance ( mr ) methodology developed to assess the myelin - associated water fraction ( mwf ) in white matter ( mackay et al . this novel technique exploits the rate of signal decay from water within specific components of tissue ; as such mwf imaging is well suited to differentiating the specific t2 water signal associated with myelin only , as opposed to water signal from extracellular , neuronal or cerebral spinal fluid compartments ( kolind et al . myelin water imaging was validated as a measure of myelin content in fixed brain samples ( laule et al . , 2006 ) , and has been used to follow myelination changes in multiple sclerosis ( laule et al . , 2003 ) and phenylketonuria ( sirrs et al . in a previous study , mwf was successfully used to obtain index measures of frontal white matter integrity in chronic schizophrenia patients and healthy normal volunteers ( flynn et al . both age and education were found to have significant positive associations with measures of myelin integrity ( flynn et al . both chronicity of illness and long - term exposure to medications may contribute to these observations , as it has been posited that some antipsychotic medications may act to at least partially restore myelin integrity ( garver et al . it is predicted that 1 ) first - episode schizophrenia patients will have reduced frontal mwf and 2 ) positive relationships between age and years of education with mwf will be present in healthy volunteers , but not in fep subjects . mwf is not expected to be associated with local white matter volume , as the mwf signal is not dependent upon white matter volume . first - episode psychosis ( fep ) subjects were recruited as part of a longitudinal study of the interactions of environment , genetics and neurodevelopment in schizophrenia ( the net - epi study : pi wg honer ) . for the current analysis of white matter integrity , a sample of 58 fep subjects and 44 age and gender - matched healthy volunteers were included . entry criteria for the current study included a current episode of psychosis , ability to give written and verbal consent in english , normal vision with or without correction , minimum iq of 80 , age 1440 , and ability to tolerate mr scanning . exclusion criteria for psychosis subjects included any prior exposure to antipsychotic medications , a history of meningitis or other cns infections , history of head injury leading to loss of consciousness for more than 5 min , dsm - iv drug dependence , severe claustrophobia , and presence of metal surgical implants or metal dental devices . healthy age and gender - matched volunteers who had no history of psychosis or any other major dsm - iv disorder were recruited by poster advertisement within the local community . demographic and clinical data ( age , gender , ethnicity , iq , years of education , diagnosis , symptom severity ) were assessed at intake ( see table 1 ) . panss scores were available for 57 of 58 subjects , as one subject was unavailable for symptom assessment at study entry . additionally , one fep subject did not complete iq assessments at time of entry . intelligence ( iq ) was assessed by the kaufman brief intelligence test ( kbit ) ( kaufman and kaufman , 1990 ) premorbid iq was assessed with the national american adult reading test ( naart ) ( blair and spreen , 1989 ) . all images were inspected for motion artifact and susceptibility artifact ( djl , ws ) to determine suitability for inclusion in the study . optimized for assessment of the mwf in white matter were acquired on a 1.5 t ge , signa echospeed scanner ( software version 5.7 ) . after a sagittal localizer ( tr = 350 ms , te = 14 ms , 15 slices ) , single - slice myelin water imaging data were acquired using a 48-echo cpmg sequence , consisting of a 90 slice selective pulse followed by 48 rectangular composite 180 pulses flanked by slice - selective crusher gradient pulses for elimination of signal from outside the selected slice ( tr = 3800 ms , echo spacing = 10 ms for the first 32 echoes and 50 ms for the next 16 echoes , bw = 31 khz , fov = 22 cm , thickness = 10 mm , matrix 256 128 , averages = 4 ) . for the 20 central lines of k - space , the repetition time was 3.8 s and was ramped linearly to 2.12 s at the extremities of k - space . the myelin water image slice was positioned transversely to the slice parallel to the anterior commissure posterior commissure line to optimize simultaneous visualization of frontal white matter , basal ganglia structures , thalamic nuclei and posterior white matter . parallel t1-weighted fspgr ir 3-d structural images were acquired with the following parameters : tr = 11.5 ms , te = 5 ms , fa 250 , b value = 1000 , fov 24 cm , nex = 1 , acquisition and reconstruction matrices = 256 192 and 256 256 respectively , voxel dimensions = 0.9375 mm 0.9375 mm 2 mm , and interslice thickness = 1 mm . ( genu , minor forceps , anterior internal capsule , posterior internal capsule , splenium and major forceps ) were manually selected by a trained rater ( ey ) and averaged over 3 trials ( see fig . , 1997 ) and myelin water fraction was defined as the signal with t2 below 50 ms divided by the total signal in the t2 distribution ( mackay et al . the fsl bet brain extraction tool ) was used to perform skull - stripping and then to register extracted whole - brain images to standard mni152 space . subsequently , using the fast v4.1 tool in fsl , all brain images were parcellated into gray matter , white matter and cerebrospinal fluid ( csf ) . preliminary comparisons included an independent t - test of age between groups , a 1-way anova to explore diagnosis by age interactions on years of education completed and chi - square analysis of gender distribution between groups . medication dosages were converted from raw lifetime doses to chlorpromazine equivalents in accordance with standard pharmacokinetic d2 occupancy rates to investigate potential relationships between medication dose and mwf ( virani et al . left and right mwf values of the major white matter regions were summed to obtain pooled measures , as preliminary comparisons by repeated measures t - tests of left and right sides did not reveal hemispheric differences for any regions of interest ( all p - values > 0.10 ) . relationships between age , education , iq , panss score and frontal white matter ( i.e. omnibus ancova models were use to compare mwf scores between groups with age entered as a co - variate and gender entered as an independent factor , as both age and gender are associated with alterations in myelination ( de bellis et al . the ancova model bonferroni correction level for comparisons of mwfs across regions was set to .025 as our a priori specifically hypothesized differences between groups in the frontal white matter ( minor forceps ) , with the investigation of other regions being more exploratory in nature . omnibus group comparisons of frontal white matter volumes included total age , gender and total brain volume as covariates . linear pearson 's correlation and univariate regression models were used to explore relationships between white matter volume and frontal mwf scores on the left and right sides in both groups . standard bonferroni correction was applied for multiple comparisons ( significance set at the p = 0.008 level ) . healthy volunteers and fep subjects were similar in age , t(df 100 ) = 1.23 , p = 0.11 , and had similar levels of educational attainment , f(df 100 ) = 0.01 , p = 0.90 ( note : education data were not available for one fep subject ) . gender distribution across groups was significantly different , with females making up a greater proportion of the healthy volunteer group compared to the fep group , (df 1 ) = 6.82 , p = 0.01 . no within group differences in mwf scores were seen between male and female fep subjects or healthy volunteers ( all p - values > .01 ) . healthy volunteers also had greater crystallized iq as assessed by the naart ; f(1,99 ) = 22.23 , p 0.0001 . no relationships between mean baseline medication dose ( chlorpromazine equivalents per day ) or total lifetime chlorpromazine dose and mwf in any of the six regions of interest were observed in the fep sample ( all p - values > 0.05 ) . analysis of covariance did not reveal differences in mwf scores for any region of interest between fep subjects and healthy volunteers ( all p - values > 0.10 ) . in healthy volunteers , significant positive relationships were found between frontal white matter mwf and age ( r - value .48 , p = .0005 ) , naart iq ( r - value .43 , p = .0045 ) and years of completed education ( r - value .52 , p = 0.0003 ) ( see fig . these relationships remained significant after bonferroni correction ( p - level .008 ) . healthy volunteers also had positive relationships between age and mwf in the anterior and posterior internal capsules , the genu , and the splenium ( all p - values < 0.008 ) , but not with years of completed education or naart iq ( all p - values > .008 ) . in fep subjects , correlation of frontal white mwf and age ( r - value .34 , p = 0.009 ) did not remain significant after bonferroni correction , nor did the correlation of frontal mwf and years of education completed ( r - value .33 , p = 0.01 ) ( see fig . however , the relationship of frontal mwf and naart iq remained significant in the fep group ( r - value .38 , p = .004 ) . additionally , a significant correlation between age and mwf of the splenium in fep subjects was observed ( r - value .35 , p = 0.0006 ) . in fep subjects , no significant relationships between age , naart iq and years of completed education were observed for all other regions of interest . no between - group differences in frontal lobe white matter volume were observed for either the left or right side ( all p - values > 0.50 ) , nor were there left versus right side difference in frontal white volumes within either group ( all p - values > 0.40 ) . neither left nor right frontal mwf was associated with ipsilateral frontal white matter volumes in either patients or healthy volunteers ( all p - values > 0.10 ) . no relationships between naart iq or age and frontal white matter volumes were observed for either left or right side in fep subjects or healthy volunteers ( all p - values > 0.30 ) . in fep subjects , positive relationships between frontal white volume and education were observed on the left ( r = .34 , f(1,55 ) = 7.40 , p = 0.009 ) and the right ( r = .28 , f(1,55 ) = 4.6 , p = 0.037 ) , however these relationships did not remain significant after bonferroni correction at the p 0.008 level . similarly , in healthy volunteers , no significant relationships of white matter volume and education on either the left or right were observed ( all p - values > 0.15 ) . in the current study of first - episode schizophrenia patients , we did not observe any significant reduction in normal mwf in the frontal white matter using t2 myelin water imaging . and ho and colleagues indicating that volumetric frontal white matter reductions are not seen at the first episode , but are progressive over the first 35 years of illness ( hirayasu et al . , 2001 ; ho et al the failure to detect significant frontal white matter mwf abnormality in the present fe sample is congruent with white matter that has not yet undergone measurable physical change . the lack of reductions in frontal white matter volumes in this first - episode cohort provides empirical support for our expectation that 1 ) mwf indices are unrelated to underlying white matter volume and 2 ) in the early stages of illness , frontal white matter volumes are preserved in schizophrenia patients . this observation is of interest as frontal areas are thought to have greater vulnerability in schizophrenia ( iwashiro et al . , 2012 ) and have been suggested to be affected at very early stages in the disease ( kanahara et al . the current data suggest a lack of significant reduction in frontal white matter mwf early in the illness , and support the hypothesis of progressive change in later schizophrenia . recently observed decreased posterior internal capsule and frontal white matter volume in untreated first episode patients . the myelin - associated fraction index offers a distinct assessment of a physical property of myelin that is independent of white matter volume in our cohort . in our study , associations between mwf and age in the anterior and posterior internal capsules , the genu , and the splenium in healthy volunteers were observed . the present results also suggest attenuated associations between years of education and iq in first - episode patients , particularly in the frontal white matter . flynn and colleagues reported significant relationships between age and frontal white mwf ( r = .47 , p = .01 ) and years of education and frontal white mwf ( r = .51 , p = 0.006 ) in healthy controls , but not in chronic schizophrenia patients . such alterations in the relationships between frontal mwf and age , iq and education suggest that subtle myelin - associated abnormalities already exist during the first - episode . with respect to age , associations between the anterior and posterior internal capsules , and the genu were observed in healthy volunteers . these findings suggest that aberrant myelination patterns in schizophrenia may result in the emergence of altered white - matter maturation early in illness . myelination of the human brain is a continuous process in the first 3540 years of life , undergoing periods of pruning and rapid expansion as part of the normal maturation process ( miller et al . this timeframe coincides with the typical age at onset for schizophrenia , suggesting that abnormal myelination in the context of post - natal neurodevelopment may be involved in the emergence of psychosis . given the accumulating evidence for widespread subtle abnormalities of white matter at illness onset , the role of white matter pathophysiology may be important for the emergence of schizophrenia ( lee et al . additionally , genetic studies of risk - variants of the myelin - associated glycoprotein ( mag ) gene have reported that specific genotypes may predispose individuals to alterations of brain morphology , surprisingly in the temporal and parietal gray matter ( felsky et al . whether some regions are more likely to be affected early in the course of illness , has not yet been established , although frontal areas appear to have greater vulnerability ( yang et al . recent work using phosphorus spectroscopy found reduced brain phospholipid content in people with schizophrenia , which could be interpreted as reduced myelin content considering the high phospholipid content in the myelin bilayer ( vostrikov , 2007 ) . in addition , the efficacy of antipsychotic medication may be partially mediated by its salutary effect on myelin and oligodendrocyte function . this finding provides additional support for a generalized underlying pathophysiology of myelin in psychosis ( ren et al . the current application of myelin water imaging in a cohort of minimally treated patients , provides further evidence of abnormalities in frontal myelination patterns with respect to normal aging , iq and educational attainment . the multiple lines of evidence suggest that aberrant myelination in schizophrenia plays a central role in the emergence of schizophrenia , and in particular , frontal myelin disruptions or deficits may be a key component of the illness .

a chromodomain ( chd ) is a protein domain involved in chromatin remodeling and the regulation of gene expression in eukaryotes ( e.g. , [ 13 ] ) . chds perform a wide range of functions , including chromatin targeting and interactions between different proteins , rna and dna . there are two major groups of chds that are found in eukaryotic chromodomain - containing proteins . the so - called classical chds carry the characteristic chromo - box motif ( y / f)-(l / f / y)-(l / i / v)-k-(w / y)-(k / r)-g ( single - letter code , capital letters standing for the most prominent aminoacid ) . classical chds are highly conserved among eukaryotes and are represented in a large number of proteins in many genomes . they are believed to have a similar three - dimensional structure , which consists of an n - terminal three - stranded -barrel capped by a c - terminal helix . three conserved residues , y24 , w45 , and y48 , are essential for aromatic pocket formation [ 6 , 7 ] . the second group of chds , shadow chromodomains , is more variable and includes chromo - related domains , which are well conserved in their central region , but they deviate significantly in other regions . the majority of the shadow chds contain the conserved residue w45 and lack y24 and y48 [ 3 , 4 ] . in comparison with the classical chds , the shadow chromodomains contain one helix at the n - terminus and another inserted before the c - terminal helix . the best - known protein with both types of chromodomains is heterochromatin protein-1 ( hp1 ) . presence is a hallmark of constitutive heterochromatin in drosophila , a condensed and highly repressive type of chromatin that organizes the repetitive pericentromeric dna . dimethyl - k9 ( h3k9me2 ) and histone h3 trimethyl - k9 ( h3k9me3 ) to help establish transcriptionally silent heterochromatin [ 911 ] . the chromodomain has been found not only in eukaryotic functional proteins but also in diverse ltr retrotransposons , which are called chromodomain - containing gypsy ltr retrotransposons or chromoviruses [ 12 , 13 ] . chromoviruses are the most widespread lineage of gypsy ltr retrotransposons and are present in the genomes of fungi , plants , and vertebrates [ 13 , 14 ] . group i chds from retrotransposons ( chds_i ) are similar to classical chds of chromodomain - containing proteins . this group of chds was found in diverse eukaryotic ltr retrotransposons , including fungal and vertebrate gypsy elements , as well as in ltr retrotransposons from moss physcomitrella patens and spike - moss selaginella moellendorffii , which belong to the tcn1 clade [ 13 , 1618 ] . the information on the role of chds_i in the retrotransposition of ltr retrotransposons is limited . the transposition activity of the maggy retrotransposon of the rice blast fungus magnaporthe oryzae dramatically decreased with the loss or alteration of the chromodomain . on the other hand , the chromointegrase of the tf1 ltr retrotransposon from schizosaccharomyces pombe that lacks the chromodomain demonstrated a significantly higher activity and a substantially reduced substrate specificity . as was demonstrated recently , the maggy chromodomain interacts with h3k9me2 and h3k9me3 in a similar way compared to the hp1 classical chromodomain . representatives of group ii chds from retrotransposons ( chds_ii ) lack the first conserved aromatic residue ( y24 ) and usually the third ( y48 ) . the mostly heterochromatic distribution of plant chromoviruses along with data describing the localization of chromodomain - yfp fused protein in heterochromatin can be used as indirect evidence for recognizing heterochromatin and directing the integration role of chds_ii . nevertheless , the actual mechanisms with which these chromodomains act are still unknown . previously , we demonstrated that our knowledge of plant chromodomain - containing ltr retrotransposons is mostly limited to knowledge about seed plants ( mostly angiosperms ) [ 1618 ] . an investigation of retrotransposons from nonseed plants could shed light on the evolutionary history of retrotransposons and their impact on the evolutionary history of plant genomes . for example , it is still not clear whether chds_i and chds_ii were acquired independently by distinct lineages of ltr retrotransposons or whether they evolved from a common ancestor . the present survey of chromodomains from diverse plants with an emphasis on the spike - moss selaginella demonstrated that a transition from chds_i to chds_ii occurred in the evolutionary history of plants approximately 500400 mya . moreover , several types of clade - specific chds_ii were found in plants ; sequence dissimilarities among these clade - specific chds hypothesized to indicate functional differences . we examined the evolutionary constraints that shaped the diversity of chds_ii in plants and demonstrated that positive selection contributed to the diversification of clade - specific ltr retrotransposon chds . we propose that the presence of chds_i or chds_ii is related to the distribution of heterochromatin / euchromatin marks and molecular differences in these marks between distinct lineages of eukaryotes , such as fungi / metazoa and plants . both the transition from chds_i to chds_ii and the diversification of clade - specific chds reflect evolutionary changes that occurred in plant chromatin organization . previously , we described sm - tcn1 chd - containing gypsy ltr retrotransposons , which presumably appeared as a result of a horizontal transfer from fungi during the early evolution of plants . several other families of ltr retroelements were identified by performing blastn and tblastn searches of the s. moellendorffii whole genome shotgun ( wgs ) database ( http://genome.jgi-psf.org/selmo1 ) using the sm - tcn1 retrotransposon as well as previously described retrotransposons from other species as queries ( see section 4 ) . the newly identified retrotransposons were classified as representatives of the same or different families based on the levels of their similarities . more than 80% identity at the nucleotide level is believed to be sufficient for the classification of retrotransposons in the same family . an additional criterion to designate retrotransposon families is a minimum of 50% nucleotide identity in ltrs . the exemplar element was retrieved or reconstructed based on copies that were available for each family . these retrotransposons were used for further classification based on comparative and phylogenetic analysis , which included known ltr retrotransposons from other plants . in total , five diverse families of chd - containing ltr retrotransposons were found in addition to the previously described sm - tcn1 . the five families were named sm1-galahad and sm2-galahad , sm - fogey , sm - diluvium , and sm - cranky ( table 1 ) . sm1-galahad and sm2-galahad are closely related to each other and form a common clade with galadriel - like retrotransposons from monocots and dicots , suggesting that this clade originated before nonvascular and vascular plants separated from a common ancestor , roughly 400 mya . among the sequences that are available in the ncbi protein database based on blastp analysis , the retroelement that is most closely related to sm1-galahad and sm2-galahad is the galadriel ltr retrotransposon from lycopersicon esculentum , which was identified in the cf-9 disease resistance gene cluster . we did not identify a putative intact copy of sm1-galahad in s. moellendorffii wgs ; thus , we used a number of copies to obtain a consensus sequence . sm1-galahad is highly repeated and represented by hundreds of copies per genome , which share 95% nucleotide similarity on average . the reconstructed sm1-galahad is 7.5 kbp in length and carries ltrs that are more than 1 kbp in length and which possess a short inverted terminal repeat ( tgca ) , typical for ltr retrotransposons and retroviruses . they also contain polyguanine tracks that vary in length ( from 11 to 25 bp ) between different copies . a 13 bp primer binding site - like sequence ( pbs ) that is complementary to the 3 end region of trna is present downstream of the 5ltr , and a polypurine tract ( ppt ) was detected immediately upstream of the 3ltr ( figure 1(b ) ) . the putative single open reading frame ( orf ) of sm1-galahad is 3885 bp in length . the hypothetical protein product of the orf of sm1-galahad ( 1295 aa ) exhibits significant similarity to both gag and pol gene products of known retroelements , especially with those of gypsy retrotransposons . a search for characteristic motifs within the polyprotein sequence identified several functional domains , such as the cysteine or zn - finger motif with cys - x2-cys - x4-his - x4-cys ( cchc ) composition , proteinase ( pr ) , reverse transcriptase ( rt ) , ribonuclease h ( rnh ) , and core integrase ( core int ) , in the order indicated , confirming that sm1-galahad belongs to the ty3/gypsy ltr retrotransposons . no chromodomain ( chd ) was found at the c - terminal end of the gag - pol protein from the reconstructed sm1-galahad . however , analysis of the dna sequence downstream of the putative orf revealed two additional short orfs ( 316 bp and 309 bp in length ) . the 309-bp orf3 encodes a protein ( 113 aa ) containing a chd group i ( chd_i ) domain . the full - length putatively intact sm2-galahad was found in scaffold_24 ( between position 1030224 and 1037576 ) . sm2-galahad is a 7.4 kbp ltr retrotransposon with a single orf ( figure 1(b ) ) . a 5 bp target site duplication ( tsd ) was also detected ( cctatcctat ) . an 11 bp pbs complementary to the trna is located just downstream of the 5ltr , and 13 bp ppt is located upstream of 3ltr . the putative orf ( 4731 bp ) encodes a fused gag - pol protein ( 1577 aa ) , which carries several functional domains , including the cchc zn - finger motif , pr , rt , rnh , core int , and chd_i . the complete retrotransposons sm - fogey and sm - diluvium are 4.9 kbp and 5 kbp long , respectively ( figure 1(b ) ) . the full - length intact copies of sm - fogey and sm - diluvium can be found in scaffold_20 ( position from 2127930 to 2132846 ) and scaffold_73 ( position from 936919 to 941957 ) . target site duplications of five base pairs mark the integration of the intact sm - fogey and sm - diluvium in the genomic sequence . both ltr retrotransposons contain four catalytic regions , pr , rt , rnh , and int of the retroviral genes gag and pol , in a continuous single open reading frame . sm - diluvium also has a chd group ii ( chd_ii ) , whereas sm - fogey does not carry a chromodomain . sm - fogey is characterized by 233 bp terminal repeats ; in contrast , the ltrs of sm - diluvium are only 112 bp in length . the ltrs of sm - fogey and sm - diluvium contain consensus short inverted terminal repeats ( tgca ) , which are important for the integration of retroviral sequences . the primer binding site ( pbs ) , which is complementary to the 3 end of trna and has a one - nucleotide spacer next to the adjacent 5 ltr , the ppt with the stretch of 14 purines for sm - fogey and sm - diluvium is located immediately before the 3 ltr . several full - length highly similar copies for both sm - fogey and sm - diluvium can be found in s. moellendorffii wgs . for example , nine putatively intact copies of sm - fogey presented in wgs share 98.5% average similarity at the dna level . all of them are flanked by 5-bp tsd . despite the fact that only one full - length copy was detected for sm - diluvium , seven full - length copies presented in the genome showed an average 99.1% dna similarity . additionally , the 5 and 3ltrs of elements have high similarity , 98.7% on average for sm - fogey and 97.3% on average for sm - diluvium . altogether , these features indicate that sm - fogey and sm - diluvium recently retrotransposed and may still be active . the putative gag - pol polyproteins of sm - fogey and sm - diluvium were compared to those reported for other plant ltr retrotransposons . the aminoacid domains show the highest similarity to the corresponding regions of the tekay - like retrotransposon shmidt that is adjacent to the disease resistance - priming gene npr1 in beta vulgaris ( ef101866 ; ) and retrotransposons from the diverse oryza species , including ltr retrotransposons from oryza sativa and oryza australiensis ( e.g. , dq365821 , dp000086 ; ) . it appears that sm - fogey and sm - diluvium are the most closely related to the tekay clade of chd - containing gypsy ltr retrotransposons from plants . they formed a common branch on the phylogenetic tree , but neither sm - fogey nor sm - diluvium can be assigned to this clade because of the low bootstrap support for this cluster ( bootstrap 77% ; figure 1(a ) ) . one more chd - containing gypsy ltr retrotransposon , sm - cranky , was found to be present with a few copies per genome . we detected only one full - length sm - cranky located in scaffold_1 ( between position 5651434 and 5657027 ) and six additional truncated copies . the retrotransposon is 5.6 kbp long with ltrs of 217 bp , terminating in the two - nucleotide inverted repeat ( tgca ) . a stretch of 12 bp located with two - nucleotide spacers downstream of the 5ltr is complementary to the 3 end of trna , probably providing a primer site for reverse transcription . the putative pseudo - orf is interrupted by three stop codons , but there are no frameshifts . this sequence can be translated to the protein that bears a resemblance to the characteristic pr , rt , int , and chd_ii motifs ( figure 1(b ) ) . the most intriguing finding that concerns the chd - containing gypsy ltr retrotransposons from the spike - moss s. moellendorffii is the presence of both types of ltr retrotransposon chromodomains , chd_i and chd_ii , in the same plant genome . the sm - tcn1 , sm1-galahad , and sm2-galahad ltr retrotransposons from selaginella carry chd_i , while sm - diluvium and sm - cranky contain chd_ii . the distribution of both retrotransposon chd types among different taxa is considered to be well known . chd_i is typical for fungal and animal ltr retrotransposons as well as for green algae ( chlamyvir clade ) , whereas only chd_ii was found in ltr retrotransposons from seed plants ( tekay , galadriel , and reina clades ) . however , it appears that phylogenetic distribution of chd_i - containing elements extends to most if not all extant green plant lineages . previously , we reported that chd_i - containing ltr retrotransposons belonging to the tcn1 clade can be found in both moss physcomitrella and spike - moss selaginella [ 16 , 18 ] . to expand our understanding of the evolution and diversity of retrotransposon chromodomains in green plants , we implemented a search throughout sequence databases including but not limited to plantgdb ( http://www.plantgdb.org/ ) and phytozome ( http://www.phytozome.net/ ) . a plant retrotransposon chd search was performed with ( tblastn ) using aminoacid sequences of known chds as queries ( see section 4 ) . altogether 114 plant species were investigated and results for some of them are presented in table 2 . the full list and primary information can be found in supporting information table 1s and table 2s . it should be noted that the majority of sequences available were derived from est databases . in fact , chd - containing retrotransposons are barely expressed , and as a rule , they are underrepresented in ests [ 13 , 16 ] . another factor that has an effect on the final result is the presence of a strong bias with respect to the species diversity that is represented in databases . more than 83% of the analyzed species ( 95 out of a total of 114 ) were angiosperms , and only a few representatives from other green plant groups are currently available . among the analyzed species , only 26 did not produce any significant hits . for 80 species , retrotransposon chds were detected in ests ; chds were also found in genomesurvey sequences ( gss ) and whole genomic sequences ( wgs ) databases for 46 investigated species . among those species for which the wgs database is available , a few did not show the presence of retrotransposon chds , including red algae ( porphyrayezoensis , galdieriasulphuraria , and cyanidioschyzonmerolae ) as well as one green algae , micromonas pusilla ccmp1545 . this result can arise from either the limited number of sources of sequences available or the loss of this type of retrotransposon . only the representatives of the chlamyvir clade showed the presence of chd_i which was found in green algae chlorella vulgaris c-169 and chlamydomonas reinhardtii ( table 2 ; ) . the source of ests as well as genomic sequences of gymnosperms is very limited , especially in comparison to those for angiosperms . nevertheless , we were able to identify a few ltr retrotransposon chds in all of the species investigated ( table 2 ; supporting information table 1s ) . comparative analysis indicated that almost all of the chromodomains of type ii can be easily classified as reina- , tekay- , or galadriel - like chds based on their sequence similarity . almost all of the plant species that produced hits in our search contain reina- and tekay - like chds . galadriel - like chds were underrepresented in the analyzed databases . the phylogenetic analysis based on the aminoacid sequences of newly identified chds and chds from known plant ltr retrotransposons support these findings with several exceptions : ( i ) galadriel - like ltr retrotransposons from selaginella have chd_i and are grouped with the tcn1 clade ; ( ii ) chds from conifers , which were previously believed to belong to reina - like ltr retrotransposons from angiosperms , actually form their own branch ; ( iii ) three chds identified in green algae grouped together with the selaginella sm - cranky ltr retrotransposon and not with chds from the chlamyvir clade ( figure 2 ; ) . additionally , tekay - like ltr retrotransposon chds from gymnosperms formed a common cluster that appears to be distinct from other tekay - like chds . it is worthwhile to note that tekay - like chds retrieved from representatives of the family poaceae formed their own branch , with fairly high bootstrap support ( bootstrap value 77% ; figure 2 ) . moreover , retrotransposon activity has been implicated as playing a major role in genome size evolution in angiosperm lineage . this has been especially well - characterized in the poaceae ( e.g. , [ 29 , 30 ] ) . comparative analysis of primary sequences and tertiary structures of retrotransposon chds and the chds from functional proteins with known function can provide important insights into the possible roles of some of the specific aminoacids and the retrotransposon chds as a whole [ 3 , 3133 ] . classical and shadow chds from cellular functional proteins , we identified changes in plant chds starting with the chd_i found in ltr retrotransposons from green algae , moss physcomitrella , and spike moss selaginella and culminating in the chd_ii that is isolated from genomes of gymnosperms and angiosperms ( figure 3 ) . the chds of ltr retrotransposons obtained from the selaginella genome represent transitional stages between chd_i and chd_ii . for example , sm2-galahad chd is close to the classical chds , whereas sm1-galahad chd contains a few substitutions in the chromo box motif ( y / f)-(l / f / y)-(l / i / v)-k-(w / y)-(k / r)-g . the chromo - box is one of the motifs that is essential for hydrophobic core formation [ 31 , 32 ] . this motif is characteristic for the classical chds of chromodomain - containing proteins . sm - diluvium , lacks y24 ( corresponding to position 1 in the multiple alignment represented in figure 3 ) , but it still has both aromatic aminoacids , w45 and y48 ( positions 28 and 31 ) , which form methyl binding cages [ 3 , 32 ] . with respect to different clades of ltr retrotransposons , galadriel - like chds lost essential aminoacids ( y24 and y48 ) and diverged significantly from chd_i . nevertheless , galadriel - like chds have the conservative motif ( y / f)-(l / y)-(i / v)-k - w - k - g ( single - letter code , capital letters represent the most prominent aminoacid ) , which is very close to the chromo box . reina - like ltr retrotransposons have traces of this motif , but tekay - like chds have lost the motif with the exception of v43 ( position 26 in the multiple alignment ) and the highly conservative residue w45 ( figure 3 ) . at the same time , tekay - like chds have a number of conservative characteristic motifs . for example , the protein motif ( k / r)-x-(l / t)-r - x-(k / r ) is present in all of the investigated tekay - like retrotransposon chds but not in reina- or galadriel - like chds . one more tekay - characteristic motif , eextwexe , is highly conserved in tekay chds . a similar motif can be found in diverse ltr retrotransposon chds ; however , this motif is not as conserved in other clades as it is in tekay . the aminoacid motif twe is extremely conserved among all of the retrotransposon chds , with a few exceptions in green algae and is believed to have important functions . interestingly , this particular motif is absent in the majority of shadow chds and in a few classical chds of chromodomain - containing proteins . this motif corresponds to the 3 strand in the tertiary structure [ 3 , 31 ] . while a majority of plant retrotransposon chds lack conserved aromatic residues y24 and y48 , they still retain high sequence similarity with known classical chds of functional proteins . this similarity is much higher than the similarity among classical and shadow chds . moreover , the analysis of tertiary structure shows the presence of all of the structural features that are characteristic of classical chds ( see below ) . for further understanding of processes that lead to the current diversity of plant retrotransposon chds , we examined evolutionary constraints that shape the essential domains of plant ltr retrotransposon polyproteins . the presence of conservative motifs in chd sequences among diverse ltr retrotransposons suggests that evolution has been strongly constrained . at the same time , the presence of conserved clade - specific motifs , as well as the transition from chd_i to chd_ii can indicate that some aminoacid changes had a selective advantage during diversification between clades and could be accumulated at a rate higher than expected under natural evolution ( positive selection ) . to distinguish between these possibilities and to indicate the positions that evolved under positive selection , we analyzed the nonsynonymous to synonymous substitutions rate ratio ( , see section 4 for details ) . only retrotransposons that maintained intact domains chd , core integrase ( int ) , and reverse transcriptase ( rt ) domains of 39 ltr retrotransposons were used as datasets . phylogenetic trees based on multiple alignments of nucleotide sequences were reconstructed for each domain separately . as expected , the major difference in tree topologies among datasets included the appearance of common clusters for galadriel - like ltr retrotransposons from selaginella and the tcn1 clade on tree reconstructions based on chd sequences . overall , rt- and chd - based phylogenies are similar , whereas the int - based tree has a different position for the reina clade ( figure 4 ) . first , we estimated the ratio averaged over all of the sites and all of the lineages using m0 model . this model yields estimates that are close to 0 : ^=0.022 for rt , ^= 0.039 for int , and ^=0.068 for the chd domain . this low rate indicated that there is a dominating role of purifying selection in the evolution of all of the domains . the clade - site test demonstrated that no events of positive selection were inferred for rt , which was expected for a highly conserved protein domain evolving under strict constraints ( e.g. , [ 3436 ] ) . unexpectedly , strong positive selection was detected by clade - site test on the branch subtending the reina clade on the int phylogenetic tree ( r branch ; figure 4(b ) ) . this result was confirmed by a branch - site test of positive selection ( modified model a modified model a with 2 = 1 fixed comparison ) that detected positive selection on the r branch with a significance level of 0.01 ( table 3 ) , taking into consideration multiple testing corrections ( see section 4 for details ) . however , it is highly unlikely , taken in consideration the nature of the analyzed sequences and the pattern of substitutions ( see figure 3 ) . the branch - site tests also revealed several events of positive selection on the chd phylogenetic tree . the evolutionary changes of chds in the galadriel - tekay - reina group most probably occurred under positive selection at the 0.05 significance level based on the mixture distribution by hommel correction procedure ( gtr branch on figure 4(c ) ) . nonsynonymous substitutions were also significantly elevated above background on branches subtending the tekay - reina group and the galadriel clade ( branch site significance level of 0.1 ; tr and g branches on figure 4(c ) , resp . ) . we found evidence for the positive selection of chds for the gtr and tr branches , with positive signals coming from a few codons ( significance level 0.1 ; figure 5 ) . specifically , three codons appeared to be under positive selection for the tr branch at the cutoff posterior probability 95% ( corresponding to aminoacid resides in positions 3 , 46 , and 48 of the multiple alignments presented in figure 5(a ) ) . proline residues that are located in positions 3 and 48 of chds from tekay and reina ltr retrotransposons are highly conserved in these clades ( see also figure 3 ) . such a high degree of conservation may indicate that these residues are functionally important for both the reina- and tekay - like chds . p3 is located in a position that corresponds to the residue v3 in classical chds and is believed to participate in the formation of a complementary surface that is responsible for histone 3 peptide ( h3 ) recognition ( based on a study of chds from histone protein 1 , dmhp1 , from drosophila melanogaster ; [ 3 , 3133 ] . the p48 and e46 residues are located in the area that corresponds to the helical structure in dmhp1 chd and other chds . the presence of p48 in the region , which is expected to form an -helix , should have significant effects on secondary structure . prolines are rarely found in and structures , because the structure 's side chain -n can form only one hydrogen bond , which would reduce the stability of such structures . at the same time , prolines are easily accommodated in a variety of turns ; for example , as a pro - x corner ( where x is a variable aminoacid residue ) . we reconstructed the tertiary structure of some representatives of plant retrotransposon chds using i - tasser . all of the representatives clearly exhibited the presence of tertiary structure similar to that of dmhp1 ( figure 5(b ) ) . however , as expected due to the presence of p48 , chds from ltr retrotransposons that belong to the reina and tekay clades bear additional helix structures in comparison to dmhp1 . the p48 is located between the two helices and appears to be crucial for the helix - helix structure formation that is specific to plant ltr retrotransposon chds . the evolutionary history of retrotransposons includes the gain ( and loss ) of functional enzymatic domains , which allows them to adapt to a constantly changing genomic environment [ 12 , 4345 ] . the chromodomain ( chd ) is believed to be a comparatively recent acquisition of ltr retrotransposons [ 12 , 45 ] . the role of the chromodomains most likely is in targeting the insertion of new ltr retrotransposon copies into heterochromatic regions by recognizing specific heterochromatic histone marks and/or other factors . as a consequence , ltr retrotransposons can easily avoid subsequent inactivation and elimination ( purifying selection ) because the chance to interfere with any coding sequence is small in heterochromatic regions . comparative and phylogenetic analysis demonstrates that plant ltr retrotransposon chds represent heterogeneous groups of enzymatic domains with a complex evolutionary history . first , it appears that plant retrotransposon chd_ii evolved from chd_i , which can still be found in genomic sequences of green algae ( chlamyvir clade ) and nonseed plants such as mosses and lycophytes ( tcn1 clade ; figure 6 ; and [ 13 , 16 , 18 ] ) . in addition , the sm1-galahad and sm2-galahad found in lycophyte selaginella carried chd_i , while belonged to the galadriel clade of plant ltr retrotransposons . lycophyte selaginella appears to be a unique model species for the investigation of chromodomains among plants ( figure 6 ) . this species is the only plant species known to have both types of retrotransposon chds in its genome . moreover , ltr retrotransposon chds found in selaginella represent transitional stages between chd_i ( found in fungi and animals ) and chd_ii ( described from angiosperms ) . interestingly , the few chds that were found in gymnosperms are also distant from typical angiosperm chd_ii domain . we believe that the evolutionary history of plant ltr retrotransposon chds and their diversity among different plant groups reflect changes that occurred in chromatin organization ( e.g. , the distribution of heterochromatin / euchromatin mark ; or molecular differences in specific heterochromatin / euchromatin marks ) from green algae to higher plants . it was proposed earlier that although the histone methylation marks are conserved among eukaryotes , the distribution of the individual marks and their functional meaning may have diverged as different phyla evolved . for example , histone h3 trimethyl - k9 ( h3k9me3 ) is a heterochromatin - specific mark in schizosaccharomyces pombe and in mammals [ 49 , 50 ] but has never been found to be associated with heterochromatin in plants ( reviewed in ) . moreover , heterochromatin - specific marks have an uneven distribution among plants : histone h3 dimethyl - k27 ( h3k27me2 ) has been shown to be a typical modification in the heterochromatic regions of arabidopsis thaliana , vicea faba , zea mays , and secale cereale , but it was not detected in species such as glycine max , plantago ovate , and hordeum vulgare [ 5258 ] ; only two species so far , s. cereale and in v. faba , showed labeling of heterochromatin with histone h3 trimethyl - k27 ( h3k27me3 ) [ 53 , 55 ] . the limited information that is available for gymnosperms indicates that their heterochromatic marks seem to be quite different from those of angiosperms . for example , histone h3 monomethyl - k9 ( h3k9me1 ) , h3k9me2 , and histone h3 monomethyl - k27 ( h3k27me1 ) modifications , which are believed to be associated with silencing and heterochromatin formation in a. thaliana , are underrepresented in picea abies and pinus sylvestris . at the same time , h3k9me3 and h3k27me3 are typical heterochromatic marks in two gymnosperm species but are not present in arabidopsis . one would expect mobile elements to be very sensitive to any changes in host genome function and organization ; they must adapt or be eliminated from the genome . divergence in the distribution of individual heterochromatin - associated histone methylation marks could trigger the evolutionary changes of ltr retrotransposon chds in plants , which would result in a shift from the original chd_i ( still present in green algae , mosses , and lycophytes ) to chd_ii ( all higher plants ) and subsequent subdivision into clade - specific chds ( tekay- , reina- , and galadriel - like chds ) . chromodomains carry diverse functions in cells , from the recognition of specific h3 histone modifications to protein dimerisation as well as dna and rna binding ( for review ) . it is believed that considerable diversity of recognition by chds is generated within the chd family through relatively few aminoacid substitutions at the aromatic cage or the peptide - binding sites . while the function of retrotransposon chds is generally unknown , it was demonstrated that chd_i of maggy ltr retrotransposon from rice - blast fungus magnaporthe oryzae targets the integration of new copies to heterochromatin by recognizing h3k9me2 and h3k9me3 modifications . although a colocalization of tfl2 , a dmhp1-like homolog in arabidopsis , and chd_ii of the tma ltr retrotransposon from a. thaliana were shown in the same study , the actual interacting factor(s ) for plant chd_ii was not found . the sequence divergence between chd_i and chd_ii is a key to the functional differences , and positive selection appears to be involved in the diversification of chds during the evolutionary history of ltr retrotransposons . it was proposed earlier that ltr retrotransposons rarely undergo substitution events that are driven by positive selection , which allows elements to remain unrecognized by the host genome and to escape silencing . however , the chd itself provides the possibility of escaping silencing by the specific targeting of heterochromatic regions when ltr retrotransposons integrate , and their rapid evolution could be advantageous . most of the genes for which positive selection has been documented are involved in interactions between the organism and the environment ( e.g. , ) and/or are subjected to genetic conflict ( e.g. , [ 6163 ] ) . one of the most attractive explanations is coevolutionary pressures ; for example , plant ltr retrotransposon chds might evolve after heterochromatin - associated histone methylation marks the host species . this scenario could explain the shift from chd_i to chd_ii after the divergence of the plant and fungi / metazoa groups as well as the divergence of chds between angiosperms and gymnosperms . it is possible that rapid adaptation coupled with subsequent strong selective pressure not only led to the adaptation of ltr retrotransposons to a changing chromatin environment in plants in general , but it also may have contributed to a functional diversification of clade - specific ltr retrotransposon chds . in other words , while galadriel- , reina- and tekay - like chds were still involved in targeted - integration of new ltr retrotransposon copies , they could possibly recognize different chromatin marks or factors . mobile elements in general , and ltr retrotransposons in particular , are important parts of plant genomes . while it is still believed that mobile elements are selected against and silenced in host genomes to prevent their harmful effects , increasing numbers of studies indicate that mobile elements have been positively selected as major components of heterochromatin ( see ) . chromodomain - containing ltr retrotransposons are the most remarkable example of mobile elements that developed the mechanism of targeted integration into heterochromatic regions . in the present study , we inferred that interactions between chromodomain - containing ltr retrotransposons and the host genome resulted in the present diversity of plant ltr retrotransposon chds and , most likely , led to the retrotransposon - enriched genome organization in plants . it is necessary to note that chromodomain - containing ltr retrotransposons in plant genomes represent a large pool of diverse chromatin remodeling domains , which also possess high evolutionary plasticity ( for a review , see ) . the potential roles of ltr retrotransposon chromodomains in genome and chromatin organization are poorly understood but should not be underestimated . selaginella moellendorffii genomic sequence is available at the doe joint genome institute (; http://genome.jgi-psf.org/selmo1/selmo1.info.html ) . we performed blastn and tblastn searches of the s. moellendorffii database ( http://genome.jgi-psf.org/selmo1/selmo1.info.html ; with default parameters ) using the sm - tcn1 retrotransposon and previously described retrotransposons from other species as queries : osr35ac068924 ; rn377 - 208ak068625 ; reina the full - length copies of newly identified gypsy ltr retrotransposons were discovered in genomic sequences and analyzed by unipro ugene software ( http://ugene.unipro.ru/ ) . the ltr retrotransposon sequences obtained during blastn and tblastn searches were localized using unipro ugene option with default parameters ( both strands , match percent : 100% , whole sequence range ) . all dna alignments were performed by clustalw with default parameters and were edited manually in unipro ugene . the ltr retrotransposon chromodomain search was carried out using blast ( blastn , tblastn , and blastp ) . blast analysis was performed using sequence databases that were accessible from the national center for biotechnology information ( ncbi ) server ( http://blast.ncbi.nlm.nih.gov/blast.cgi ; blastp and megablast with default parameters ) , the u.s . department of energy joint genome institute ( http://genome.jgi-psf.org/ ) , plantgdb ( http://www.plantgdb.org/ ; blastn , tblastn and blastp with default parameters ) , and phytozome , a tool for green plant comparative genomics ( http://www.phytozome.net/ ; blastn with default parameters ) . the full list of species investigated and primary information can be found in supporting information table 1s and table 2s . aminoacid sequences of the known chds were used as queries : maggy l35053 ; tcn1xm_571377 ; retrosor2af061282 ; tma all multiple alignments were performed by clustalw and were edited manually in unipro ugene ( http://ugene.unipro.ru/ ) . phylogenetic analyses were performed using the maximum likelihood ( ml ) method in the phyml 3.0 program . statistical support for the nj tree was evaluated by bootstrapping ( number of replications , 1000 ) . statistical support for the ml tree was evaluated by approximate likelihood ratio test ( alrt ; figure 2 ) and by bootstrapping ( number of replications , 100 ; figure 4 ) [ 69 , 70 ] . the tertiary structures of investigated chd peptides were predicted using i - tasser server with default parameters ( http://zhanglab.ccmb.med.umich.edu/i-tasser/ ) . the tertiary structure of dmhp1 from drosophila melanogaster is available in protein data bank ( http://www.rcsb.org/pdb/home/home.do ) under the id1q3l . the multiple alignment of chd sequences was performed using clustalw available at the revtrans 1.4 server (; revtrans takes a set of dna sequences , virtually translates them , aligns the peptide sequences , and uses this as a scaffold for constructing the corresponding dna multiple alignment . the phylogenetic trees of domains ( figure 4 ) were obtained with the maximum likelihood algorithm implemented in the phyml 3.0 program . from nucleotide sequence alignments for each domain the phyml tree searching algorithm was chosen as the best of subtree pruning and regrafting ( spr ) and nearest neighbor interchange ( nni ) for more thorough explorations of the space of topologies . to assess the reliability of the reconstructed phylogenies , we performed 100 bootstrap reconstructions for each domain . the nonsynonymous to synonymous substitution rate ratio ( = dn / ds ) provides a measure of natural selection at the protein level , with = 1 , > 1 , and < 1 , indicating neutral evolution , purifying selection , and positive selection , respectively . we use codeml program ( the paml package ) to perform lineage and clade - specific analyses of dn / ds ratios ( ) [ 7274 ] . positive selection is tested using a likelihood ratio test ( lrt ) comparing a null model that does not allow > 1 with an alternative model that does . when two models are nested , twice the log - likelihood difference between the two models can be compared with the distribution , with the difference in the number of parameters between the two models as the degrees of freedom ( df ) . at first , to evaluate whether any of three chosen domains from four diverse clades are undergoing positive selection affecting all sites over prolonged time , the simplest one - ratio site model ( m0 ) was used ( codeml parameters used were as follows : model = 0 , nssites = 0 ) . one should keep in mind , since our null hypothesis was always the absence of positive selection failing to reject a null hypothesis and/or providing a null hypothesis were interpreted as an absence of positive selection in either case . at the second stage , we tested each of the clades / groups for a signature of positive selection using clade - site test , this compares the modified clade model c ( model = 3 , nssites = 2 ) with the neutral m1a model ( model = 0 , nssites = 0 ) . for this comparison df we conducted one specific test for each domain using extension of clade model c which allows for more than two branch types . branches leading to appropriate clade / group were labeled : tekay : t ; reina : r ; galadriel : g ; tekay - reina : tr ; galadriel - tekay - reina : gtr ; reina - galadriel : rg ; tekay - reina - galadriel : trg , other branches were used as background . the main purpose of the test was to identify whether there is at least one branch potentially under positive selection . all maximum likelihood estimates for rt branches of the site class 2 ratio were close to 0 , thus taking in the consideration previous results we conclude that there are not any events of positive selection . nevertheless , this clade - based test does not directly examine whether any ratio is significantly greater than one . finally , we applied the lrt based on two branch - site models with one , where was to be estimated and other with fixed to 1 , to test every of five aforementioned branch of the chd and int trees for evidence of positive selection , this test is also known as the branch - site test of positive selection . branch - site models of codon substitution allow to vary both among sites in the protein and across branches on the tree and provide a means to detect short episodes of molecular adaptation affecting just a few sites . in these models one of two branch - site models presented in codeml modified model a ( model = 2 , nssites = 2 ) was used for comparison with null hypothesis . site class 0 includes codons that are conserved throughout the tree , with 0 < 0 < 1 estimated . site class 1 includes codons that are evolving neutrally throughout the tree with 1 = 1 . site classes 2a and 2b include codons that are conserved or neutral on the background branches , but become under positive selection on the foreground branches with 2 > 1 estimated . the model involves four parameters in the distribution : p0 , p1 , 0 , and 2 . the null hypothesis was also modified model a but with 2 = 1 fixed ( codeml options were switched from fix_omega = 0 to fix_omega = 1 and omega = 1 ) . a likelihood ratio test ( lrt ) based on models was found to have satisfactory accuracy and reasonable power [ 7579 ] . branch - site models allow only two types of branches thus the most common approach to test several branches on the tree is to treat every branch as foreground in turn . the probability of rejecting falsely at least one of null hypotheses in such tests can be high . the hommel procedure that controls family - wise error rate ( fwer ) was used as correction method . the results of bayes empirical bayes ( beb ) approach which accommodates uncertainties in the maximum likelihood estimates were used to identify sites under positive selection if the likelihood ratio test after correction procedure was significant . table 3 summarizes maximum likelihood estimates and test statistics for lrts corresponding to the every of 5 branches leading to appropriate clades / groups of the chd tree ( see supporting information protocol s1 for details ) .

chromodomain - containing ltr retrotransposons are one of the most successful groups of mobile elements in plant genomes . previously , we demonstrated that two types of chromodomains ( chds ) are carried by plant ltr retrotransposons . chromodomains from group i ( chd_i ) were detected only in tcn1-like ltr retrotransposons from nonseed plants such as mosses ( including the model moss species physcomitrella ) and lycophytes ( the selaginella species ) . ltr retrotransposon chromodomains from group ii ( chd_ii ) have been described from a wide range of higher plants . in the present study , we performed computer - based mining of plant ltr retrotransposon chds from diverse plants with an emphasis on spike - moss selaginella . our extended comparative and phylogenetic analysis demonstrated that two types of chds are present only in the selaginella genome , which puts this species in a unique position among plants . it appears that a transition from chd_i to chd_ii and further diversification occurred in the evolutionary history of plant ltr retrotransposons at approximately 400 mya and most probably was associated with the evolution of chromatin organization .

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

chds perform a wide range of functions , including chromatin targeting and interactions between different proteins , rna and dna . there are two major groups of chds that are found in eukaryotic chromodomain - containing proteins . the chromodomain has been found not only in eukaryotic functional proteins but also in diverse ltr retrotransposons , which are called chromodomain - containing gypsy ltr retrotransposons or chromoviruses [ 12 , 13 ] . chromoviruses are the most widespread lineage of gypsy ltr retrotransposons and are present in the genomes of fungi , plants , and vertebrates [ 13 , 14 ] . group i chds from retrotransposons ( chds_i ) are similar to classical chds of chromodomain - containing proteins . this group of chds was found in diverse eukaryotic ltr retrotransposons , including fungal and vertebrate gypsy elements , as well as in ltr retrotransposons from moss physcomitrella patens and spike - moss selaginella moellendorffii , which belong to the tcn1 clade [ 13 , 1618 ] . representatives of group ii chds from retrotransposons ( chds_ii ) lack the first conserved aromatic residue ( y24 ) and usually the third ( y48 ) . the mostly heterochromatic distribution of plant chromoviruses along with data describing the localization of chromodomain - yfp fused protein in heterochromatin can be used as indirect evidence for recognizing heterochromatin and directing the integration role of chds_ii . previously , we demonstrated that our knowledge of plant chromodomain - containing ltr retrotransposons is mostly limited to knowledge about seed plants ( mostly angiosperms ) [ 1618 ] . an investigation of retrotransposons from nonseed plants could shed light on the evolutionary history of retrotransposons and their impact on the evolutionary history of plant genomes . the present survey of chromodomains from diverse plants with an emphasis on the spike - moss selaginella demonstrated that a transition from chds_i to chds_ii occurred in the evolutionary history of plants approximately 500400 mya . we examined the evolutionary constraints that shaped the diversity of chds_ii in plants and demonstrated that positive selection contributed to the diversification of clade - specific ltr retrotransposon chds . both the transition from chds_i to chds_ii and the diversification of clade - specific chds reflect evolutionary changes that occurred in plant chromatin organization . previously , we described sm - tcn1 chd - containing gypsy ltr retrotransposons , which presumably appeared as a result of a horizontal transfer from fungi during the early evolution of plants . these retrotransposons were used for further classification based on comparative and phylogenetic analysis , which included known ltr retrotransposons from other plants . in total , five diverse families of chd - containing ltr retrotransposons were found in addition to the previously described sm - tcn1 . sm1-galahad and sm2-galahad are closely related to each other and form a common clade with galadriel - like retrotransposons from monocots and dicots , suggesting that this clade originated before nonvascular and vascular plants separated from a common ancestor , roughly 400 mya . among the sequences that are available in the ncbi protein database based on blastp analysis , the retroelement that is most closely related to sm1-galahad and sm2-galahad is the galadriel ltr retrotransposon from lycopersicon esculentum , which was identified in the cf-9 disease resistance gene cluster . a search for characteristic motifs within the polyprotein sequence identified several functional domains , such as the cysteine or zn - finger motif with cys - x2-cys - x4-his - x4-cys ( cchc ) composition , proteinase ( pr ) , reverse transcriptase ( rt ) , ribonuclease h ( rnh ) , and core integrase ( core int ) , in the order indicated , confirming that sm1-galahad belongs to the ty3/gypsy ltr retrotransposons . the 309-bp orf3 encodes a protein ( 113 aa ) containing a chd group i ( chd_i ) domain . target site duplications of five base pairs mark the integration of the intact sm - fogey and sm - diluvium in the genomic sequence . both ltr retrotransposons contain four catalytic regions , pr , rt , rnh , and int of the retroviral genes gag and pol , in a continuous single open reading frame . sm - diluvium also has a chd group ii ( chd_ii ) , whereas sm - fogey does not carry a chromodomain . the primer binding site ( pbs ) , which is complementary to the 3 end of trna and has a one - nucleotide spacer next to the adjacent 5 ltr , the ppt with the stretch of 14 purines for sm - fogey and sm - diluvium is located immediately before the 3 ltr . the aminoacid domains show the highest similarity to the corresponding regions of the tekay - like retrotransposon shmidt that is adjacent to the disease resistance - priming gene npr1 in beta vulgaris ( ef101866 ; ) and retrotransposons from the diverse oryza species , including ltr retrotransposons from oryza sativa and oryza australiensis ( e.g. it appears that sm - fogey and sm - diluvium are the most closely related to the tekay clade of chd - containing gypsy ltr retrotransposons from plants . the most intriguing finding that concerns the chd - containing gypsy ltr retrotransposons from the spike - moss s. moellendorffii is the presence of both types of ltr retrotransposon chromodomains , chd_i and chd_ii , in the same plant genome . however , it appears that phylogenetic distribution of chd_i - containing elements extends to most if not all extant green plant lineages . previously , we reported that chd_i - containing ltr retrotransposons belonging to the tcn1 clade can be found in both moss physcomitrella and spike - moss selaginella [ 16 , 18 ] . to expand our understanding of the evolution and diversity of retrotransposon chromodomains in green plants , we implemented a search throughout sequence databases including but not limited to plantgdb ( http://www.plantgdb.org/ ) and phytozome ( http://www.phytozome.net/ ) . for 80 species , retrotransposon chds were detected in ests ; chds were also found in genomesurvey sequences ( gss ) and whole genomic sequences ( wgs ) databases for 46 investigated species . nevertheless , we were able to identify a few ltr retrotransposon chds in all of the species investigated ( table 2 ; supporting information table 1s ) . the phylogenetic analysis based on the aminoacid sequences of newly identified chds and chds from known plant ltr retrotransposons support these findings with several exceptions : ( i ) galadriel - like ltr retrotransposons from selaginella have chd_i and are grouped with the tcn1 clade ; ( ii ) chds from conifers , which were previously believed to belong to reina - like ltr retrotransposons from angiosperms , actually form their own branch ; ( iii ) three chds identified in green algae grouped together with the selaginella sm - cranky ltr retrotransposon and not with chds from the chlamyvir clade ( figure 2 ; ) . additionally , tekay - like ltr retrotransposon chds from gymnosperms formed a common cluster that appears to be distinct from other tekay - like chds . comparative analysis of primary sequences and tertiary structures of retrotransposon chds and the chds from functional proteins with known function can provide important insights into the possible roles of some of the specific aminoacids and the retrotransposon chds as a whole [ 3 , 3133 ] . classical and shadow chds from cellular functional proteins , we identified changes in plant chds starting with the chd_i found in ltr retrotransposons from green algae , moss physcomitrella , and spike moss selaginella and culminating in the chd_ii that is isolated from genomes of gymnosperms and angiosperms ( figure 3 ) . the chds of ltr retrotransposons obtained from the selaginella genome represent transitional stages between chd_i and chd_ii . sm - diluvium , lacks y24 ( corresponding to position 1 in the multiple alignment represented in figure 3 ) , but it still has both aromatic aminoacids , w45 and y48 ( positions 28 and 31 ) , which form methyl binding cages [ 3 , 32 ] . with respect to different clades of ltr retrotransposons , galadriel - like chds lost essential aminoacids ( y24 and y48 ) and diverged significantly from chd_i . reina - like ltr retrotransposons have traces of this motif , but tekay - like chds have lost the motif with the exception of v43 ( position 26 in the multiple alignment ) and the highly conservative residue w45 ( figure 3 ) . interestingly , this particular motif is absent in the majority of shadow chds and in a few classical chds of chromodomain - containing proteins . while a majority of plant retrotransposon chds lack conserved aromatic residues y24 and y48 , they still retain high sequence similarity with known classical chds of functional proteins . for further understanding of processes that lead to the current diversity of plant retrotransposon chds , we examined evolutionary constraints that shape the essential domains of plant ltr retrotransposon polyproteins . at the same time , the presence of conserved clade - specific motifs , as well as the transition from chd_i to chd_ii can indicate that some aminoacid changes had a selective advantage during diversification between clades and could be accumulated at a rate higher than expected under natural evolution ( positive selection ) . as expected , the major difference in tree topologies among datasets included the appearance of common clusters for galadriel - like ltr retrotransposons from selaginella and the tcn1 clade on tree reconstructions based on chd sequences . this low rate indicated that there is a dominating role of purifying selection in the evolution of all of the domains . the evolutionary changes of chds in the galadriel - tekay - reina group most probably occurred under positive selection at the 0.05 significance level based on the mixture distribution by hommel correction procedure ( gtr branch on figure 4(c ) ) . proline residues that are located in positions 3 and 48 of chds from tekay and reina ltr retrotransposons are highly conserved in these clades ( see also figure 3 ) . p3 is located in a position that corresponds to the residue v3 in classical chds and is believed to participate in the formation of a complementary surface that is responsible for histone 3 peptide ( h3 ) recognition ( based on a study of chds from histone protein 1 , dmhp1 , from drosophila melanogaster ; [ 3 , 3133 ] . however , as expected due to the presence of p48 , chds from ltr retrotransposons that belong to the reina and tekay clades bear additional helix structures in comparison to dmhp1 . the p48 is located between the two helices and appears to be crucial for the helix - helix structure formation that is specific to plant ltr retrotransposon chds . the evolutionary history of retrotransposons includes the gain ( and loss ) of functional enzymatic domains , which allows them to adapt to a constantly changing genomic environment [ 12 , 4345 ] . comparative and phylogenetic analysis demonstrates that plant ltr retrotransposon chds represent heterogeneous groups of enzymatic domains with a complex evolutionary history . first , it appears that plant retrotransposon chd_ii evolved from chd_i , which can still be found in genomic sequences of green algae ( chlamyvir clade ) and nonseed plants such as mosses and lycophytes ( tcn1 clade ; figure 6 ; and [ 13 , 16 , 18 ] ) . in addition , the sm1-galahad and sm2-galahad found in lycophyte selaginella carried chd_i , while belonged to the galadriel clade of plant ltr retrotransposons . lycophyte selaginella appears to be a unique model species for the investigation of chromodomains among plants ( figure 6 ) . this species is the only plant species known to have both types of retrotransposon chds in its genome . moreover , ltr retrotransposon chds found in selaginella represent transitional stages between chd_i ( found in fungi and animals ) and chd_ii ( described from angiosperms ) . we believe that the evolutionary history of plant ltr retrotransposon chds and their diversity among different plant groups reflect changes that occurred in chromatin organization ( e.g. moreover , heterochromatin - specific marks have an uneven distribution among plants : histone h3 dimethyl - k27 ( h3k27me2 ) has been shown to be a typical modification in the heterochromatic regions of arabidopsis thaliana , vicea faba , zea mays , and secale cereale , but it was not detected in species such as glycine max , plantago ovate , and hordeum vulgare [ 5258 ] ; only two species so far , s. cereale and in v. faba , showed labeling of heterochromatin with histone h3 trimethyl - k27 ( h3k27me3 ) [ 53 , 55 ] . for example , histone h3 monomethyl - k9 ( h3k9me1 ) , h3k9me2 , and histone h3 monomethyl - k27 ( h3k27me1 ) modifications , which are believed to be associated with silencing and heterochromatin formation in a. thaliana , are underrepresented in picea abies and pinus sylvestris . divergence in the distribution of individual heterochromatin - associated histone methylation marks could trigger the evolutionary changes of ltr retrotransposon chds in plants , which would result in a shift from the original chd_i ( still present in green algae , mosses , and lycophytes ) to chd_ii ( all higher plants ) and subsequent subdivision into clade - specific chds ( tekay- , reina- , and galadriel - like chds ) . while the function of retrotransposon chds is generally unknown , it was demonstrated that chd_i of maggy ltr retrotransposon from rice - blast fungus magnaporthe oryzae targets the integration of new copies to heterochromatin by recognizing h3k9me2 and h3k9me3 modifications . although a colocalization of tfl2 , a dmhp1-like homolog in arabidopsis , and chd_ii of the tma ltr retrotransposon from a. thaliana were shown in the same study , the actual interacting factor(s ) for plant chd_ii was not found . the sequence divergence between chd_i and chd_ii is a key to the functional differences , and positive selection appears to be involved in the diversification of chds during the evolutionary history of ltr retrotransposons . one of the most attractive explanations is coevolutionary pressures ; for example , plant ltr retrotransposon chds might evolve after heterochromatin - associated histone methylation marks the host species . this scenario could explain the shift from chd_i to chd_ii after the divergence of the plant and fungi / metazoa groups as well as the divergence of chds between angiosperms and gymnosperms . it is possible that rapid adaptation coupled with subsequent strong selective pressure not only led to the adaptation of ltr retrotransposons to a changing chromatin environment in plants in general , but it also may have contributed to a functional diversification of clade - specific ltr retrotransposon chds . mobile elements in general , and ltr retrotransposons in particular , are important parts of plant genomes . chromodomain - containing ltr retrotransposons are the most remarkable example of mobile elements that developed the mechanism of targeted integration into heterochromatic regions . in the present study , we inferred that interactions between chromodomain - containing ltr retrotransposons and the host genome resulted in the present diversity of plant ltr retrotransposon chds and , most likely , led to the retrotransposon - enriched genome organization in plants . it is necessary to note that chromodomain - containing ltr retrotransposons in plant genomes represent a large pool of diverse chromatin remodeling domains , which also possess high evolutionary plasticity ( for a review , see ) . the potential roles of ltr retrotransposon chromodomains in genome and chromatin organization are poorly understood but should not be underestimated . we performed blastn and tblastn searches of the s. moellendorffii database ( http://genome.jgi-psf.org/selmo1/selmo1.info.html ; with default parameters ) using the sm - tcn1 retrotransposon and previously described retrotransposons from other species as queries : osr35ac068924 ; rn377 - 208ak068625 ; reina the full - length copies of newly identified gypsy ltr retrotransposons were discovered in genomic sequences and analyzed by unipro ugene software ( http://ugene.unipro.ru/ ) . the phylogenetic trees of domains ( figure 4 ) were obtained with the maximum likelihood algorithm implemented in the phyml 3.0 program . to assess the reliability of the reconstructed phylogenies , we performed 100 bootstrap reconstructions for each domain . at the second stage , we tested each of the clades / groups for a signature of positive selection using clade - site test , this compares the modified clade model c ( model = 3 , nssites = 2 ) with the neutral m1a model ( model = 0 , nssites = 0 ) . all maximum likelihood estimates for rt branches of the site class 2 ratio were close to 0 , thus taking in the consideration previous results we conclude that there are not any events of positive selection . branch - site models allow only two types of branches thus the most common approach to test several branches on the tree is to treat every branch as foreground in turn .

incorporating pharmaceutical compounds into a biodegradable polymer carrier is an effective strategy for forming a drug delivery system and has been given increasing attention.15 yin and yates reported that drug - entrapped biodegradable microcapsules , with sustained drug release , were prepared by spraying a suspension of hollow poly(dl - lactide ) particles in procaine hydrochloride aqueous solution into plasticizing solvents.6 a further report by zvonar et al described use of a self - microemulsifying system to fabricate ca - pectinate microcapsules to improve the release and permeability of furosemide.7 drug - loaded polymer microspheres have been variously prepared by phase separation,8 interfacial polymerization,9 spray - drying,10 freeze - drying,11 and emulsion evaporation.12 however , these conventional strategies always either involve an intense operating process or preparation of particles with a wide particle size distribution and a relatively large amount of organic solvent residue . compared with these traditional methodologies , supercritical co2-based technology offers a number of advantages , including mild critical conditions ( tc = 304.1 k , pc = 7.38 mpa ) , no toxicity , a nonorganic residue , are not flammable , and all at a relatively lower price.1315 the supercritical antisolvent process has been widely utilized to fabricate drug - loaded polymer microparticles.1618 in the supercritical antisolvent process , an organic solution is sprayed into supercritical co2 to effect immediate contact between the two media . extremely rapid extraction of the organic solvent into supercritical co2 leads to higher supersaturation of the solution , creating fast nucleation and growth , and finally micronization of the solute . similarly , when the drug and polymer are both dissolved to obtain a homogeneous organic solution and sprayed into supercritical co2 , the drug and polymer are coprecipitated due to a higher supersaturation caused by diffusion of the organic solvent into the supercritical co2 and vice versa,19,20 known as the coprecipitation process . however , the coprecipitation process generates drug - loaded polymer microspheres accompanied by relatively low drug loading , low encapsulation efficiency , and no sustained release of the drug , which can be partially explained by the fact that the drug is precipitated onto or just loosely attached to the surface of the polymer microspheres.19,21 to overcome the drawbacks of the coprecipitation process , ie , low drug loading and low encapsulation efficiency , the microencapsulation process was developed19 whereby nanoparticles of a drug are first prepared , suspended in an organic polymer solution , and then sprayed into supercritical co2 using a high - performance liquid chromatography ( hplc ) pump . due to precipitation of the polymer from the sprayed suspension , drug nanoparticles are entrapped by the polymer with a matrix or core - shell structure.19,22 in order to strengthen mutual diffusion between the sprayed solution and the supercritical co2 , a solutionenhanced dispersion by supercritical fluid ( seds ) process was developed and has been widely used in supercritical fluid techniques.23 during this process , the substrate(s ) solution and supercritical co2 are copulverized through a coaxial nozzle , in which supercritical co2 acts as an antisolvent and spraying enhancer.2427 however , when seds is used in the microencapsulation process , ie , driving , delivering , and spraying the suspension into the high - pressure reaction vessel , the delivery system , especially the one - way valve , is easily damaged or obstructed by the suspended particles . to tackle this problem , a suspension - enhanced dispersion by supercritical fluid ( speds ) process was developed in the present study based on seds . with speds , supercritical co2 and a suspension consisting of drug nanoparticles and polymer solution are simultaneously sprayed through a stainless steel coaxial nozzle , whereby spraying and dispersion of the suspension is enhanced by spraying of supercritical co2 . to avoid obstruction and guarantee smooth delivery of the suspension , a stainless steel cylindrical container equipped with a piston working like an injector was introduced and added into the previous seds apparatus . this study attempted to prepare drug - loaded polymer microspheres using a supercritical microencapsulation process with improved drug loading , encapsulation efficiency , and sustained release , compared with the coprecipitation process . methotrexate is an antifolate drug which is nearly insoluble in water , is effective in the treatment of a range of malignant and autoimmune diseases,28 and was selected as the drug model in this study . the drug was micronized by seds , suspended in a plla - peg - plla solution containing dichloromethane , and microencapsulated by plla - peg - plla via speds . for the purpose of comparison , a parallel study involving the coprecipitation process the products were characterized by scanning electron microscopy ( sem ) and fourier transform infrared spectroscopy ( ftir ) . drug loading , encapsulation efficiency , and drug release profiles as well as release kinetics were also studied . the plla - peg - plla triblock polymer with a gross molecular weight of 77 kda and 8% ( w / w ) peg ( 6000 kda ) was obtained from jianbaokaiyuan biomaterials co , ltd ( jinan , china ) . dichloromethane and dimethyl sulfoxide were purchased from sinopharm chemical reagent co , ltd ( beijing , china ) . co2 ( purity 99.9% ) was provided by rihong industrial development co , ltd ( xiamen , china ) . methotrexate nanoparticles were prepared according to a method used in our previous study.29 specifically , methotrexate was first dissolved in dimethyl sulfoxide , and acetone was then added to obtain a homogeneous solution in which acetone acted as an organic nonsolvent for methotrexate and thus increased the saturation ratio and reduced its concentration . the final concentration of methotrexate was 0.5% ( w / v ) ; the dimethyl sulfoxide to acetone ratio was 1:4 ( v / v ) . the methotrexate nanoparticles were micronized by seds , whereby the pressure and temperature of co2 were stabilized at the desired levels , the solution of methotrexate in dimethyl sulfoxide and acetone began being delivered by a hplc pump to the coaxial nozzle and sprayed simultaneously with the supercritical co2 , and methotrexate nanoparticles were generated due to extremely rapid precipitation of methotrexate . in the preparation process , the operating pressure , temperature , flow rate of co2 , and flow rate of the methotrexate solution were stabilized at 12 mpa , 306 k , 2000 standard liters per hour , and 1 ml per minute , respectively . figure 1 is a schematic diagram of the speds apparatus for microencapsulating methotrexate into plla - peg - plla . the apparatus comprised three main sections , ie , a co2 delivery system , a particle suspension delivery system , and a high pressure reaction vessel , the volume of which was 500 ml . an injector was used to avoid obstruction of the pump used for suspension delivery . the seds apparatus has all the same components as those for speds , except for the injector . the injector consists of three stainless steel components , ie , a cylinder ( inside diameter 25.0 mm , outside diameter 35.0 mm , length 155.0 mm ) , an inbuilt columnar piston ( height 20.0 mm ) girdled by a rubber ring , and two nuts ( outside diameter 50 mm , insider diameter 35.0 mm , depth 18 mm ) , one of which is connected to the coaxial nozzle with a stainless conduit and the other with an hplc pump . the stainless steel cylinder is divided into two chambers by the piston . for our experiment , the front chamber was filled with a suspension consisting of methotrexate nanoparticles and a solution of plla - peg - plla containing dichloromethane , and the back chamber was filled with water . when the hplc pump was turned on , the piston started to be driven by water from the hplc pump , pushing the suspension smoothly forward through the stainless conduit to the stainless steel coaxial nozzle . the suspension placed in the front chamber of the injector was obtained by suspending methotrexate nanoparticles in the plla - peg - plla solution containing dichloromethane by ultrasonic dispersion , with a final methotrexate concentration of 0.5% ( w / v ) and a methotrexate to plla - peg - plla ratio of 1:9 ( w / w ) . in the microencapsulation experiment , the chosen pressure and temperature were first achieved and kept stable , and the methotrexate nanoparticle suspension was then driven by the designed injector and sprayed into the high - pressure vessel through a stainless steel coaxial nozzle . the operating pressure , temperature , flow rate of co2 , and flow rate of the methotrexate solution were stabilized at 12 mpa , 306 k , 2000 standard liters per hour , and 1 ml per minute , respectively . after spraying on the suspension , fresh co2 was continuously poured in , with the operating conditions unchanged , to wash the products for 30 minutes and completely remove the residual organic solvent . when washing was completed , the high - pressure vessel was slowly depressurized and the products were collected for subsequent analysis . in order to make comparisons , a coprecipitation process involving methotrexate and plla - peg - plla was carried out in parallel under the same operating conditions ( pressure 12 mpa , temperature 306 k , co2 flow rate 2000 standard liters per hour , and a 1 ml per minute flow rate for the methotrexate solution ) . briefly , methotrexate was first dissolved in dimethyl sulfoxide , and plla - peg - plla solution containing dichloromethane was then added homogeneously , with a final plla concentration of 0.5% ( w / v ) , methotrexate to plla - peg - plla ratios of 1:19 , 1:9 , and 3:17 ( w / w ) , and a dimethyl sulfoxide to dichloromethane ratio of 1:4 ( v / v ) . the surface morphology of the samples was investigated by sem ( hitachi s-4800 , tokyo , japan ) . before observation , the sample was directly absorbed onto a piece of carbon film that was self - adhered on an aluminum stub . the particle size and particle size distribution ftir spectra of the original methotrexate and plla - peg - plla , and the methotrexate ( mtx)-plla - peg - plla microspheres prepared by the microencapsulation and coprecipitation processes were performed using an ftir spectrometer ( ftir 8400s , hitachi ) in transmission mode , with a wavenumber range of 4000400 cm . drug loading and encapsulation efficiency are both significant factors when evaluating the properties of drug - loaded polymer microspheres . to determine drug loading , 10 mg of mtx - plla - peg - plla microspheres was dissolved in 5 ml of dichloromethane ; 30 ml of phosphate - buffered solution buffer ( ph 7.4 ) was added , and the solution was gently heated with magnetic stirring to evaporate the dichloromethane . the solution obtained was filtered through a 0.22 m membrane , and the concentration of methotrexate was measured using an ultraviolet spectrophotometer ( unic uv-2800 , culver city , ca ) at 302.8 nm and comparing this with a standard concentration curve . for calculating the encapsulation efficiency , another 10 mg of mtx - plla - peg - plla microspheres was dispersed in 10 ml of a dilute aqueous solution of sodium hydroxide ( 0.1 mol / l ) to wash off the unencapsulated and loosely surface - bound methotrexate ; the resulting suspension was filtered through a 0.22 m membrane and the amount of methotrexate was determined using the ultraviolet spectrophotometer at 302.8 nm and calculating according to the standard concentration curve . drug loading and encapsulation efficiency were calculated using the following two equations : where w1 , w2 , and w3 represent the weight of methotrexate in the microspheres , the gross weight of the microspheres , and the weight of methotrexate in the microspheres with the unencapsulated methotrexate washed off , respectively . each experiment was performed in triplicate . to simulate and monitor the sustained release of methotrexate in the microspheres , first , 10 mg of mtx - plla - peg - plla microspheres was placed in a pretreated dialysis bag . the bag was tied tightly , inserted in a sealed bottle containing 50 ml of phosphate - buffered solution ( ph 7.4 ) , and incubated in a shaking waterbath at 310 k and 60 rpm . at preset time points ( hours 0.5 , 1 , 2 , 3 onwards ) , 5 ml of the incubated solution was periodically removed , with subsequent addition of 5 ml of fresh phosphate - buffered solution ( ph 7.4 ) . the concentration of methotrexate in the aqueous solution was measured using the ultraviolet spectrophotometer at 302.8 nm and calculating according to the standard concentration curve . the sustained - release profiles of methotrexate were calculated in terms of the cumulative release percentage of methotrexate ( w / w ) with incubation time . the plla - peg - plla triblock polymer with a gross molecular weight of 77 kda and 8% ( w / w ) peg ( 6000 kda ) was obtained from jianbaokaiyuan biomaterials co , ltd ( jinan , china ) . dichloromethane and dimethyl sulfoxide were purchased from sinopharm chemical reagent co , ltd ( beijing , china ) . co2 ( purity 99.9% ) was provided by rihong industrial development co , ltd ( xiamen , china ) . methotrexate nanoparticles were prepared according to a method used in our previous study.29 specifically , methotrexate was first dissolved in dimethyl sulfoxide , and acetone was then added to obtain a homogeneous solution in which acetone acted as an organic nonsolvent for methotrexate and thus increased the saturation ratio and reduced its concentration . the final concentration of methotrexate was 0.5% ( w / v ) ; the dimethyl sulfoxide to acetone ratio was 1:4 ( v / v ) . the methotrexate nanoparticles were micronized by seds , whereby the pressure and temperature of co2 were stabilized at the desired levels , the solution of methotrexate in dimethyl sulfoxide and acetone began being delivered by a hplc pump to the coaxial nozzle and sprayed simultaneously with the supercritical co2 , and methotrexate nanoparticles were generated due to extremely rapid precipitation of methotrexate . in the preparation process , the operating pressure , temperature , flow rate of co2 , and flow rate of the methotrexate solution were stabilized at 12 mpa , 306 k , 2000 standard liters per hour , and 1 ml per minute , respectively . figure 1 is a schematic diagram of the speds apparatus for microencapsulating methotrexate into plla - peg - plla . the apparatus comprised three main sections , ie , a co2 delivery system , a particle suspension delivery system , and a high pressure reaction vessel , the volume of which was 500 ml . an injector was used to avoid obstruction of the pump used for suspension delivery . the seds apparatus has all the same components as those for speds , except for the injector . the injector consists of three stainless steel components , ie , a cylinder ( inside diameter 25.0 mm , outside diameter 35.0 mm , length 155.0 mm ) , an inbuilt columnar piston ( height 20.0 mm ) girdled by a rubber ring , and two nuts ( outside diameter 50 mm , insider diameter 35.0 mm , depth 18 mm ) , one of which is connected to the coaxial nozzle with a stainless conduit and the other with an hplc pump . the stainless steel cylinder is divided into two chambers by the piston . for our experiment , the front chamber was filled with a suspension consisting of methotrexate nanoparticles and a solution of plla - peg - plla containing dichloromethane , and the back chamber was filled with water . when the hplc pump was turned on , the piston started to be driven by water from the hplc pump , pushing the suspension smoothly forward through the stainless conduit to the stainless steel coaxial nozzle . the suspension placed in the front chamber of the injector was obtained by suspending methotrexate nanoparticles in the plla - peg - plla solution containing dichloromethane by ultrasonic dispersion , with a final methotrexate concentration of 0.5% ( w / v ) and a methotrexate to plla - peg - plla ratio of 1:9 ( w / w ) . in the microencapsulation experiment , the chosen pressure and temperature were first achieved and kept stable , and the methotrexate nanoparticle suspension was then driven by the designed injector and sprayed into the high - pressure vessel through a stainless steel coaxial nozzle . the operating pressure , temperature , flow rate of co2 , and flow rate of the methotrexate solution were stabilized at 12 mpa , 306 k , 2000 standard liters per hour , and 1 ml per minute , respectively . after spraying on the suspension , fresh co2 was continuously poured in , with the operating conditions unchanged , to wash the products for 30 minutes and completely remove the residual organic solvent . when washing was completed , the high - pressure vessel was slowly depressurized and the products were collected for subsequent analysis . in order to make comparisons , a coprecipitation process involving methotrexate and plla - peg - plla was carried out in parallel under the same operating conditions ( pressure 12 mpa , temperature 306 k , co2 flow rate 2000 standard liters per hour , and a 1 ml per minute flow rate for the methotrexate solution ) . briefly , methotrexate was first dissolved in dimethyl sulfoxide , and plla - peg - plla solution containing dichloromethane was then added homogeneously , with a final plla concentration of 0.5% ( w / v ) , methotrexate to plla - peg - plla ratios of 1:19 , 1:9 , and 3:17 ( w / w ) , and a dimethyl sulfoxide to dichloromethane ratio of 1:4 ( v / v ) . the surface morphology of the samples was investigated by sem ( hitachi s-4800 , tokyo , japan ) . before observation , the sample was directly absorbed onto a piece of carbon film that was self - adhered on an aluminum stub . the particle size and particle size distribution ftir spectra of the original methotrexate and plla - peg - plla , and the methotrexate ( mtx)-plla - peg - plla microspheres prepared by the microencapsulation and coprecipitation processes were performed using an ftir spectrometer ( ftir 8400s , hitachi ) in transmission mode , with a wavenumber range of 4000400 cm . drug loading and encapsulation efficiency are both significant factors when evaluating the properties of drug - loaded polymer microspheres . to determine drug loading , 10 mg of mtx - plla - peg - plla microspheres was dissolved in 5 ml of dichloromethane ; 30 ml of phosphate - buffered solution buffer ( ph 7.4 ) was added , and the solution was gently heated with magnetic stirring to evaporate the dichloromethane . the solution obtained was filtered through a 0.22 m membrane , and the concentration of methotrexate was measured using an ultraviolet spectrophotometer ( unic uv-2800 , culver city , ca ) at 302.8 nm and comparing this with a standard concentration curve . for calculating the encapsulation efficiency , another 10 mg of mtx - plla - peg - plla microspheres was dispersed in 10 ml of a dilute aqueous solution of sodium hydroxide ( 0.1 mol / l ) to wash off the unencapsulated and loosely surface - bound methotrexate ; the resulting suspension was filtered through a 0.22 m membrane and the amount of methotrexate was determined using the ultraviolet spectrophotometer at 302.8 nm and calculating according to the standard concentration curve . drug loading and encapsulation efficiency were calculated using the following two equations : where w1 , w2 , and w3 represent the weight of methotrexate in the microspheres , the gross weight of the microspheres , and the weight of methotrexate in the microspheres with the unencapsulated methotrexate washed off , respectively . to simulate and monitor the sustained release of methotrexate in the microspheres , release studies were carried out as follows . first , 10 mg of mtx - plla - peg - plla microspheres was placed in a pretreated dialysis bag . the bag was tied tightly , inserted in a sealed bottle containing 50 ml of phosphate - buffered solution ( ph 7.4 ) , and incubated in a shaking waterbath at 310 k and 60 rpm . at preset time points ( hours 0.5 , 1 , 2 , 3 onwards ) , 5 ml of the incubated solution was periodically removed , with subsequent addition of 5 ml of fresh phosphate - buffered solution ( ph 7.4 ) . the concentration of methotrexate in the aqueous solution was measured using the ultraviolet spectrophotometer at 302.8 nm and calculating according to the standard concentration curve . the sustained - release profiles of methotrexate were calculated in terms of the cumulative release percentage of methotrexate ( w / w ) with incubation time . using the optimized operating parameters , together with addition of acetone as a nonsolvent to enhance the degree of saturation and reduce the methotrexate concentration in the initial solution , nanoparticles of methotrexate were successfully fabricated by seds . the surface morphology seen on sem is shown in figure 2 , in which methotrexate nanoparticles can be observed to have a smooth surface . when observed by the human eye , the methotrexate nanoparticles had a fluffy and soft appearance resembling cotton - candy . this macroscopic surface morphology can be explained by the fact that methotrexate when processed by seds exists not in the form of connected aggregates but as free nanoparticles . 30 furthermore , methotrexate nanoparticles prepared by seds were shown to have a spherical shape with a relatively narrow particle size distribution , ie , a span of 0.73 and an average size of 86 nm , as shown in table 1 . during the microencapsulation process , a suspension consisting of methotrexate nanoparticles and plla - peg - plla in dichloromethane solution was driven by an injector and sprayed into a high - pressure vessel through a coaxial nozzle , where the jet stream of supercritical co2 strengthened spraying of the suspension . when the droplets of suspension came into contact with supercritical co2 , a nonsolvent for plla - peg - plla , extensive mutual diffusion into and out of the suspension droplets occurred due to the high solubility of dichloromethane in supercritical co2 . with regard to the plla - peg - plla dichloromethane solution , the crystallization pathway shifted from the liquid - solution region into the solid - liquid region , via the liquid - solid and liquid - fluid two - phase region and the solid - liquid - fluid three - phase region , thus crossing a series of tie lines . when supersaturation of the plla - peg - plla dichloromethane solution was approached , extraction of dichloromethane from the suspension droplet led to an extremely rapid phase change , with formation of a solid phase and precipitation of plla - peg - plla onto the surface of the methotrexate nanoparticles . figure 3a and b illustrate the surface morphology of mtx - plla - peg - plla microspheres manufactured by the microencapsulation process and the coprecipitation process , respectively . the particle size of the mtx - plla - peg - plla microspheres , prepared by either the coprecipitation process or the microencapsulation process , appeared much larger than that of the methotrexate nanoparticles , indicating that most of the methotrexate nanoparticles were successfully encapsulated by plla - peg - plla . the particle size of the mtx - plla - peg - plla composite microspheres prepared by the microencapsulation process was somewhat larger than that of the composite microspheres generated by the coprecipitation process . this is because , in the microencapsulation process , not one but two or more methotrexate nanoparticles were encapsulated into plla - peg - plla , generating relatively larger microspheres in which plla - peg - plla acted as a matrix to entrap methotrexate nanoparticles , ie , the composite microspheres obtained in the microencapsulation process have an inlaymatrix structure rather than a typical core - shell structure . judging from the span , mtx - plla - peg - plla composite microspheres prepared by the microencapsulation and coprecipitation processes both had a narrow particle size distribution and there was no significant difference between them . ftir is a widely used and very useful tool in chemistry for analyzing chemical components and identifying characteristic groups . as shown in the ftir spectra of figure 4 , there are three major regions of the ftir spectra for plla - peg - plla , ie , a c = o stretching vibration region at 18201700 cm , a region at 15001000 cm attributable to ch3 , ch bending , and c o c stretching vibration , and a region at 980820 cm for c c backbone stretching vibration . among these three regions , the major peak at 1759 cm is due to the stretching vibration of the carbonyl group in plla - peg - plla and can be used to identify pllapeg - plla ; this major peak can also be observed in the mtx - plla - peg - plla microspheres prepared by the two different processes . in the mtx - plla - peg - plla microsphere spectrum , a weak peak of plla - peg - plla at 1620 cm was considerably enhanced due to the existence of characteristic peaks of methotrexate at 1718 cm and 1604 cm , representing c = o stretching vibration and c = c benzene backbone stretching vibration , respectively . apart from that , no significant difference and no new peaks were found in the ftir spectrum of the composite microspheres prepared by either the microencapsulation process or the coprecipitation process , ie , no chemical reaction occurred in the two kinds of process . this all demonstrates that speds modified on the basis of seds is a typically physical process causing microencapsulation of drug nanoparticles via a biodegradable polymer , and that this process is favorable for drugs , because chemical interaction may alter the properties of drugs and reduce their effectiveness . figure 5 illustrates the drug loading and encapsulation efficiency of mtx - plla - peg - plla microspheres prepared by the microencapsulation process and by the coprecipitation process . when the drug dose was set at 10% ( w / w ) , drug loading of mtx - plla - peg - plla microspheres prepared by the microencapsulation process was 13.7% 1.8% , which was higher than the theoretical drug loading ( 10% , w / w ) , whereas drug loading ( 8.3% 2.4% ) was lower than the theoretical one for the coprecipitation process . this phenomenon indicates that plla - peg - plla was partially dissolved in supercritical co2 and flushed out by running fluid in the microencapsulation process , and methotrexate nanoparticles were hardly lost . however , more methotrexate than plla - peg - plla was lost during the coprecipitation process , which could be explained by the fact that precipitation of plla - peg - plla was faster and easier than that of methotrexate . furthermore , the encapsulation efficiency of mtx - plla - peg- plla microspheres prepared by the microencapsulation process was 39.2% 5.9% , indicating that more methotrexate was encapsulated in the composite microspheres . a relatively lower encapsulation efficiency of 20.4% 6.3% was obtained in the coprecipitation process , indicating that more methotrexate was precipitated and absorbed or combined loosely onto the surface of the composite microspheres . apart from that , when different drug doses were used in the coprecipitation process , drug loading increased and encapsulation efficiency decreased with the increase in drug dose . when drug doses were set at 5% , 10% , and 15% , the corresponding drug loading was 6.1% 0.1% , 8.3% 0.4% , and 13.1% 1.4% , together with an encapsulation efficiency of 51.3% 3.0% , 20.4% 6.3% , and 16.6% 8.0% , respectively . when the drug dose was larger , more methotrexate would be precipitated at any given time point . furthermore , the precipitation speed of methotrexate was lower than that of plla - peg - plla in supercritical co2 , ie , the precipitated plla - peg - plla acted as a crystal nucleus and in turn induced coprecipitation of methotrexate and plla - peg - plla . these both contributed to the precipitation of more methotrexate on the surface of the preexisting mtx - plla - peg - plla composite microspheres , generating microspheres with higher drug loading and lower encapsulation efficiency . methotrexate is a poorly water - soluble drug with a short half - life in vivo . an effective way to lengthen its half - life and enhance its therapeutic efficacy is to entrap it with biodegradable polymers to supply a sustained - release effect . figure 6 illustrates the release profiles of mtx - plla - peg - plla microspheres manufactured by the microencapsulation process and by the coprecipitation process using different drug doses . when the drug dose was set at 10% ( w / w ) , in the first 30 minutes , 39.7% of the total methotrexate was released from the microspheres prepared by the microencapsulation process , while microspheres fabricated by the coprecipitation process released 42.3% of its methotrexate , both with an initial burst release . the methotrexate released rapidly at an early stage was unencapsulated or loosely adhered onto the microsphere surface and thus easily extracted , which again confirmed the result obtained by measurement of drug loading and encapsulation efficiency . after 30 hours , the cumulative percentage of the methotrexate released from the corresponding two kinds of microspheres went up to 86.0% and 89.8% , respectively . in the following hours , there was a typical plateau period . at that time , the amount of drug remaining was rather limited , with sustained and slow drug release . when the plateau period was extended to 144 hours , the cumulative drug release percentage of the two kinds of composite microspheres rose to 91.7% and 94.1% , respectively . with regard to the coprecipitation process , samples with a drug dose of 5% ( w / w ) released methotrexate more slowly and showed more desirable sustained release at the late stage , compared with the other two samples containing drug doses of 10% ( w / w ) and 15% ( w / w ) , between which there was no significant difference . it can also be seen in figure 6 that drug release within 12 hours is a first - order process ( correlation coefficient 0.99011 , standard deviation 1.4709 ) for the mtx - plla - peg - plla microspheres prepared by the microencapsulation process,31 and after 12 hours , methotrexate started to be released in a zero - order kinetics manner , with the correlation coefficient and standard deviation being 0.97960 and 0.3393 , respectively . this result demonstrates that drug release from mtx - plla - peg - plla microspheres prepared by the microencapsulation process was dominated by drug diffusion for the first 12 hours and mainly by polymer erosion thereafter , which can be explained by the fact that relatively large quantities of methotrexate nanoparticles had been encapsulated by plla - peg - plla . methotrexate is an antimetabolite and antifolate drug than can prevent formation of thymine within the cell by inhibiting dihydrofolate reductase , rendering it incapable of dna replication . methotrexate has been widely used as a chemotherapy agent for cancer and arthritis . as shown in figure 6 , mtx - plla - peg - plla prepared by the microencapsulation process can provide desirable sustained in vitro release of methotrexate , which can be used in the treatment of ectopic pregnancy32 and leukemia33 by intravenous injection , in breast cancer34 by pulmonary delivery , and in psoriasis35 and rheumatoid arthritis36 by percutaneous administration . first - order kinetics are obeyed for drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process ( the corresponding correlation coefficients and standard deviations for different drug doses are shown in table 2 ) . this indicates that drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process is dominated by drug diffusion , confirming that most methotrexate nanoparticles are absorbed or combined into the microsphere surface . overall , cumulative release of mtx - plla - peg - plla microspheres prepared by the microencapsulation process appeared to be more smooth and slower than that of microspheres prepared by the coprecipitation process . using the optimized operating parameters , together with addition of acetone as a nonsolvent to enhance the degree of saturation and reduce the methotrexate concentration in the initial solution , nanoparticles of methotrexate were successfully fabricated by seds . the surface morphology seen on sem is shown in figure 2 , in which methotrexate nanoparticles can be observed to have a smooth surface . when observed by the human eye , the methotrexate nanoparticles had a fluffy and soft appearance resembling cotton - candy . this macroscopic surface morphology can be explained by the fact that methotrexate when processed by seds exists not in the form of connected aggregates but as free nanoparticles . 30 furthermore , methotrexate nanoparticles prepared by seds were shown to have a spherical shape with a relatively narrow particle size distribution , ie , a span of 0.73 and an average size of 86 nm , as shown in table 1 . during the microencapsulation process , a suspension consisting of methotrexate nanoparticles and plla - peg - plla in dichloromethane solution was driven by an injector and sprayed into a high - pressure vessel through a coaxial nozzle , where the jet stream of supercritical co2 strengthened spraying of the suspension . when the droplets of suspension came into contact with supercritical co2 , a nonsolvent for plla - peg - plla , extensive mutual diffusion into and out of the suspension droplets occurred due to the high solubility of dichloromethane in supercritical co2 . with regard to the plla - peg - plla dichloromethane solution , the crystallization pathway shifted from the liquid - solution region into the solid - liquid region , via the liquid - solid and liquid - fluid two - phase region and the solid - liquid - fluid three - phase region , thus crossing a series of tie lines . when supersaturation of the plla - peg - plla dichloromethane solution was approached , extraction of dichloromethane from the suspension droplet led to an extremely rapid phase change , with formation of a solid phase and precipitation of plla - peg - plla onto the surface of the methotrexate nanoparticles . this encapsulation mechanism is briefly described in figure 1 . figure 3a and b illustrate the surface morphology of mtx - plla - peg - plla microspheres manufactured by the microencapsulation process and the coprecipitation process , respectively . the corresponding particle size and particle size distribution the particle size of the mtx - plla - peg - plla microspheres , prepared by either the coprecipitation process or the microencapsulation process , appeared much larger than that of the methotrexate nanoparticles , indicating that most of the methotrexate nanoparticles were successfully encapsulated by plla - peg - plla . the particle size of the mtx - plla - peg - plla composite microspheres prepared by the microencapsulation process was somewhat larger than that of the composite microspheres generated by the coprecipitation process . this is because , in the microencapsulation process , not one but two or more methotrexate nanoparticles were encapsulated into plla - peg - plla , generating relatively larger microspheres in which plla - peg - plla acted as a matrix to entrap methotrexate nanoparticles , ie , the composite microspheres obtained in the microencapsulation process have an inlaymatrix structure rather than a typical core - shell structure . judging from the span , mtx - plla - peg - plla composite microspheres prepared by the microencapsulation and coprecipitation processes both had a narrow particle size distribution and there was no significant difference between them . ftir is a widely used and very useful tool in chemistry for analyzing chemical components and identifying characteristic groups . as shown in the ftir spectra of figure 4 , there are three major regions of the ftir spectra for plla - peg - plla , ie , a c = o stretching vibration region at 18201700 cm , a region at 15001000 cm attributable to ch3 , ch bending , and c o c stretching vibration , and a region at 980820 cm for c c backbone stretching vibration . among these three regions , the major peak at 1759 cm is due to the stretching vibration of the carbonyl group in plla - peg - plla and can be used to identify pllapeg - plla ; this major peak can also be observed in the mtx - plla - peg - plla microspheres prepared by the two different processes . in the mtx - plla - peg - plla microsphere spectrum , a weak peak of plla - peg - plla at 1620 cm was considerably enhanced due to the existence of characteristic peaks of methotrexate at 1718 cm and 1604 cm , representing c = o stretching vibration and c = c benzene backbone stretching vibration , respectively . apart from that , no significant difference and no new peaks were found in the ftir spectrum of the composite microspheres prepared by either the microencapsulation process or the coprecipitation process , ie , no chemical reaction occurred in the two kinds of process . this all demonstrates that speds modified on the basis of seds is a typically physical process causing microencapsulation of drug nanoparticles via a biodegradable polymer , and that this process is favorable for drugs , because chemical interaction may alter the properties of drugs and reduce their effectiveness . figure 5 illustrates the drug loading and encapsulation efficiency of mtx - plla - peg - plla microspheres prepared by the microencapsulation process and by the coprecipitation process . when the drug dose was set at 10% ( w / w ) , drug loading of mtx - plla - peg - plla microspheres prepared by the microencapsulation process was 13.7% 1.8% , which was higher than the theoretical drug loading ( 10% , w / w ) , whereas drug loading ( 8.3% 2.4% ) was lower than the theoretical one for the coprecipitation process . this phenomenon indicates that plla - peg - plla was partially dissolved in supercritical co2 and flushed out by running fluid in the microencapsulation process , and methotrexate nanoparticles were hardly lost . however , more methotrexate than plla - peg - plla was lost during the coprecipitation process , which could be explained by the fact that precipitation of plla - peg - plla was faster and easier than that of methotrexate . furthermore , the encapsulation efficiency of mtx - plla - peg- plla microspheres prepared by the microencapsulation process was 39.2% 5.9% , indicating that more methotrexate was encapsulated in the composite microspheres . a relatively lower encapsulation efficiency of 20.4% 6.3% was obtained in the coprecipitation process , indicating that more methotrexate was precipitated and absorbed or combined loosely onto the surface of the composite microspheres . apart from that , when different drug doses were used in the coprecipitation process , drug loading increased and encapsulation efficiency decreased with the increase in drug dose . when drug doses were set at 5% , 10% , and 15% , the corresponding drug loading was 6.1% 0.1% , 8.3% 0.4% , and 13.1% 1.4% , together with an encapsulation efficiency of 51.3% 3.0% , 20.4% 6.3% , and 16.6% 8.0% , respectively . when the drug dose was larger , more methotrexate would be precipitated at any given time point . furthermore , the precipitation speed of methotrexate was lower than that of plla - peg - plla in supercritical co2 , ie , the precipitated plla - peg - plla acted as a crystal nucleus and in turn induced coprecipitation of methotrexate and plla - peg - plla . these both contributed to the precipitation of more methotrexate on the surface of the preexisting mtx - plla - peg - plla composite microspheres , generating microspheres with higher drug loading and lower encapsulation efficiency . methotrexate is a poorly water - soluble drug with a short half - life in vivo . an effective way to lengthen its half - life and enhance its therapeutic efficacy is to entrap it with biodegradable polymers to supply a sustained - release effect . figure 6 illustrates the release profiles of mtx - plla - peg - plla microspheres manufactured by the microencapsulation process and by the coprecipitation process using different drug doses . when the drug dose was set at 10% ( w / w ) , in the first 30 minutes , 39.7% of the total methotrexate was released from the microspheres prepared by the microencapsulation process , while microspheres fabricated by the coprecipitation process released 42.3% of its methotrexate , both with an initial burst release . the methotrexate released rapidly at an early stage was unencapsulated or loosely adhered onto the microsphere surface and thus easily extracted , which again confirmed the result obtained by measurement of drug loading and encapsulation efficiency . after 30 hours , the cumulative percentage of the methotrexate released from the corresponding two kinds of microspheres went up to 86.0% and 89.8% , respectively . in the following hours , there was a typical plateau period . at that time , the amount of drug remaining was rather limited , with sustained and slow drug release . when the plateau period was extended to 144 hours , the cumulative drug release percentage of the two kinds of composite microspheres rose to 91.7% and 94.1% , respectively . with regard to the coprecipitation process , samples with a drug dose of 5% ( w / w ) released methotrexate more slowly and showed more desirable sustained release at the late stage , compared with the other two samples containing drug doses of 10% ( w / w ) and 15% ( w / w ) , between which there was no significant difference . it can also be seen in figure 6 that drug release within 12 hours is a first - order process ( correlation coefficient 0.99011 , standard deviation 1.4709 ) for the mtx - plla - peg - plla microspheres prepared by the microencapsulation process,31 and after 12 hours , methotrexate started to be released in a zero - order kinetics manner , with the correlation coefficient and standard deviation being 0.97960 and 0.3393 , respectively . this result demonstrates that drug release from mtx - plla - peg - plla microspheres prepared by the microencapsulation process was dominated by drug diffusion for the first 12 hours and mainly by polymer erosion thereafter , which can be explained by the fact that relatively large quantities of methotrexate nanoparticles had been encapsulated by plla - peg - plla . methotrexate is an antimetabolite and antifolate drug than can prevent formation of thymine within the cell by inhibiting dihydrofolate reductase , rendering it incapable of dna replication . methotrexate has been widely used as a chemotherapy agent for cancer and arthritis . as shown in figure 6 , mtx - plla - peg - plla prepared by the microencapsulation process can provide desirable sustained in vitro release of methotrexate , which can be used in the treatment of ectopic pregnancy32 and leukemia33 by intravenous injection , in breast cancer34 by pulmonary delivery , and in psoriasis35 and rheumatoid arthritis36 by percutaneous administration . first - order kinetics are obeyed for drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process ( the corresponding correlation coefficients and standard deviations for different drug doses are shown in table 2 ) . this indicates that drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process is dominated by drug diffusion , confirming that most methotrexate nanoparticles are absorbed or combined into the microsphere surface . overall , cumulative release of mtx - plla - peg - plla microspheres prepared by the microencapsulation process appeared to be more smooth and slower than that of microspheres prepared by the coprecipitation process . with the addition of an injector in the suspension delivery system , speds was effective in preventing damage to the hplc pump and obstruction of the one - way valve , and was shown to be a successfully modified version of the original seds procedure . by suspending methotrexate nanoparticles in an plla - peg - plla solution containing dichloromethane , composite microspheres were manufactured by a microencapsulation process , with higher drug loading and encapsulation efficiency and more desirable sustained release than those produced by the coprecipitation process . this research demonstrates that microencapsulation of drugs with a biodegradable polymer via speds affords great potential for designing a controlled - release drug system .

backgroundthe aim of this study was to improve the drug loading , encapsulation efficiency , and sustained - release properties of supercritical co2-based drug - loaded polymer carriers via a process of suspension - enhanced dispersion by supercritical co2 ( speds ) , which is an advanced version of solution - enhanced dispersion by supercritical co2 ( seds).methodsmethotrexate nanoparticles were successfully microencapsulated into poly ( l - lactide)-poly(ethylene glycol)-poly(l - lactide ) ( plla - peg - plla ) by speds . methotrexate nanoparticles were first prepared by seds , then suspended in plla - peg - plla solution , and finally microencapsulated into plla - peg - plla via speds , where an injector was utilized in the suspension delivery system.resultsafter microencapsulation , the composite methotrexate ( mtx)-plla - peg - plla microspheres obtained had a mean particle size of 545 nm , drug loading of 13.7% , and an encapsulation efficiency of 39.2% . after an initial burst release , with around 65% of the total methotrexate being released in the first 3 hours , the mtx - plla - peg - plla microspheres released methotrexate in a sustained manner , with 85% of the total methotrexate dose released within 23 hours and nearly 100% within 144 hours.conclusioncompared with a parallel study of the coprecipitation process , microencapsulation using speds offered greater potential to manufacture drug - loaded polymer microspheres for a drug delivery system .

Introduction Materials and methods Materials Preparation of methotrexate nanoparticles by SEDS Microencapsulation of methotrexate into PLLA-PEG-PLLA by SpEDS Surface morphology and particle size distribution FTIR analysis Drug loading and encapsulation efficiency Drug release studies Results and discussion Preparation of methotrexate nanoparticles Surface morphology and particle size of MTX-PLLA-PEG-PLLA FTIR analysis Drug load and efficiency of encapsulation Drug release profiles Conclusion

incorporating pharmaceutical compounds into a biodegradable polymer carrier is an effective strategy for forming a drug delivery system and has been given increasing attention.15 yin and yates reported that drug - entrapped biodegradable microcapsules , with sustained drug release , were prepared by spraying a suspension of hollow poly(dl - lactide ) particles in procaine hydrochloride aqueous solution into plasticizing solvents.6 a further report by zvonar et al described use of a self - microemulsifying system to fabricate ca - pectinate microcapsules to improve the release and permeability of furosemide.7 drug - loaded polymer microspheres have been variously prepared by phase separation,8 interfacial polymerization,9 spray - drying,10 freeze - drying,11 and emulsion evaporation.12 however , these conventional strategies always either involve an intense operating process or preparation of particles with a wide particle size distribution and a relatively large amount of organic solvent residue . compared with these traditional methodologies , supercritical co2-based technology offers a number of advantages , including mild critical conditions ( tc = 304.1 k , pc = 7.38 mpa ) , no toxicity , a nonorganic residue , are not flammable , and all at a relatively lower price.1315 the supercritical antisolvent process has been widely utilized to fabricate drug - loaded polymer microparticles.1618 in the supercritical antisolvent process , an organic solution is sprayed into supercritical co2 to effect immediate contact between the two media . however , the coprecipitation process generates drug - loaded polymer microspheres accompanied by relatively low drug loading , low encapsulation efficiency , and no sustained release of the drug , which can be partially explained by the fact that the drug is precipitated onto or just loosely attached to the surface of the polymer microspheres.19,21 to overcome the drawbacks of the coprecipitation process , ie , low drug loading and low encapsulation efficiency , the microencapsulation process was developed19 whereby nanoparticles of a drug are first prepared , suspended in an organic polymer solution , and then sprayed into supercritical co2 using a high - performance liquid chromatography ( hplc ) pump . due to precipitation of the polymer from the sprayed suspension , drug nanoparticles are entrapped by the polymer with a matrix or core - shell structure.19,22 in order to strengthen mutual diffusion between the sprayed solution and the supercritical co2 , a solutionenhanced dispersion by supercritical fluid ( seds ) process was developed and has been widely used in supercritical fluid techniques.23 during this process , the substrate(s ) solution and supercritical co2 are copulverized through a coaxial nozzle , in which supercritical co2 acts as an antisolvent and spraying enhancer.2427 however , when seds is used in the microencapsulation process , ie , driving , delivering , and spraying the suspension into the high - pressure reaction vessel , the delivery system , especially the one - way valve , is easily damaged or obstructed by the suspended particles . this study attempted to prepare drug - loaded polymer microspheres using a supercritical microencapsulation process with improved drug loading , encapsulation efficiency , and sustained release , compared with the coprecipitation process . the drug was micronized by seds , suspended in a plla - peg - plla solution containing dichloromethane , and microencapsulated by plla - peg - plla via speds . the suspension placed in the front chamber of the injector was obtained by suspending methotrexate nanoparticles in the plla - peg - plla solution containing dichloromethane by ultrasonic dispersion , with a final methotrexate concentration of 0.5% ( w / v ) and a methotrexate to plla - peg - plla ratio of 1:9 ( w / w ) . briefly , methotrexate was first dissolved in dimethyl sulfoxide , and plla - peg - plla solution containing dichloromethane was then added homogeneously , with a final plla concentration of 0.5% ( w / v ) , methotrexate to plla - peg - plla ratios of 1:19 , 1:9 , and 3:17 ( w / w ) , and a dimethyl sulfoxide to dichloromethane ratio of 1:4 ( v / v ) . the particle size and particle size distribution ftir spectra of the original methotrexate and plla - peg - plla , and the methotrexate ( mtx)-plla - peg - plla microspheres prepared by the microencapsulation and coprecipitation processes were performed using an ftir spectrometer ( ftir 8400s , hitachi ) in transmission mode , with a wavenumber range of 4000400 cm . to determine drug loading , 10 mg of mtx - plla - peg - plla microspheres was dissolved in 5 ml of dichloromethane ; 30 ml of phosphate - buffered solution buffer ( ph 7.4 ) was added , and the solution was gently heated with magnetic stirring to evaporate the dichloromethane . to simulate and monitor the sustained release of methotrexate in the microspheres , first , 10 mg of mtx - plla - peg - plla microspheres was placed in a pretreated dialysis bag . the methotrexate nanoparticles were micronized by seds , whereby the pressure and temperature of co2 were stabilized at the desired levels , the solution of methotrexate in dimethyl sulfoxide and acetone began being delivered by a hplc pump to the coaxial nozzle and sprayed simultaneously with the supercritical co2 , and methotrexate nanoparticles were generated due to extremely rapid precipitation of methotrexate . the suspension placed in the front chamber of the injector was obtained by suspending methotrexate nanoparticles in the plla - peg - plla solution containing dichloromethane by ultrasonic dispersion , with a final methotrexate concentration of 0.5% ( w / v ) and a methotrexate to plla - peg - plla ratio of 1:9 ( w / w ) . briefly , methotrexate was first dissolved in dimethyl sulfoxide , and plla - peg - plla solution containing dichloromethane was then added homogeneously , with a final plla concentration of 0.5% ( w / v ) , methotrexate to plla - peg - plla ratios of 1:19 , 1:9 , and 3:17 ( w / w ) , and a dimethyl sulfoxide to dichloromethane ratio of 1:4 ( v / v ) . the particle size and particle size distribution ftir spectra of the original methotrexate and plla - peg - plla , and the methotrexate ( mtx)-plla - peg - plla microspheres prepared by the microencapsulation and coprecipitation processes were performed using an ftir spectrometer ( ftir 8400s , hitachi ) in transmission mode , with a wavenumber range of 4000400 cm . to determine drug loading , 10 mg of mtx - plla - peg - plla microspheres was dissolved in 5 ml of dichloromethane ; 30 ml of phosphate - buffered solution buffer ( ph 7.4 ) was added , and the solution was gently heated with magnetic stirring to evaporate the dichloromethane . for calculating the encapsulation efficiency , another 10 mg of mtx - plla - peg - plla microspheres was dispersed in 10 ml of a dilute aqueous solution of sodium hydroxide ( 0.1 mol / l ) to wash off the unencapsulated and loosely surface - bound methotrexate ; the resulting suspension was filtered through a 0.22 m membrane and the amount of methotrexate was determined using the ultraviolet spectrophotometer at 302.8 nm and calculating according to the standard concentration curve . 30 furthermore , methotrexate nanoparticles prepared by seds were shown to have a spherical shape with a relatively narrow particle size distribution , ie , a span of 0.73 and an average size of 86 nm , as shown in table 1 . during the microencapsulation process , a suspension consisting of methotrexate nanoparticles and plla - peg - plla in dichloromethane solution was driven by an injector and sprayed into a high - pressure vessel through a coaxial nozzle , where the jet stream of supercritical co2 strengthened spraying of the suspension . figure 3a and b illustrate the surface morphology of mtx - plla - peg - plla microspheres manufactured by the microencapsulation process and the coprecipitation process , respectively . the particle size of the mtx - plla - peg - plla microspheres , prepared by either the coprecipitation process or the microencapsulation process , appeared much larger than that of the methotrexate nanoparticles , indicating that most of the methotrexate nanoparticles were successfully encapsulated by plla - peg - plla . the particle size of the mtx - plla - peg - plla composite microspheres prepared by the microencapsulation process was somewhat larger than that of the composite microspheres generated by the coprecipitation process . this is because , in the microencapsulation process , not one but two or more methotrexate nanoparticles were encapsulated into plla - peg - plla , generating relatively larger microspheres in which plla - peg - plla acted as a matrix to entrap methotrexate nanoparticles , ie , the composite microspheres obtained in the microencapsulation process have an inlaymatrix structure rather than a typical core - shell structure . among these three regions , the major peak at 1759 cm is due to the stretching vibration of the carbonyl group in plla - peg - plla and can be used to identify pllapeg - plla ; this major peak can also be observed in the mtx - plla - peg - plla microspheres prepared by the two different processes . figure 5 illustrates the drug loading and encapsulation efficiency of mtx - plla - peg - plla microspheres prepared by the microencapsulation process and by the coprecipitation process . when the drug dose was set at 10% ( w / w ) , drug loading of mtx - plla - peg - plla microspheres prepared by the microencapsulation process was 13.7% 1.8% , which was higher than the theoretical drug loading ( 10% , w / w ) , whereas drug loading ( 8.3% 2.4% ) was lower than the theoretical one for the coprecipitation process . this phenomenon indicates that plla - peg - plla was partially dissolved in supercritical co2 and flushed out by running fluid in the microencapsulation process , and methotrexate nanoparticles were hardly lost . furthermore , the encapsulation efficiency of mtx - plla - peg- plla microspheres prepared by the microencapsulation process was 39.2% 5.9% , indicating that more methotrexate was encapsulated in the composite microspheres . when the drug dose was set at 10% ( w / w ) , in the first 30 minutes , 39.7% of the total methotrexate was released from the microspheres prepared by the microencapsulation process , while microspheres fabricated by the coprecipitation process released 42.3% of its methotrexate , both with an initial burst release . it can also be seen in figure 6 that drug release within 12 hours is a first - order process ( correlation coefficient 0.99011 , standard deviation 1.4709 ) for the mtx - plla - peg - plla microspheres prepared by the microencapsulation process,31 and after 12 hours , methotrexate started to be released in a zero - order kinetics manner , with the correlation coefficient and standard deviation being 0.97960 and 0.3393 , respectively . this result demonstrates that drug release from mtx - plla - peg - plla microspheres prepared by the microencapsulation process was dominated by drug diffusion for the first 12 hours and mainly by polymer erosion thereafter , which can be explained by the fact that relatively large quantities of methotrexate nanoparticles had been encapsulated by plla - peg - plla . as shown in figure 6 , mtx - plla - peg - plla prepared by the microencapsulation process can provide desirable sustained in vitro release of methotrexate , which can be used in the treatment of ectopic pregnancy32 and leukemia33 by intravenous injection , in breast cancer34 by pulmonary delivery , and in psoriasis35 and rheumatoid arthritis36 by percutaneous administration . first - order kinetics are obeyed for drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process ( the corresponding correlation coefficients and standard deviations for different drug doses are shown in table 2 ) . this indicates that drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process is dominated by drug diffusion , confirming that most methotrexate nanoparticles are absorbed or combined into the microsphere surface . overall , cumulative release of mtx - plla - peg - plla microspheres prepared by the microencapsulation process appeared to be more smooth and slower than that of microspheres prepared by the coprecipitation process . during the microencapsulation process , a suspension consisting of methotrexate nanoparticles and plla - peg - plla in dichloromethane solution was driven by an injector and sprayed into a high - pressure vessel through a coaxial nozzle , where the jet stream of supercritical co2 strengthened spraying of the suspension . figure 3a and b illustrate the surface morphology of mtx - plla - peg - plla microspheres manufactured by the microencapsulation process and the coprecipitation process , respectively . the corresponding particle size and particle size distribution the particle size of the mtx - plla - peg - plla microspheres , prepared by either the coprecipitation process or the microencapsulation process , appeared much larger than that of the methotrexate nanoparticles , indicating that most of the methotrexate nanoparticles were successfully encapsulated by plla - peg - plla . the particle size of the mtx - plla - peg - plla composite microspheres prepared by the microencapsulation process was somewhat larger than that of the composite microspheres generated by the coprecipitation process . this is because , in the microencapsulation process , not one but two or more methotrexate nanoparticles were encapsulated into plla - peg - plla , generating relatively larger microspheres in which plla - peg - plla acted as a matrix to entrap methotrexate nanoparticles , ie , the composite microspheres obtained in the microencapsulation process have an inlaymatrix structure rather than a typical core - shell structure . among these three regions , the major peak at 1759 cm is due to the stretching vibration of the carbonyl group in plla - peg - plla and can be used to identify pllapeg - plla ; this major peak can also be observed in the mtx - plla - peg - plla microspheres prepared by the two different processes . figure 5 illustrates the drug loading and encapsulation efficiency of mtx - plla - peg - plla microspheres prepared by the microencapsulation process and by the coprecipitation process . when the drug dose was set at 10% ( w / w ) , drug loading of mtx - plla - peg - plla microspheres prepared by the microencapsulation process was 13.7% 1.8% , which was higher than the theoretical drug loading ( 10% , w / w ) , whereas drug loading ( 8.3% 2.4% ) was lower than the theoretical one for the coprecipitation process . this phenomenon indicates that plla - peg - plla was partially dissolved in supercritical co2 and flushed out by running fluid in the microencapsulation process , and methotrexate nanoparticles were hardly lost . furthermore , the encapsulation efficiency of mtx - plla - peg- plla microspheres prepared by the microencapsulation process was 39.2% 5.9% , indicating that more methotrexate was encapsulated in the composite microspheres . when the drug dose was set at 10% ( w / w ) , in the first 30 minutes , 39.7% of the total methotrexate was released from the microspheres prepared by the microencapsulation process , while microspheres fabricated by the coprecipitation process released 42.3% of its methotrexate , both with an initial burst release . it can also be seen in figure 6 that drug release within 12 hours is a first - order process ( correlation coefficient 0.99011 , standard deviation 1.4709 ) for the mtx - plla - peg - plla microspheres prepared by the microencapsulation process,31 and after 12 hours , methotrexate started to be released in a zero - order kinetics manner , with the correlation coefficient and standard deviation being 0.97960 and 0.3393 , respectively . this result demonstrates that drug release from mtx - plla - peg - plla microspheres prepared by the microencapsulation process was dominated by drug diffusion for the first 12 hours and mainly by polymer erosion thereafter , which can be explained by the fact that relatively large quantities of methotrexate nanoparticles had been encapsulated by plla - peg - plla . as shown in figure 6 , mtx - plla - peg - plla prepared by the microencapsulation process can provide desirable sustained in vitro release of methotrexate , which can be used in the treatment of ectopic pregnancy32 and leukemia33 by intravenous injection , in breast cancer34 by pulmonary delivery , and in psoriasis35 and rheumatoid arthritis36 by percutaneous administration . first - order kinetics are obeyed for drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process ( the corresponding correlation coefficients and standard deviations for different drug doses are shown in table 2 ) . this indicates that drug release from mtx - plla - peg - plla microspheres prepared by the coprecipitation process is dominated by drug diffusion , confirming that most methotrexate nanoparticles are absorbed or combined into the microsphere surface . overall , cumulative release of mtx - plla - peg - plla microspheres prepared by the microencapsulation process appeared to be more smooth and slower than that of microspheres prepared by the coprecipitation process . by suspending methotrexate nanoparticles in an plla - peg - plla solution containing dichloromethane , composite microspheres were manufactured by a microencapsulation process , with higher drug loading and encapsulation efficiency and more desirable sustained release than those produced by the coprecipitation process .

a distinctive feature of cancer cells is their ability to dissociate from the primary tumor and establish metastases at various sites . the common routes by which cancer cells spread include local invasion , and lymphatic , hematogenous , and peritoneal dissemination . the hematogenous and lymphatic pathways are well - established routes of metastatic spread . however , although routes along nerves have been described in the literature since the mid-1800s , they have received relatively little research attention . recently , it has become increasingly evident that cancer - stromal interactions play a critical role in cancer growth and progression . the involvement of peripheral nerves has been overlooked for a long time ; these nerves have been considered inert bystanders in solid malignancies , but they are now gaining recognition as potentially important components of the cancer microenvironment . cancer cells invade both the epineurium and the perineurium , and may reach the endoneurium , becoming intimately associated with schwann cells and nerve axons . perineural invasion was first reported in the english literature by doctors who described head and neck cancers with a predilection for growth along nerves , as they made their way toward the intracranial fossa . d'amico et al showed that perineural invasion is an independent prognostic factor for prostate cancer recurrence . however , freedland et al reported that perineural invasion did not correlate with extracapsular extension or biochemical recurrence . we compared the preoperative prediction of perineural invasion in a prostate biopsy ( pnib ) with that of perineural invasion in a prostatectomy specimen ( pnip ) and investigated the clinical and prognostic significance of pnip in prostate cancer patients . we reviewed the medical records of 361 consecutive patients with prostate adenocarcinomas who had received no hormonal therapy or radiation therapy before or after a retropubic radical prostatectomy or laparoscopic radical prostatectomy , performed between 1999 and 2010 . data pertaining to the demographics , staging , pathology , and outcomes of each patient were reviewed and entered into a comprehensive database . all procedures involving study participants were approved by the institutional review board ( irb approved protocol number : e2010 - 014 ) . the period of observation of this unselected cohort was the interval between the date of the surgical resection and the last contact ( death or last follow - up ) . the mean duration of follow - up was 42.433.6 months ( range , 6.5 - 141.6 months ) . the mean patient age at the time of radical prostatectomy was 69.0 years ( range , 49 - 94 years ) . prostate biopsies were recommended for men with a serum prostate - specific antigen ( psa ) level of 3.0 ng / ml or higher . we evaluated the primary gleason grade , secondary gleason grade , gleason score , occurrence of a high gleason grade ( > 3 ) , percentage of positive biopsy cores , percentage of tumor cells in the positive cores , bilaterality , and perineural invasion in a prostate biopsy . the percentage of positive biopsy cores was defined as : ( number of positive biopsy cores / total number of biopsy cores ) 100 . we analyzed the association between the preoperative parameters cited above and pnip to evaluate the preoperative prediction of pnip . either a medical or a surgical oncologist who was a member of our institution 's multidisciplinary tumor board at the time of the patient 's treatment established the tumor stage postoperatively , according to the tumor , lymph node , metastasis ( tnm ) classification of the international union against cancer ( uicc 2002 ) and the gleason system . we reviewed the primary gleason grade , secondary gleason grade , gleason score , presence of a high gleason grade ( > 3 ) , presence of gleason grade 5 , t - stage , tumor volume , lymphovascular invasion , surgical margin status , perineural invasion , pelvic lymph - node metastasis at radical prostatectomy , and nadir psa . tumor volume was defined as the volume of cancer cells relative to the total resected prostate volume . perineural invasion was defined as tumor cells within any layer of the nerve sheath or tumor cells in the perineural space that involved at least one third of the nerve circumference . lymphovascular invasion was defined as the presence of cancer cells within an endothelium - lined space without a muscular wall . we also followed up the serum psa every 3 months during the first year after surgery , semiannually in years 2 to 5 , and annually thereafter . the cutoff value for biochemical serum psa recurrence was defined as 0.2 ng / ml . survival records were obtained from the korean national statistics registry database , and the cancer - specific survival data were analyzed . all radical prostatectomy specimens were evaluated in a standard manner . after eliminating the apical and bladder neck margins , the specimens were sectioned transversely at 5 mm intervals from the apex to the base . the seminal vesicles were evaluated at the intersection at which they entered the prostate gland . the apical and bladder neck margins and the anterior , radial , posterior , and posterolateral surgical margins were defined as positive if the tumor was in direct contact with the indicated inked surface of the prostate in the sections . the original h&e stained slides from the tumor resections were collected from the pathology department . all slides containing tumor cells were reviewed for pnip by a single pathologist with expertise in pnip , who was blinded to all patient data , including the stage of the disease and its outcome . the associations between the preoperative parameters and pnip were calculated with a binary logistic regression model and the mantel - haenszel test . the association of pnip with various clinicopathological characteristics was assessed by using the ( two - tailed ) pearson correlation test . the influence of pnip on biochemical recurrence and overall and cancer - specific survival were estimated by using the kaplan - meier method with a log - rank test . the effects of pnip and other clinicopathological parameters on biochemical recurrence and cancer - specific survival were analyzed by using the cox regression model . we reviewed the medical records of 361 consecutive patients with prostate adenocarcinomas who had received no hormonal therapy or radiation therapy before or after a retropubic radical prostatectomy or laparoscopic radical prostatectomy , performed between 1999 and 2010 . data pertaining to the demographics , staging , pathology , and outcomes of each patient were reviewed and entered into a comprehensive database . all procedures involving study participants were approved by the institutional review board ( irb approved protocol number : e2010 - 014 ) . the period of observation of this unselected cohort was the interval between the date of the surgical resection and the last contact ( death or last follow - up ) . the mean duration of follow - up was 42.433.6 months ( range , 6.5 - 141.6 months ) . the mean patient age at the time of radical prostatectomy was 69.0 years ( range , 49 - 94 years ) . prostate biopsies were recommended for men with a serum prostate - specific antigen ( psa ) level of 3.0 ng / ml or higher . we evaluated the primary gleason grade , secondary gleason grade , gleason score , occurrence of a high gleason grade ( > 3 ) , percentage of positive biopsy cores , percentage of tumor cells in the positive cores , bilaterality , and perineural invasion in a prostate biopsy . the percentage of positive biopsy cores was defined as : ( number of positive biopsy cores / total number of biopsy cores ) 100 . we analyzed the association between the preoperative parameters cited above and pnip to evaluate the preoperative prediction of pnip . either a medical or a surgical oncologist who was a member of our institution 's multidisciplinary tumor board at the time of the patient 's treatment established the tumor stage postoperatively , according to the tumor , lymph node , metastasis ( tnm ) classification of the international union against cancer ( uicc 2002 ) and the gleason system . we reviewed the primary gleason grade , secondary gleason grade , gleason score , presence of a high gleason grade ( > 3 ) , presence of gleason grade 5 , t - stage , tumor volume , lymphovascular invasion , surgical margin status , perineural invasion , pelvic lymph - node metastasis at radical prostatectomy , and nadir psa . tumor volume was defined as the volume of cancer cells relative to the total resected prostate volume . perineural invasion was defined as tumor cells within any layer of the nerve sheath or tumor cells in the perineural space that involved at least one third of the nerve circumference . lymphovascular invasion was defined as the presence of cancer cells within an endothelium - lined space without a muscular wall . we also followed up the serum psa every 3 months during the first year after surgery , semiannually in years 2 to 5 , and annually thereafter . the cutoff value for biochemical serum psa recurrence was defined as 0.2 ng / ml . survival records were obtained from the korean national statistics registry database , and the cancer - specific survival data were analyzed . all radical prostatectomy specimens were evaluated in a standard manner . after eliminating the apical and bladder neck margins , the specimens were sectioned transversely at 5 mm intervals from the apex to the base . the seminal vesicles were evaluated at the intersection at which they entered the prostate gland . the apical and bladder neck margins and the anterior , radial , posterior , and posterolateral surgical margins were defined as positive if the tumor was in direct contact with the indicated inked surface of the prostate in the sections . the original h&e stained slides from the tumor resections were collected from the pathology department . all slides containing tumor cells were reviewed for pnip by a single pathologist with expertise in pnip , who was blinded to all patient data , including the stage of the disease and its outcome . the associations between the preoperative parameters and pnip were calculated with a binary logistic regression model and the mantel - haenszel test . the association of pnip with various clinicopathological characteristics was assessed by using the ( two - tailed ) pearson correlation test . the influence of pnip on biochemical recurrence and overall and cancer - specific survival were estimated by using the kaplan - meier method with a log - rank test . the effects of pnip and other clinicopathological parameters on biochemical recurrence and cancer - specific survival were analyzed by using the cox regression model . fifty - two percent of patients ( 188 of 361 patients ) had a positive pnip ( fig . only 7.4% of these patients ( 14 of 188 patients ) were identified as having positive pnib , demonstrating the underreporting of this pathology in prostate biopsies . in evaluating the relations between the preoperative variables and pnip , pnip was related to preoperative psa ( p=0.002 ) , the primary gleason grade ( p<0.001 ) , secondary gleason grade ( p=0.003 ) , gleason score ( p<0.001 ) , a gleason grade > 3 ( p<0.001 ) , the number of positive cores ( p<0.001 ) , the percentage of positive cores ( p<0.001 ) , and the percentage of tumor cells in positive - core prostate biopsies ( p<0.001 ) , but not to age ( p=0.069 ) , bilaterality ( p=0.175 ) , or perineural invasion at prostate biopsy ( p=0.277 ) . in a multivariate analysis , pnip was related to the primary gleason grade ( p=0.020 , hazard ratio=2.040 , 95% confidence interval [ ci ] 1.119 - 3.719 ) , the number of positive cores ( p=0.008 , hazard ratio=1.303 , 95% ci : 1.072 - 1.582 ) , and the percentage of tumor cells in positive cores ( p=0.021 , hazard ratio=1.015 , 95% ci : 1.002 - 1.029 ) at prostate biopsy . in a univariate analysis , pnip was correlated with the pathological stage of the prostatectomy specimen ( p<0.001 ) . perineural invasion was observed in 50% of t2a tumors , 11.1% of t2b tumors , 48.3% of t2c tumors , 79.4% of t3a tumors , 91.9% of t3b tumors , and 100% of t4 tumors . the gleason score and the primary and secondary gleason grades were also significantly related to pnip ( p<0.001 , p=0.019 , and p<0.001 , respectively ) . pnip was observed in 39.3% of patients with a gleason grade of 3 and in 71.5% of patients with a gleason grade of > 3 . the tumor volume of the prostate cancer was higher in the pnip - positive specimens than in the pnip - negative specimens ( 27.7% vs 14.5% , respectively ; p<0.001 ) . pnip was observed almost twice as frequently in surgical - margin - positive specimens at the time of radical prostatectomy than in the surgical - margin - negative specimens ( 53.5% vs 35.7% , respectively ; odds ratio 2.069 , p=0.003 ) . pnip showed a slight correlation with lymph - node metastasis ( 5.3% vs 0.8% , respectively ; odds ratio : 7.022 , p=0.055 ) . in a multivariate analysis , pnip was significantly related to the gleason score ( p=0.010 , hazard ratio=1.959 , 95% ci : 1.178 - 3.256 ) , t - stage ( p=0.015 , hazard ratio=1.387 , 95% ci : 1.065 - 1.807 ) , and lymphovascular invasion ( p=0.019 , hazard ratio=3.863 , 95% ci : 1.247 - 11.967 ) . the relations between pnip and other clinical and pathological parameters were investigated with the pearson correlation . age ( p=0.022 ) , primary gleason grade ( p<0.001 ) , gleason score ( p<0.001 ) , tumor stage ( p<0.001 ) , tumor volume of the prostate ( p<0.001 ) , lymphovascular invasion ( p<0.001 ) , pnip ( p=0.018 ) , surgical margin status ( p=0.018 ) , number of positive lymph nodes ( p=0.004 ) , nadir psa ( p<0.001 ) , the first psa after surgery ( p=0.041 ) , psa at 1 year after surgery ( p<0.001 ) , and psa velocity ( p=0.049 ) were significantly related to biochemical serum psa recurrence . using the log - rank test , we found no significant difference in biochemical serum psa recurrence between patients with pnip and those without pnip ( p=0.597 ; fig . 2 ) . in a multivariate analysis , nadir psa ( p<0.001 , hazard ratio=61.746 , 95% ci : 15.363 - 248.174 ) and psa at 1 year after surgery ( p<0.001 , hazard ratio=2.878 , 95% ci : 1.893 - 4.375 ) were significantly related to biochemical serum psa recurrence . however , pnip was not correlated with biochemical recurrence ( p=0.364 ) . in a kaplan - meier survival analysis , the gleason score , preoperative psa , and nadir psa were significantly related to cancer - specific survival ( p<0.001 , p<0.001 , and p<0.001 , respectively ) . however , pnip did not correlate with cancer - specific survival ( p=0.726 ; fig . a cox proportional multivariate analysis was used to assess the influence of all the significant parameters on cancer - specific survival and 5-year psa - recurrence - free survival . the gleason score was significantly associated with a poor prognosis ( p=0.047 , hazard ratio=1.412 , 95% ci : 1.005 - 1.986 ) . the presence of pnip did not correlate with biochemical recurrence or cancer - specific survival . fifty - two percent of patients ( 188 of 361 patients ) had a positive pnip ( fig . only 7.4% of these patients ( 14 of 188 patients ) were identified as having positive pnib , demonstrating the underreporting of this pathology in prostate biopsies . in evaluating the relations between the preoperative variables and pnip , pnip was related to preoperative psa ( p=0.002 ) , the primary gleason grade ( p<0.001 ) , secondary gleason grade ( p=0.003 ) , gleason score ( p<0.001 ) , a gleason grade > 3 ( p<0.001 ) , the number of positive cores ( p<0.001 ) , the percentage of positive cores ( p<0.001 ) , and the percentage of tumor cells in positive - core prostate biopsies ( p<0.001 ) , but not to age ( p=0.069 ) , bilaterality ( p=0.175 ) , or perineural invasion at prostate biopsy ( p=0.277 ) . in a multivariate analysis , pnip was related to the primary gleason grade ( p=0.020 , hazard ratio=2.040 , 95% confidence interval [ ci ] 1.119 - 3.719 ) , the number of positive cores ( p=0.008 , hazard ratio=1.303 , 95% ci : 1.072 - 1.582 ) , and the percentage of tumor cells in positive cores ( p=0.021 , hazard ratio=1.015 , 95% ci : 1.002 - 1.029 ) at prostate biopsy . in a univariate analysis , pnip was correlated with the pathological stage of the prostatectomy specimen ( p<0.001 ) . perineural invasion was observed in 50% of t2a tumors , 11.1% of t2b tumors , 48.3% of t2c tumors , 79.4% of t3a tumors , 91.9% of t3b tumors , and 100% of t4 tumors . the gleason score and the primary and secondary gleason grades were also significantly related to pnip ( p<0.001 , p=0.019 , and p<0.001 , respectively ) . pnip was observed in 39.3% of patients with a gleason grade of 3 and in 71.5% of patients with a gleason grade of > 3 . the tumor volume of the prostate cancer was higher in the pnip - positive specimens than in the pnip - negative specimens ( 27.7% vs 14.5% , respectively ; p<0.001 ) . pnip was observed almost twice as frequently in surgical - margin - positive specimens at the time of radical prostatectomy than in the surgical - margin - negative specimens ( 53.5% vs 35.7% , respectively ; odds ratio 2.069 , p=0.003 ) . pnip showed a slight correlation with lymph - node metastasis ( 5.3% vs 0.8% , respectively ; odds ratio : 7.022 , p=0.055 ) . in a multivariate analysis , pnip was significantly related to the gleason score ( p=0.010 , hazard ratio=1.959 , 95% ci : 1.178 - 3.256 ) , t - stage ( p=0.015 , hazard ratio=1.387 , 95% ci : 1.065 - 1.807 ) , and lymphovascular invasion ( p=0.019 , hazard ratio=3.863 , 95% ci : 1.247 - 11.967 ) . the relations between pnip and other clinical and pathological parameters were investigated with the pearson correlation . age ( p=0.022 ) , primary gleason grade ( p<0.001 ) , gleason score ( p<0.001 ) , tumor stage ( p<0.001 ) , tumor volume of the prostate ( p<0.001 ) , lymphovascular invasion ( p<0.001 ) , pnip ( p=0.018 ) , surgical margin status ( p=0.018 ) , number of positive lymph nodes ( p=0.004 ) , nadir psa ( p<0.001 ) , the first psa after surgery ( p=0.041 ) , psa at 1 year after surgery ( p<0.001 ) , and psa velocity ( p=0.049 ) were significantly related to biochemical serum psa recurrence . using the log - rank test , we found no significant difference in biochemical serum psa recurrence between patients with pnip and those without pnip ( p=0.597 ; fig . 2 ) . in a multivariate analysis , nadir psa ( p<0.001 , hazard ratio=61.746 , 95% ci : 15.363 - 248.174 ) and psa at 1 year after surgery ( p<0.001 , hazard ratio=2.878 , 95% ci : 1.893 - 4.375 ) were significantly related to biochemical serum psa recurrence . in a kaplan - meier survival analysis , the gleason score , preoperative psa , and nadir psa were significantly related to cancer - specific survival ( p<0.001 , p<0.001 , and p<0.001 , respectively ) . however , pnip did not correlate with cancer - specific survival ( p=0.726 ; fig . a cox proportional multivariate analysis was used to assess the influence of all the significant parameters on cancer - specific survival and 5-year psa - recurrence - free survival . the gleason score was significantly associated with a poor prognosis ( p=0.047 , hazard ratio=1.412 , 95% ci : 1.005 - 1.986 ) . the presence of pnip did not correlate with biochemical recurrence or cancer - specific survival . perineural invasion has become an increasingly relevant , yet understudied , aspect of tumor biology in several cancers , including prostate cancer . because the incidence of prostate cancer is increasing rapidly in korea , we sought to determine the impact of perineural invasion on prostate cancer in our patient population . previous studies have reported incidences of pnib of 7 - 43% and of pnip of 31.9 - 79.0% [ 3,8 - 12 ] . in the present study , pnib and pnip were observed in 7.4% and 52.8% of patients , respectively , which means that pnib has only limited prognostic value and more attention should be directed to pathological examinations . however , pnip was related to the primary gleason grade , secondary gleason grade , gleason score , gleason grade of > 3 , the number of positive cores , the percentage of positive cores , and the percentage of tumor cells in the positive cores on prostate biopsies . this result indicates that biopsy specimens do not represent the whole prostate pathology , even when multiple prostate biopsies are performed . some reports have shown that pnib does not predict biochemical psa recurrence after radical prostatectomy . however , other reports have demonstrated that pnib is significantly related to postoperative biochemical recurrence . loeb et al showed that pnib is an independent risk factor for aggressive pathological features and a nonindependent risk factor for biochemical progression after radical prostatectomy . a limitation of our study was that we could not assess the exact predictive value of pnib for biochemical recurrence because the number of patients with positive pnib was small . lee et al showed a statistically significant association between the presence of pnip and adverse preoperative risk parameters , including a higher clinical t - stage , higher gleason score at biopsy , and higher preoperative serum psa . another report also demonstrated that pnip was significantly related to preoperative serum psa and psa density . in our study , we found that pnip was significantly related to the primary gleason grade , the percentage of positive cores , and the percentage of positive cores on prostate biopsy . recently , jeon and colleagues also reported that pnip was related to a higher gleason score , extracapsular extension , seminal vesicle invasion , and a positive surgical margin . stone et al reported that pnip predicted pelvic lymph node metastasis in men with prostate cancer . in the present study , we also found that pnip correlated with several clinicopathological parameters , including the pathological t - stage , gleason score , and lymphovascular invasion . stone et al showed that pnip is an independent predictor of lymph node metastasis in prostate cancer patients . however , miyake et al reported that pnip is not an independent predictor of biochemical recurrence and might not provide any extra useful information when the presence of perineural invasion is considered in predicting the prognosis of men undergoing radical prostatectomy if there are other conventional parameters available . our data indicate that pnip did not correlate with biochemical serum psa recurrence , although it was related to other known prognostic factors . there are a few reports of the significance of perineural invasion for the survival of patients with prostate cancer . however , most of these reports have limitations , because the studies included patients who had undergone radiation therapy , so they evaluated the presence of pnib , but not pnip . therefore , we could not compare our results with previous reports , including biochemical serum psa recurrence or the survival rate , in patients who had undergone radical prostatectomy . moreover , most studies have defined biochemical recurrence as the only study endpoint , whereas we evaluated the prognostic value of pnip for cancer - specific survival . in our study , pnip was not significantly related to cancer - specific survival . in this study , the relationship between pnip and biochemical recurrence did not show statistical significance according to t stage and gleason score . one of the limitations of our study was that the number of patients with positive pnib was very small , so we were unable to estimate the value of pnib as a prognostic factor . we recorded the presence or absence of pnib in all cases , but did not quantify the extent or laterality of pnib in some patients . because the gleason grading system has been changed , all the specimen slides were reviewed by a single pathologist . however , we had only the pathology reports for some patients who had undergone prostate biopsies at another hospital . before 2005 , we performed sextant prostate biopsies , whereas after 2005 , extended multisite biopsies were performed . the rates of pnib in the sextant biopsies and extended biopsies were 6.25% ( 4/64 ) and 3.37% ( 10/297 ) , respectively . therefore , we considered that the number of biopsy cores did not affect the detection of perineural invasion at prostate biopsy . pnip was significantly related to biologically aggressive tumor patterns but was not a prognostic factor for biochemical serum psa recurrence or cancer - specific survival in patients with prostate cancer . the prognostic value of pnip lacks statistical significance for biochemical serum psa recurrence and cancer - specific survival .

purposethe prognostic significance of perineural invasion by prostate cancer is debated . we investigated the association between perineural invasion and clinicopathological factors and the effect of perineural invasion on survival in patients with prostate cancer.materials and methodsa total of 361 patients with prostate cancer without any neoadjuvant therapies prior to surgery from 1999 to 2010 were analyzed retrospectively . whole - mount sections of surgical specimens from all patients who underwent radical prostatectomy were evaluated . positive perineural invasion was defined as infiltration of cancer cells in the perineurium or neural fascicles . the relationship of perineural invasion with clinicopathological features and prognosis of prostate cancer was studied . we also researched preoperative factors that were associated with perineural invasion.resultsperineural invasion in a prostatectomy specimen ( pnip ) was positive in 188 of 361 patients ( 52.1% ) . in the multivariate analysis of the preoperative variables , pnip was related to the primary gleason grade ( p=0.020 ) , the number of positive cores ( p=0.008 ) , and the percentage of tumor cells in positive cores ( p=0.021 ) , but not to perineural invasion of a prostate biopsy . in the evaluation between pnip and pathologic findings of the prostatectomy specimen , pnip was related to the gleason score ( p=0.010 ) , t - stage ( p=0.015 ) , and lymphovascular invasion ( p=0.019 ) . however , by multivariate analysis , the pnip was not an independent prognostic factor of biochemical serum recurrence ( p=0.364 ) or cancer - specific survival ( p=0.726).conclusionspnip was significantly related to biologically aggressive tumor patterns but was not a prognostic factor for biochemical serum psa recurrence or cancer - specific survival in patients with prostate cancer .

INTRODUCTION MATERIALS AND METHODS 1. Patients and preoperative parameters 2. Assessment of clinicopathologic factors 3. Histopathological analysis 4. Statistical analysis RESULTS 1. Incidence of perineural invasion in prostate cancer 2. Preoperative prediction of PNIp ( 3. Relation between PNIp and known prognostic factors in prostatectomy specimens ( 4. Role of PNIp in biochemical recurrence of prostate cancer ( 5. PNIp is not an independent prognostic factor for outcome in prostate cancer ( DISCUSSION CONCLUSIONS

cancer cells invade both the epineurium and the perineurium , and may reach the endoneurium , becoming intimately associated with schwann cells and nerve axons . d'amico et al showed that perineural invasion is an independent prognostic factor for prostate cancer recurrence . we compared the preoperative prediction of perineural invasion in a prostate biopsy ( pnib ) with that of perineural invasion in a prostatectomy specimen ( pnip ) and investigated the clinical and prognostic significance of pnip in prostate cancer patients . we reviewed the medical records of 361 consecutive patients with prostate adenocarcinomas who had received no hormonal therapy or radiation therapy before or after a retropubic radical prostatectomy or laparoscopic radical prostatectomy , performed between 1999 and 2010 . we evaluated the primary gleason grade , secondary gleason grade , gleason score , occurrence of a high gleason grade ( > 3 ) , percentage of positive biopsy cores , percentage of tumor cells in the positive cores , bilaterality , and perineural invasion in a prostate biopsy . the percentage of positive biopsy cores was defined as : ( number of positive biopsy cores / total number of biopsy cores ) 100 . either a medical or a surgical oncologist who was a member of our institution 's multidisciplinary tumor board at the time of the patient 's treatment established the tumor stage postoperatively , according to the tumor , lymph node , metastasis ( tnm ) classification of the international union against cancer ( uicc 2002 ) and the gleason system . we reviewed the primary gleason grade , secondary gleason grade , gleason score , presence of a high gleason grade ( > 3 ) , presence of gleason grade 5 , t - stage , tumor volume , lymphovascular invasion , surgical margin status , perineural invasion , pelvic lymph - node metastasis at radical prostatectomy , and nadir psa . tumor volume was defined as the volume of cancer cells relative to the total resected prostate volume . perineural invasion was defined as tumor cells within any layer of the nerve sheath or tumor cells in the perineural space that involved at least one third of the nerve circumference . lymphovascular invasion was defined as the presence of cancer cells within an endothelium - lined space without a muscular wall . we also followed up the serum psa every 3 months during the first year after surgery , semiannually in years 2 to 5 , and annually thereafter . the cutoff value for biochemical serum psa recurrence was defined as 0.2 ng / ml . survival records were obtained from the korean national statistics registry database , and the cancer - specific survival data were analyzed . the apical and bladder neck margins and the anterior , radial , posterior , and posterolateral surgical margins were defined as positive if the tumor was in direct contact with the indicated inked surface of the prostate in the sections . the influence of pnip on biochemical recurrence and overall and cancer - specific survival were estimated by using the kaplan - meier method with a log - rank test . the effects of pnip and other clinicopathological parameters on biochemical recurrence and cancer - specific survival were analyzed by using the cox regression model . we reviewed the medical records of 361 consecutive patients with prostate adenocarcinomas who had received no hormonal therapy or radiation therapy before or after a retropubic radical prostatectomy or laparoscopic radical prostatectomy , performed between 1999 and 2010 . we evaluated the primary gleason grade , secondary gleason grade , gleason score , occurrence of a high gleason grade ( > 3 ) , percentage of positive biopsy cores , percentage of tumor cells in the positive cores , bilaterality , and perineural invasion in a prostate biopsy . the percentage of positive biopsy cores was defined as : ( number of positive biopsy cores / total number of biopsy cores ) 100 . either a medical or a surgical oncologist who was a member of our institution 's multidisciplinary tumor board at the time of the patient 's treatment established the tumor stage postoperatively , according to the tumor , lymph node , metastasis ( tnm ) classification of the international union against cancer ( uicc 2002 ) and the gleason system . we reviewed the primary gleason grade , secondary gleason grade , gleason score , presence of a high gleason grade ( > 3 ) , presence of gleason grade 5 , t - stage , tumor volume , lymphovascular invasion , surgical margin status , perineural invasion , pelvic lymph - node metastasis at radical prostatectomy , and nadir psa . tumor volume was defined as the volume of cancer cells relative to the total resected prostate volume . perineural invasion was defined as tumor cells within any layer of the nerve sheath or tumor cells in the perineural space that involved at least one third of the nerve circumference . lymphovascular invasion was defined as the presence of cancer cells within an endothelium - lined space without a muscular wall . we also followed up the serum psa every 3 months during the first year after surgery , semiannually in years 2 to 5 , and annually thereafter . the cutoff value for biochemical serum psa recurrence was defined as 0.2 ng / ml . survival records were obtained from the korean national statistics registry database , and the cancer - specific survival data were analyzed . the apical and bladder neck margins and the anterior , radial , posterior , and posterolateral surgical margins were defined as positive if the tumor was in direct contact with the indicated inked surface of the prostate in the sections . the influence of pnip on biochemical recurrence and overall and cancer - specific survival were estimated by using the kaplan - meier method with a log - rank test . the effects of pnip and other clinicopathological parameters on biochemical recurrence and cancer - specific survival were analyzed by using the cox regression model . fifty - two percent of patients ( 188 of 361 patients ) had a positive pnip ( fig . in evaluating the relations between the preoperative variables and pnip , pnip was related to preoperative psa ( p=0.002 ) , the primary gleason grade ( p<0.001 ) , secondary gleason grade ( p=0.003 ) , gleason score ( p<0.001 ) , a gleason grade > 3 ( p<0.001 ) , the number of positive cores ( p<0.001 ) , the percentage of positive cores ( p<0.001 ) , and the percentage of tumor cells in positive - core prostate biopsies ( p<0.001 ) , but not to age ( p=0.069 ) , bilaterality ( p=0.175 ) , or perineural invasion at prostate biopsy ( p=0.277 ) . in a multivariate analysis , pnip was related to the primary gleason grade ( p=0.020 , hazard ratio=2.040 , 95% confidence interval [ ci ] 1.119 - 3.719 ) , the number of positive cores ( p=0.008 , hazard ratio=1.303 , 95% ci : 1.072 - 1.582 ) , and the percentage of tumor cells in positive cores ( p=0.021 , hazard ratio=1.015 , 95% ci : 1.002 - 1.029 ) at prostate biopsy . in a univariate analysis , pnip was correlated with the pathological stage of the prostatectomy specimen ( p<0.001 ) . the gleason score and the primary and secondary gleason grades were also significantly related to pnip ( p<0.001 , p=0.019 , and p<0.001 , respectively ) . the tumor volume of the prostate cancer was higher in the pnip - positive specimens than in the pnip - negative specimens ( 27.7% vs 14.5% , respectively ; p<0.001 ) . in a multivariate analysis , pnip was significantly related to the gleason score ( p=0.010 , hazard ratio=1.959 , 95% ci : 1.178 - 3.256 ) , t - stage ( p=0.015 , hazard ratio=1.387 , 95% ci : 1.065 - 1.807 ) , and lymphovascular invasion ( p=0.019 , hazard ratio=3.863 , 95% ci : 1.247 - 11.967 ) . age ( p=0.022 ) , primary gleason grade ( p<0.001 ) , gleason score ( p<0.001 ) , tumor stage ( p<0.001 ) , tumor volume of the prostate ( p<0.001 ) , lymphovascular invasion ( p<0.001 ) , pnip ( p=0.018 ) , surgical margin status ( p=0.018 ) , number of positive lymph nodes ( p=0.004 ) , nadir psa ( p<0.001 ) , the first psa after surgery ( p=0.041 ) , psa at 1 year after surgery ( p<0.001 ) , and psa velocity ( p=0.049 ) were significantly related to biochemical serum psa recurrence . using the log - rank test , we found no significant difference in biochemical serum psa recurrence between patients with pnip and those without pnip ( p=0.597 ; fig . in a multivariate analysis , nadir psa ( p<0.001 , hazard ratio=61.746 , 95% ci : 15.363 - 248.174 ) and psa at 1 year after surgery ( p<0.001 , hazard ratio=2.878 , 95% ci : 1.893 - 4.375 ) were significantly related to biochemical serum psa recurrence . however , pnip was not correlated with biochemical recurrence ( p=0.364 ) . in a kaplan - meier survival analysis , the gleason score , preoperative psa , and nadir psa were significantly related to cancer - specific survival ( p<0.001 , p<0.001 , and p<0.001 , respectively ) . however , pnip did not correlate with cancer - specific survival ( p=0.726 ; fig . a cox proportional multivariate analysis was used to assess the influence of all the significant parameters on cancer - specific survival and 5-year psa - recurrence - free survival . the gleason score was significantly associated with a poor prognosis ( p=0.047 , hazard ratio=1.412 , 95% ci : 1.005 - 1.986 ) . the presence of pnip did not correlate with biochemical recurrence or cancer - specific survival . fifty - two percent of patients ( 188 of 361 patients ) had a positive pnip ( fig . in evaluating the relations between the preoperative variables and pnip , pnip was related to preoperative psa ( p=0.002 ) , the primary gleason grade ( p<0.001 ) , secondary gleason grade ( p=0.003 ) , gleason score ( p<0.001 ) , a gleason grade > 3 ( p<0.001 ) , the number of positive cores ( p<0.001 ) , the percentage of positive cores ( p<0.001 ) , and the percentage of tumor cells in positive - core prostate biopsies ( p<0.001 ) , but not to age ( p=0.069 ) , bilaterality ( p=0.175 ) , or perineural invasion at prostate biopsy ( p=0.277 ) . in a multivariate analysis , pnip was related to the primary gleason grade ( p=0.020 , hazard ratio=2.040 , 95% confidence interval [ ci ] 1.119 - 3.719 ) , the number of positive cores ( p=0.008 , hazard ratio=1.303 , 95% ci : 1.072 - 1.582 ) , and the percentage of tumor cells in positive cores ( p=0.021 , hazard ratio=1.015 , 95% ci : 1.002 - 1.029 ) at prostate biopsy . in a univariate analysis , pnip was correlated with the pathological stage of the prostatectomy specimen ( p<0.001 ) . the gleason score and the primary and secondary gleason grades were also significantly related to pnip ( p<0.001 , p=0.019 , and p<0.001 , respectively ) . the tumor volume of the prostate cancer was higher in the pnip - positive specimens than in the pnip - negative specimens ( 27.7% vs 14.5% , respectively ; p<0.001 ) . pnip was observed almost twice as frequently in surgical - margin - positive specimens at the time of radical prostatectomy than in the surgical - margin - negative specimens ( 53.5% vs 35.7% , respectively ; odds ratio 2.069 , p=0.003 ) . in a multivariate analysis , pnip was significantly related to the gleason score ( p=0.010 , hazard ratio=1.959 , 95% ci : 1.178 - 3.256 ) , t - stage ( p=0.015 , hazard ratio=1.387 , 95% ci : 1.065 - 1.807 ) , and lymphovascular invasion ( p=0.019 , hazard ratio=3.863 , 95% ci : 1.247 - 11.967 ) . age ( p=0.022 ) , primary gleason grade ( p<0.001 ) , gleason score ( p<0.001 ) , tumor stage ( p<0.001 ) , tumor volume of the prostate ( p<0.001 ) , lymphovascular invasion ( p<0.001 ) , pnip ( p=0.018 ) , surgical margin status ( p=0.018 ) , number of positive lymph nodes ( p=0.004 ) , nadir psa ( p<0.001 ) , the first psa after surgery ( p=0.041 ) , psa at 1 year after surgery ( p<0.001 ) , and psa velocity ( p=0.049 ) were significantly related to biochemical serum psa recurrence . using the log - rank test , we found no significant difference in biochemical serum psa recurrence between patients with pnip and those without pnip ( p=0.597 ; fig . in a multivariate analysis , nadir psa ( p<0.001 , hazard ratio=61.746 , 95% ci : 15.363 - 248.174 ) and psa at 1 year after surgery ( p<0.001 , hazard ratio=2.878 , 95% ci : 1.893 - 4.375 ) were significantly related to biochemical serum psa recurrence . in a kaplan - meier survival analysis , the gleason score , preoperative psa , and nadir psa were significantly related to cancer - specific survival ( p<0.001 , p<0.001 , and p<0.001 , respectively ) . however , pnip did not correlate with cancer - specific survival ( p=0.726 ; fig . a cox proportional multivariate analysis was used to assess the influence of all the significant parameters on cancer - specific survival and 5-year psa - recurrence - free survival . the gleason score was significantly associated with a poor prognosis ( p=0.047 , hazard ratio=1.412 , 95% ci : 1.005 - 1.986 ) . the presence of pnip did not correlate with biochemical recurrence or cancer - specific survival . because the incidence of prostate cancer is increasing rapidly in korea , we sought to determine the impact of perineural invasion on prostate cancer in our patient population . however , pnip was related to the primary gleason grade , secondary gleason grade , gleason score , gleason grade of > 3 , the number of positive cores , the percentage of positive cores , and the percentage of tumor cells in the positive cores on prostate biopsies . however , other reports have demonstrated that pnib is significantly related to postoperative biochemical recurrence . loeb et al showed that pnib is an independent risk factor for aggressive pathological features and a nonindependent risk factor for biochemical progression after radical prostatectomy . a limitation of our study was that we could not assess the exact predictive value of pnib for biochemical recurrence because the number of patients with positive pnib was small . lee et al showed a statistically significant association between the presence of pnip and adverse preoperative risk parameters , including a higher clinical t - stage , higher gleason score at biopsy , and higher preoperative serum psa . another report also demonstrated that pnip was significantly related to preoperative serum psa and psa density . in our study , we found that pnip was significantly related to the primary gleason grade , the percentage of positive cores , and the percentage of positive cores on prostate biopsy . recently , jeon and colleagues also reported that pnip was related to a higher gleason score , extracapsular extension , seminal vesicle invasion , and a positive surgical margin . in the present study , we also found that pnip correlated with several clinicopathological parameters , including the pathological t - stage , gleason score , and lymphovascular invasion . however , miyake et al reported that pnip is not an independent predictor of biochemical recurrence and might not provide any extra useful information when the presence of perineural invasion is considered in predicting the prognosis of men undergoing radical prostatectomy if there are other conventional parameters available . our data indicate that pnip did not correlate with biochemical serum psa recurrence , although it was related to other known prognostic factors . there are a few reports of the significance of perineural invasion for the survival of patients with prostate cancer . however , most of these reports have limitations , because the studies included patients who had undergone radiation therapy , so they evaluated the presence of pnib , but not pnip . therefore , we could not compare our results with previous reports , including biochemical serum psa recurrence or the survival rate , in patients who had undergone radical prostatectomy . moreover , most studies have defined biochemical recurrence as the only study endpoint , whereas we evaluated the prognostic value of pnip for cancer - specific survival . in our study , pnip was not significantly related to cancer - specific survival . in this study , the relationship between pnip and biochemical recurrence did not show statistical significance according to t stage and gleason score . one of the limitations of our study was that the number of patients with positive pnib was very small , so we were unable to estimate the value of pnib as a prognostic factor . therefore , we considered that the number of biopsy cores did not affect the detection of perineural invasion at prostate biopsy . pnip was significantly related to biologically aggressive tumor patterns but was not a prognostic factor for biochemical serum psa recurrence or cancer - specific survival in patients with prostate cancer . the prognostic value of pnip lacks statistical significance for biochemical serum psa recurrence and cancer - specific survival .

neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections . invading microorganisms there , neutrophils destroy the microorganism by a series of mechanisms , mainly phagocytosis , release of antimicrobial substances , and the formation of neutrophil extracellular traps ( nets ) . activated neutrophils also release proteinases into the surrounding tissue , causing damage to the host . in addition , neutrophils are capable of producing many cytokines and chemokines , which can influence the inflammatory response , as well as the immune response [ 4 , 5 ] . besides this classical role in antimicrobial functions , neutrophils early studies suggested that these tumor - associated neutrophils ( tans ) were mere bystanders because it was hard to imagine that neutrophils , being short - lived cells , could have an effect on chronic and progressive diseases such as cancer . however , more recently it is becoming clear that tans have relevant roles in malignant disease . this renewed interest comes in part from the recognition that cancer - related inflammation is an important feature for the development of many tumors and it is a hallmark of cancer . the various antimicrobial and cytotoxic compounds contained in granules can destroy malignant cells , and cytokines and chemokines secreted by neutrophils can also recruit other cells with antitumor activity [ 5 , 9 ] . however , an increasing number of clinical observations and laboratory studies have shown that presence of neutrophils in tumors correlates with poor prognosis . this has been well documented for bronchoalveolar carcinoma , melanoma , renal carcinoma , and head and neck squamous cell carcinoma ( hnscc ) . in all these cases , tans are different from circulating neutrophils ( as discussed later ) , and , in untreated tumors of murine models , they can display a protumorigenic phenotype . the mechanisms for this phenotype are just beginning to be elucidated , but some of them involve genotoxicity , angiogenesis , and immunosuppression . these two types of tans described in mice have been named n1 and n2 in a similar manner as antitumor and protumor macrophages ( tams ) . it is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor , for neutrophils support to the tumor , and for neutrophil activation to enhance their antitumor functions and in the future improve cancer immunotherapy . our knowledge on the role of neutrophils in human cancers is relatively small . from an initial interest in the 1980s , the number of publications on neutrophils in cancer - related studies has been steadily going down . however , this trend is now beginning to change with the realization that neutrophils are indeed important players in cancer development , as reflected by several recent reviews [ 1618 ] , and as we will see next . in many patients with advanced cancer , elevated counts of neutrophils in blood are found . how tumors induce neutrophilia is uncertain , but production of granulocyte - macrophage colony - stimulating factor ( gm - csf ) is a possible mechanism in several types of cancer . in addition , other cytokines such as granulocyte colony - stimulating factor ( g - csf ) , interleukin- ( il- ) 1 , and il-6 produced by tumors seem to contribute to elevated neutrophil numbers in blood . this neutrophilia is associated with poor prognosis in several types of cancers , such as lung , melanoma , and renal carcinomas [ 11 , 21 , 22 ] . in agreement with this , the presence of neutrophils within certain tumors seems also to be an indicator of poor prognosis . reduced recurrence - free time and overall survival were reported for neutrophil - infiltrated tumors in renal carcinomas , hnscc , pancreatic adenocarcinomas , and liver carcinoma . because neutrophilia is frequently associated with inflammatory responses to infections and tissue damage , neutrophilia represents evidence for the concept of cancer - related inflammation inducing tumor progression . the relation of neutrophil numbers in blood to other leukocyte counts has been suggested to serve as a prognostic factor for cancer . thus , the neutrophil - to - lymphocyte ratio ( nlr ) was introduced as prognostic factor for colorectal cancer . due to its simplicity , nlr has shown to be a readily available and inexpensive biomarker for many types of tumors including non - small - cell lung cancer , hepatocellular carcinoma , nasopharyngeal carcinoma , colorectal cancer , melanoma , and breast cancer [ 29 , 30 ] . in general , the blood nlr is elevated in patients with more advanced or aggressive disease , as indicated by increased tumor size , nodal stage , and number of metastatic lesions . also , a high nlr correlates with adverse overall survival in many solid tumors [ 32 , 33 ] . despite the clinical evidence from the many studies mentioned above , neutrophilia ( larger numbers of neutrophils in blood as a consequence of elevated egress of cells from the bone marrow ) is not always a bad indicator for cancer progression . in some types of tumors , for example in fact , the capacity of neutrophils to directly kill tumor cells both in vitro and in vivo was reported long time ago [ 3537 ] . also , neutrophils from tumor - bearing animals were reported to have enhanced cytotoxic activity [ 38 , 39 ] . and recently , neutrophils isolated from blood of some healthy individuals presented direct cytotoxicity against several tumor cell lines . therefore , the exact role of neutrophils within the tumor is a controversial matter [ 14 , 41 ] . in addition to the elevated number of neutrophils in blood , an increase in the frequency of immature myeloid cells at earlier stages of differentiation has also been detected in several types of tumors , including terminal patients with lung , breast , and gastrointestinal cancer . these immature cells consist of a heterogeneous population of immunosuppressive cells defined as myeloid - derived suppressor cells ( mdscs ) . these mdscs can be divided phenotypically into granulocytic ( g - mdsc ) and monocytic ( mo - mdsc ) subgroups [ 45 , 46 ] and are found in great numbers in the spleens of tumor - bearing animals , where they display an immunosuppressive phenotype helping tumor progression [ 47 , 48 ] . the g - mdscs have immature neutrophil morphology and the consensus phenotype cd33/cd11b / hla - dr / cd15 in humans . they have been found in peripheral blood of patients with glioblastoma , multiple myeloma , hodgkin lymphoma , or head and neck cancer . the main mechanism involves production of reactive oxygen species ( ros ) by the respiratory burst of these cells . in advanced cancer patients , the hydrogen peroxide ( h2o2 ) produced by activated granulocytes reduced expression of the t cell receptor ( tcr ) cd3 chain and decreased cytokine production by patients ' t cells . these oxidized human t cells had defective chemotaxis and presented impaired f - actin remodeling . the effect was found to be mediated by oxidation of the actin - remodeling protein cofilin . cofilin is activated through dephosphorylation at ser3 , and then it mediates severing and depolymerization of f - actin for formation of the immune synapse and t cell activation . cofilin oxidation induced formation of an intramolecular disulfide bridge that prevents its activation , thus leading to impaired t cell activation . also , long - term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic - like programmed cell death takes place in human t cells . in addition , exposure of ros to memory / effector cd45ro t cells results in inhibition of nf-b activation and reduction in th1 cytokines production . furthermore , mdsc - produced ros can lead to cd8 t cell tolerance by another mechanism involving peroxynitrite . ros can combine with nitric oxide and form peroxynitrite , which is highly reactive at short distances . during mdsc - t cell contact , this process makes cd8-expressing t cells unable to bind peptide - mhc complexes and to respond to the specific peptide . another mechanism for t cell suppression is production of arginase 1 ( arg1 ) by mdscs . arg1 inhibits t cell proliferation by degrading extracellular arginine , which results in decreased responsiveness of t cells to cd3/tcr stimulation . for example , in patients with non - small cell lung cancer , tans had reduced intracellular arg1 and tumor - infiltrating lymphocytes showed reduced proliferation in response to cd3/tcr stimulation . all non - small cell lung cancer cell lines secreted il-8 , and il-8 was effective in triggering arg1 release . also , in patients with glioblastoma , degranulated neutrophils associated with elevated levels of serum arg1 correlated with decreased t cell cd3 zeta chain expression in peripheral blood t cells , resulting in immunosuppression . together , these mechanisms explain how mdsc - produced ros and arg1 mediate t cell suppression in cancer settings . because , g - mdscs share many properties with neutrophils but seem to be functionally different from mature neutrophils [ 13 , 62 ] , a transcriptomic analysis was conducted to compare in tumor - bearing animals circulating neutrophils with tans and with mdscs . however , at present it is not clear whether tans are mature neutrophils or represent a special category of cells such as immature neutrophils with protumor properties . depending on the phenotype displayed by tans , they have been classified in tumor - bearing mice as n1 or n2 . similarly to antitumor tumor - infiltrating macrophages ( m1 ) , n1 cells display proinflammatory and antitumorigenic functions . tans seem to be different from circulating neutrophils and also from g - mdsc in the bone marrow and spleen [ 44 , 63 ] . upon transforming growth factor - beta ( tgf- ) blockade , murine cd11b / ly6 g neutrophils recruited to tumors were hypersegmented and more cytotoxic to tumor cells and expressed higher levels of proinflammatory cytokines . in contrast , depletion of these neutrophils decreased tumor growth and resulted in more activated cd8 t cells intratumorally . thus , it seems that tgf- within the tumor microenvironment induces a population of tans with a protumor phenotype . in support of this idea , in two models of murine tumor cancer cell lines ( lewis lung carcinoma and ab12 mesothelioma ) , neutrophils were found primarily at the periphery of the tumor at early stages of tumor development . these tans were more cytotoxic toward tumor cells and produced higher levels of tumor necrosis factor - alpha ( tnf- ) , no , and h2o2 . in contrast , tans in established tumors had these functions downregulated and presented a more protumorigenic phenotype . these results showed that neutrophils enter the tumor and become more protumor with tumor progression . therefore , murine tans can have an antitumorigenic ( n1 ) phenotype but also a protumorigenic ( n2 ) phenotype capable of supporting tumor growth and suppressing the antitumor immune responses [ 14 , 41 ] , depending on the tumor microenvironment . despite this classification , the nature and function of tans in the cancer microenvironment remain largely unknown , particularly with human tumors . however , two recent publications describe the phenotype of neutrophils infiltrated into human tumors . in one study of surgically resected lung cancer patients , tans were isolated from digested human lung tumors and constituted 5%25% of the cells in the tumor . these tans presented an activated phenotype ( cd62l / cd54 ) with expression of a distinct repertoire of chemokine receptors that included ccr5 , ccr7 , cxcr3 , and cxcr4 . in addition , tans produced larger quantities of the proinflammatory factors mcp-1 , il-8 , mip-1 , and il-6 than blood neutrophils did . these results indicate that , in the earliest stages of lung cancer , tans are not immunosuppressive but rather stimulate t cell responses . in the second study , the role of chronic inflammation , particularly via il-23 and il-17 , in developing human colorectal cancer authors found that innate t ( t17 ) cells were the major cellular source of il-17 in colorectal cancer . tumor growth led to epithelial barrier disruption allowing microbial products to induce inflammatory dendritic cell accumulation and t17 polarization in human tumors . these activated dendritic cells induced t17 cells to secrete il-8 , tnf- , and gm - csf , thus leading to accumulation of neutrophils in the tumor . these tans were characterized by cd45/lin / hladr / cd11b / cd33/cd66b and displayed typical polymorphonuclear morphology . thus , they were described as g - mdsc . these tans ( g - mdsc ) produced much more arg1 and ros than autologous neutrophils and inhibited proliferation of activated autologous t cells and ifn- production . the tans described in these reports show that in human tumors the dual role of neutrophils is also observed . in early tumors , tans seem to be able to stimulate t cell responses , but later in established tumors tans are immunosuppressive . these important reports are just the tip of the iceberg in our understanding of the origin and function of tans . many questions remain for example , are tans in early tumors mature neutrophils with antitumor properties and tans in established tumors immature cells ( g - mdscs ) with immunosuppressive properties directly recruited from the circulation ? or are tans mature neutrophils that develop a more protumor phenotype with tumor progression ? as suggested by several tumor animal models and cancer patients [ 17 , 64 ] . a very recent publication identifies several subpopulations of neutrophils in the blood of tumor - bearing mice and in human cancer patients and describes several relationships of these cells in connection to cancer progression . in this study , the other subpopulation has lower density neutrophils ( ldns ) that copurify with the low - density mononuclear cells layer formed when separating leukocytes by density gradient centrifugation . in tumor - free mice , most neutrophils were hdns , but in tumor - bearing animals ldns increased progressively and often became the dominant neutrophil type in circulation . the hdns from cancer animals , which were previously reported as tumor - entrained neutrophils ( tens ) , displayed high cytotoxicity toward tumor cells in culture , whereas ldns were not cytotoxic . also , the ldns had reduced expression of various chemokines ( cxcl1 , cxcl2 , cxcl10 , ccl2 , and ccl3 ) and chemokine receptors ( cxcr2 and ccr5 ) , consistent with a reduced inflammatory state . the ldn subpopulation consists of large mature ( fully formed lobulated nucleus ) neutrophils and also of immature neutrophils , similar to g - mdsc . the authors then showed by brdu labeling that ldns rapidly accumulate in the circulation , whereas hdns appear in the circulation much later . this is consistent with the idea that some of the ldns are indeed immature neutrophils . in addition , authors showed that hdns are capable of becoming ldns upon treatment with tgf- . it is interesting to note that tgf- was able to induce the change of hdns from tumor - bearing mice into ldns , but it had no effect on hdns from tumor - free mice . this indicates that other stimuli are also needed for this change in animals with cancer . for example , treatment of nave healthy mice with recombinant g - csf protein elicited g - mdsc similar to those induced in tumor - bearing animals . together , all these results support a model proposed by the authors , in which neutrophils are present in three subpopulations in cancer : normal high - density neutrophils , immature low - density neutrophils ( g - mdsc ) , and large mature low - density neutrophils . while the hdns are antitumor and the ldns are protumor , they can change under the influence of the different chemokines and cytokines in the tumor microenvironment . solid tumors are composed of several cell types , including tumor cells and stromal cells . the tumor stroma contains fibroblasts , endothelial cells of blood vessels , and in many cases immune cells . these tumor - infiltrating immune cells highlight the inflammatory microenvironment that is commonly associated with tumor progression . in addition to lymphocytes and macrophages , neutrophils are found in great numbers in a wide variety of tumors [ 12 , 13 , 23 , 24 ] . clearly , neutrophils are recruited to the tumor by the action of neutrophil - attracting chemokines that can be produced not only by other immune cells but also directly by several tumor cells ( figure 1 ) . it was found that oncogenic ras induced il-8 expression , and ras - expressing mouse adenomas produced kc / cxcl1 and mip-2/cxcl2 , the murine equivalents of il-8 , to attract tans . accordingly , increased il-8 levels were found in hnscc patients , and elimination of neutrophils in cancer murine models reduced tumor burden and metastasis . deleting il-8 receptors also reduced tumor growth . these findings support the notion that tumor - produced il-8 is important for neutrophil recruitment to help tumor progression . however , the il-8 receptors cxcr1 and cxcr2 are also expressed on other cell types including endothelial cells and tumor cells . thus , determining the extent of neutrophil involvement in il-8-mediated tumor progression will require future studies . using the same cxcr1 and cxcr2 receptors , neutrophils can also respond to other chemokines such as cxcl1 , cxcl2 , cxcl5 , cxcl6 , and cxcl7 ( figure 1 ) . cxcl2 can induce neutrophil infiltration in tumors , and it was suggested that this is an autocrine effect . supporting this idea , it was also found that in tans the expression of cxcl2 , and also cxcl1 , was upregulated more than 150-fold . therefore , it seems that neutrophils activate a positive feedback mechanism by releasing neutrophil chemokines that attract more neutrophils into the tumor , similarly to neutrophil recruitment into sites of infection . the role of ena-78/cxcl5 in appearance of tans in carcinoma of the liver was investigated in 919 patients with hepatocellular carcinoma . cxcl5 was found to be overexpressed in patients with recurrent tumors , and the levels of cxcl5 correlated with greater appearance of tans and with shorter overall survival . another chemokine that also participates in neutrophil recruitment to tumors is gcp-2/cxcl6 . in a melanoma mouse model , specific anti - cxcl6 monoclonal antibodies reduced the number of tans and also tumor size . in addition , migration inhibitory factor ( mif ) , another tumor - derived chemokine for neutrophils , was identified in hnscc tumors . mif was described as an inhibitor of macrophage migration in vitro , but it is now known that it also binds cxcr2 ( figure 1 ) . tumor - derived mif levels correlated with higher tans levels and poor survival of these patients . many tumor cells can directly produce chemokines for neutrophils , but various other cells within the tumor may also be the source for these chemokines and other cytokines . in particular , activated t cells are known to produce gm - csf , cxcl1 , cxcl2 , tnf- , and ifn- . although the influence of activated t cells in neutrophil recruitment to tumors is not known , regulatory t cells ( treg ) seem to be important for neutrophil infiltrating tumors . in one study , treg were found to inhibit neutrophil recruitment to a tumor site by reducing the expression of cxcl1 and cxcl2 . in contrast , in another study , treg promoted neutrophil infiltration to tumors by producing il-8 . thus , the influence of t cell function on the appearance of tans needs to be further explored . murine tans secrete ccl17 , a potent chemokine for treg , at higher levels than circulating or splenic neutrophils ( figure 1 ) . moreover , the amounts of ccl17 increased progressively during tumor progression . a large body of clinical evidence indicates that neutrophils are involved in cancer development and tumor progression . in most cases , large numbers of tans are associated with advanced disease and poor prognosis for cancer patients . this negative association has been reported for several solid tumors , such as melanoma , hepatocellular carcinoma , non - small cell lung carcinoma , glioma , hnscc , adenocarcinoma , and colon cancer [ 41 , 88 ] . these functions involve the same molecules neutrophils use to destroy microorganisms and to modulate inflammation . important molecules that can modify growth and invasiveness of tumors involve granule proteins , matrix - degrading proteinases , reactive oxygen species ( ros ) , chemokines , and cytokines . recent reports describe how tans use these molecules to affect cell proliferation , angiogenesis , metastasis , and immune surveillance ( figure 2 ) . neutrophil elastase ( ne ) is a major protein of azurophilic granules that is released upon cell degranulation . the main physiologic function of ne seems to be elimination of invading microorganisms , but it also has important inflammatory effects . ne is a serine protease with a broad range of substrates ; among them are neutrophil - derived antibacterial proteins , extracellular matrix proteins , integrins , cytokines , and cytokine receptors . in addition to its roles in inflammation and bacteria destruction , ne has presented various protumor effects both in vivo and in vitro . ne was found to directly promote a459 tumor cell proliferation when murine neutrophils were cocultured with this lung carcinoma cell line . this effect was markedly reduced when tumor cells were cocultured with ne neutrophils , or in the presence of an ne inhibitor . the effect of ne on tumor growth was dependent on phosphatidylinositol 3-kinase ( pi-3k ) , since it was also reduced in the presence of a pi-3k inhibitor . staining experiments showed that ne got inside the tumor cells via clathrin - coated pits and localized at early endosomes . once inside the cell , ne acted on insulin receptor substrate-1 ( irs-1 ) . because irs-1 binds to the regulatory unit of pi-3k , its degradation by ne led to more pi-3k available to enhance the proliferation pathway . similar results have been found with other types of tumor cells , including esophageal cancer , gastric cancer , and breast cancer . in these cases , ne mediated release of transforming growth factor - alpha ( tgf- ) from the cell surface . coculture of human neutrophils with pancreatic ductal adenocarcinoma cells ( pdac ) resulted in dyshesion of cells from the monolayer . the same effect was observed by adding ne to pdac cultures and correlated with loss of surface expression of e - cadherin . cathepsin g is a peptidase from azurophilic granules that participates in degradation of phagocytosed microorganisms and in remodeling of extracellular matrix ( ecm ) proteins . first cathepsin g binds to the tumor cell surface , independently of its catalytic site , and then induces cell aggregation , which is dependent on its enzymatic activity ( figure 2 ) . cathepsin g degrades ecm molecules such as fibronectin and attenuates binding between integrins and fibronectin . this leads to e - cadherin - mediated homotypic cell - cell adhesion , which is protease - resistant . the formation of these tumor cell aggregates would allow tumor cells to disseminate via the circulation to distant sites and establish new metastases . once at the new site , tumor cells would need new vasculature . in a model of breast cancer metastasis to the bone , it was also found that cathepsin g enhanced tgf- signaling and upregulated vascular endothelial growth factor ( vegf ) to promote angiogenesis . together , these reports indicate that tans - derived cathepsin g may induce ecm remodeling and promote tumor progression and metastasis [ 102 , 103 ] . matrix metalloproteinase-9 ( mmp-9/gelatinase b ) is released from secondary ( specific ) granules and is believed to help neutrophils in the process of extravasation via degradation of ecm proteins . mmp-9 was found to promote tumor proliferation in a human papilloma virus- ( hpv- ) 16 skin carcinogenesis model . mmp-9 mice showed reduced keratinocyte proliferation , but this phenotype was reversed when bone marrow - derived leukocytes were transplanted into irradiated mice . also , immunostaining of mmp-9 in squamous cell carcinoma tumors showed that mmp-9 was found only in tumor infiltrating leukocytes and not in tumor cells . in addition , mmp-9 has been shown to inhibit apoptosis of tumor cells in the lung . thus , mmp-9 supplied by bone marrow - derived cells is responsible for enhancing tumor proliferation via both increased proliferation and reduced apoptosis of tumor cells . the vascular endothelial growth factor ( vegf ) is sequestered in the ecm after it is produced by cells ( figure 2 ) . the proteolytic release of vegf from tissue ecm via mmps is regarded as a prerequisite for in vivo induced angiogenesis [ 106 , 107 ] . melanoma cells were transfected to overexpress the gcp-2/cxcl6 chemokine and then implanted into nude mice . the new cxcl6-melanoma tumors grew larger and with a well - developed vasculature than wild type ( wt ) melanomas . these larger tumors also presented higher levels of mmp-9 and induced a strong influx of tans . similarly , in a model of pancreatic adenocarcinoma , new dysplastic lesions that develop into carcinomas are formed with enhanced angiogenesis . this process has been named the angiogenic switch . in these new lesions , mmp-2 and mmp-9 were upregulated . mmp inhibitors and genetic ablation of mmp-9 reduced the angiogenic switching , tumor number , and tumor growth , indicating that mmp-9 can render normal islets angiogenic . in addition , malignant keratinocyte transplantation resulted in tumors with neutrophils expressing predominantly mmp-9 and stromal cells expressing mainly mmp-2 and mmp-3 . these reports suggested a direct role for mmp-9 in tumor angiogenesis , but they did not identify the cell type producing this protease . reconstitution of tumor - bearing mmp-9 mice with wild type , mmp-9-competent hematopoietic cells demonstrated that tumor - infiltrating myeloid cells were the source for mmp-9 [ 111 , 112 ] . in a murine model of pancreatic islet carcinogenesis , mmp-9-expressing neutrophils were predominantly found inside angiogenic islet dysplasias and tumors , whereas mmp-9-expressing macrophages were localized along the periphery of such lesions . transient depletion of neutrophils significantly reduced the frequency of initial angiogenic switching in dysplasias . also tans in melanoma or fibrosarcoma tumors expressed high levels of mmp-9 and vegf , and elimination of these tans resulted in reduced tumor growth . also , reducing tans in prostate carcinoma tumors reduced angiogenesis and tumor cell intravasation . moreover , in cancer patients , neutrophils expressing high levels of mmp-9 have also been found . in hnscc , expression of mmp-9 was larger by tans than by any other cell type in the tumor , and in hepatocellular carcinoma larger numbers of tans correlated with more angiogenesis [ 117 , 118 ] . direct proof for neutrophils being the major tumor - associated leukocyte type expressing mmp-9 was recently provided in a study employing human xenografts and syngeneic murine tumors . when tumors or isolated tams and tans were double - stained for mmp-9 and for respective macrophage- or neutrophil - specific antigens , only tans gave a strong signal for mmp-9 [ 119 , 120 ] . in addition , it was calculated that 1 10 neutrophils or tans could release approximately 100200 ng prommp-9 within 1 - 2 h of incubation . in contrast , 1 10 macrophages or tams would require several weeks to produce the same amount of prommp-9 [ 119 , 120 ] . hence , neutrophil - derived mmp-9 is responsible for enhancing angiogenesis via release of vegf from the ecm in many types of tumors ( figure 2 ) . the unusual angiogenic potency of neutrophil mmp-9 is related to its unique way of production . in other cell types , the zymogen prommp-9 is released together with the inhibitor of metalloprotease 1 ( timp-1 ) , which slows the activation of mmp-9 and can also inhibit the proteolytic activity of the once activated enzyme . therefore , the timp-1-free prommp-9 from neutrophils can be activated easier and function much longer than mmp-9 from other cell types [ 122 , 123 ] . neutrophils are efficient producers of reactive oxygen species ( ros ) for destruction of microorganisms . first , neutrophils generate hydrogen peroxide ( h2o2 ) , which is next converted to hypochlorous acid ( hocl ) by myeloperoxidase ( mpo ) . hocl can then activate several ecm - degrading mmps , including mmp-2 , mmp-7 , mmp-8 , and mmp-9 . also , hocl can block timp-1 and in this manner potentiate the proteolytic activity of mmps [ 124 , 125 ] . finally , as indicated above , mmp activity leads to enhanced tumor progression by inducing proliferation and angiogenesis . nevertheless , a more potent and direct effect of ros on tumor cells is genotoxicity , which might lead to carcinogenesis ( figure 2 ) . although neutrophil - derived ros and hocl can directly damage and destroy tumor cells , they can also cause genotoxicity in circumstances when they do not kill cells . ros - mediated genotoxicity is induced by two major pathways : oxidative dna damage and mpo catalyzed activation of chemical carcinogens . point mutations and dna strand breaks are induced in many different cell types when cocultured with neutrophils , and hocl has been reported to be mutagenic in lung epithelial a549 cells . upon release from neutrophil granules , arg1 gets activated to degrade extracellular arginine , an essential amino acid for proper activation of t cells . thus , degranulation of neutrophils may exert an immunosuppressive effect in tumors by inhibiting t cells in a similar manner to the one described for g - mdsc . in fact , depletion of tans in tumor - bearing animals increased the numbers of activated cd8 t cells and promoted smaller tumors . similarly , non - small cell lung cancer cells stimulated neutrophils through il-8 to release arg1 , and in tumors tans had reduced levels of arg1 . more recently , the same group found that arg1 released from gelatinase granules was inactive at physiological ph unless activated by factor(s ) stored in azurophil granules . thus , tans can induce arg1-dependent immunosuppression through concomitant exocytosis of gelatinase and azurophil granules ( figure 2 ) . neutrophils can also produce cytokines or growth factors , which increase the tumorigenic potential of cancer cells . two clear examples have been described for oncostatin - m [ 128130 ] and for hepatocyte growth factor [ 10 , 131 , 132 ] . breast cancer cells can stimulate neutrophils to release oncostatin - m , an il-6-like cytokine . oncostatin - m in turn stimulated breast cancer cells to secrete vegf ( figure 2 ) . similarly , hepatocellular carcinoma cells stimulated neutrophils to release hepatocyte growth factor ( hgf ) . in turn , hgf stimulated tumor cells to become more invasive ( figure 2 ) . neutrophils can also influence the migration potential of cancer cells . in several types of cancer these tumors include skin squamous cell carcinoma , melanoma , adenocarcinomas , hnscc , and breast cancer . the way neutrophils augment the migratory capacity of tumor cells involves many different mechanisms that are just beginning to be elucidated . in hnscc , tumor - derived mif not only recruits tans but also induced these cells to display promigratory effects on the tumor cells . similar responses have been documented for different cancer cell lines but through a different mediator . various tumor cells release hyaluronan , which can then activate neutrophils via tlr4 and the pi-3k / akt signaling pathway . in turn , neutrophils induce enhanced migration of the tumor cells . very early reports suggested that tans release enzymes that degrade the basement membrane and promote tumor cell invasion through the basement membrane ( figure 3 ) . in vitro studies showed that human neutrophils assist the human breast tumor cell line mda - mb-231 to cross a monolayer of endothelial cells . tumor cell - conditioned medium downregulated neutrophil cytotoxicity and upregulated expression of adhesion molecules , facilitating tumor cell migration . also , in the presence of neutrophils , melanoma cell adhesion and transmigration through an endothelial cell monolayer were increased [ 141 , 142 ] ( figure 3 ) . this process seems to involve at least in part the protease ne , which can induce severe tissue damage , and as mentioned before correlates with poor prognosis . elevated amounts of ne in various types of cancer can induce tumor invasion and metastasis by degrading ecm proteins . in support of this once in circulation , neutrophils can also help tumor cells to survive by inducing tumor cell aggregation ( figure 3 ) . in patients with breast and prostate cancers , tumor cell clusters in blood have been associated with poor survival , and in animal models , injection of tumor cell clusters resulted in more metastases than injection of dispersed tumor cells . at least , for breast cancer mcf-7 cells , neutrophils can promote aggregation in vitro [ 99 , 101 ] . however , metastasis induced by neutrophil - mediated aggregation of tumor cells has not yet been directly demonstrated in vivo . circulating tumor cells directly adhere to the vascular endothelium promoting extravasation for establishing new metastases . at the site of exit , lung cancer tumor cells have been observed in close association with neutrophils . in this process , neutrophils enhance tumor cell retention and in consequence induce more metastasis ( figure 3 ) . direct cell - cell interaction of neutrophils with breast carcinoma cells has been shown to involve the adhesion molecule icam-1 on the tumor cells and 2 integrins on neutrophils . neutrophils bound tumor cells engaging integrins and inducing icam-1 clustering on the tumor cell ( figure 3 ) . this activated in the tumor cell a signaling pathway involving focal adhesion kinase ( fak ) and p38-mapk that resulted in enhanced migration . in addition , this enhanced migration was shown in vivo to result in increased metastasis to the liver . here , the cancer cells adhered directly on top of arrested neutrophils , which acted as a bridge to facilitate interactions between the tumor cells and the liver parenchyma . moreover , neutrophils seem to participate in facilitating metastasis even before the tumor cells arrive to the new site , the metastatic niche . this is a potential metastatic site where leukocytes create a permissive growth environment prior to the arrival of tumor cells [ 150 , 151 ] . vegfr1-positive bone marrow - derived cells are found in premetastatic niches of organs involved in metastasis of particular tumor types . once at the metastatic niche , these bone marrow - derived cells secrete factors that promote tumor cell growth [ 152 , 153 ] ( figure 3 ) . in lungs of mice bearing mammary adenocarcinomas , these granulocytic cells had decreased ifn- production and increased mmp-9 production , thus promoting angiogenesis . in addition , coinjection of with 4t1 tumor cells with these gr-1cd11b cells , isolated from tumors and spleens of 4t1 mammary tumor - bearing mice , resulted in increased metastases to lungs . but because these gr-1cd11b cells are a heterogeneous population of cells , including neutrophils , macrophages , dendritic cells , and other immature myeloid cells , the particular cell type(s ) needed to promote metastasis remains unclear . however , neutrophils are a good candidate because it has been reported that circulating neutrophils augment in number with increasing metastatic potential of various rat mammary adenocarcinomas , and tumors secreting il-8 also have an increased metastatic potential . clearly , the mechanisms tans use to promote tumor cell migration and metastasis are diverse and complex ( figure 3 ) . despite the large amount of evidence for a negative role of neutrophils during tumor progression , there is also clear evidence for a positive role of neutrophils in carcinogenesis . early murine neutrophils infiltrating tumors have been named n1 since they clearly display an active proinflammatory and an antitumor phenotype . in fact , the antitumor capacity of neutrophils has been recognized for more than three decades . for example , a colon adenocarcinoma cell line transfected to express g - csf lost tumorigenic activity after considerable concentration of neutrophils at the tumor site . interestingly , neutrophils could discriminate between g - csf - producing and g - csf - nonproducing cells and directly inhibited only g - csf - producing tumor cells . this antitumor effect of activated neutrophils can also be transferred to other animals , as demonstrated with spontaneous regression / complete resistance ( sr / cr ) mice . sr / cr mice resist very high doses of cancer cells that are lethal to wt mice even at low doses . the genetic , cellular , and molecular effector mechanisms in this model are largely unknown . however , purified neutrophils from the sr / cr mice independently killed cancer cells in vitro and completely transferred resistance to wt recipient mice . also , the cancer disappeared gradually following infiltration of a large number of neutrophils and few lymphocytes into the remaining tumor tissues . the importance of n1 type tans in antitumor responses is also highlighted by reports showing that depletion of murine neutrophils results in enhanced tumor growth [ 15 , 160 , 161 ] . despite the evidence presented before on neutrophils helping metastasis by preparing the metastatic niche , a complete opposite effect has also been demonstrated for metastatic breast cancer and renal carcinoma . in both models , neutrophils prevented metastasis to the lung . in the breast cancer model , the tumor cells produced ccl2 that induced neutrophil ros production , while , in the renal carcinoma model , tumor - derived il-8 recruited tumor cytotoxic neutrophils . this goes against the majority of reports implicating il-8 in protumor functions of neutrophils . nevertheless , these findings underline the dual antitumor and protumor potential of neutrophils and suggest that neutrophils could be induced to enhance their antitumor responses . the mechanisms by which neutrophils accomplish this function are numerous and not completely understood , but they involve many of the same antimicrobial and immune regulatory functions of neutrophils ( figure 4 ) . early reports indicated that neutrophils from tumor - bearing animals displayed enhanced superoxide anion generation and phagocytosis . this led to reduced tumors and less metastatic foci in lungs [ 38 , 39 ] . also , it has been shown that indeed ros produced by neutrophils can induce tumor cell lysis , through hocl delivered directly at the cell membrane . although ros could be genotoxic for tumor cells , it is clear that , in the case of rapidly growing tumors , activated neutrophils producing sufficient singlet oxygen can eliminate tumor cells at the early phase of tumor development ( figure 4 ) . because neutrophils require close contact mediated by integrins to induce killing , it is also possible that neutrophils may induce direct lysis of tumor cells by a mechanism similar to the one used by nk cells via the enzymes perforin and granzyme . neutrophils induced apoptosis of human breast cancer cells , when stimulated by antibodies targeted to her-2 . neutrophils have another way of eliminating tumor cells by inducing apoptosis of the malignant cell . this effect is mediated by the tumor necrosis factor - related apoptosis inducing ligand ( trail ) ( figure 4 ) . for a long time , carcinoma in situ of the bladder has been treated with intravesical administration of mycobacterium bovis bacillus calmette - gurin ( bcg ) . this kind of immunotherapy is very effective for treatment of this type of cancer , but the mechanism is only partially known . it was then found that neutrophils in urine of patients with carcinoma of the bladder and under bcg immunotherapy expressed high levels of trail . trail is expressed on these neutrophils at high levels both as a type ii membrane protein ( intracellular amino terminal portion and carboxyl terminus outside the cell ) and as a biologically active soluble form , which is released from intracellular stores after interaction with components of the bcg cell wall . trail is a member of the tnf family of molecules , known to have apoptosis - inducing functions . trail binds to target cells through two death receptors ( drs ) ( dr4/trail - r1 and dr5/trail - r2 ) and three decoy receptors ( dcrs ) [ dcr1/trail - r3 , dcr2/trail - r4 , and osteoprotegerin ] . drs activate the formation of a death - inducing signaling complex for caspase activation and initiation of apoptosis . an important feature of neutrophil trail - induced apoptosis is that it can kill tumorigenic and transformed cells but not normal cells and tissues [ 170 , 178 ] . for this reason , trail is becoming a major physiologic weapon against cancer , and several research laboratories and pharmaceutical companies are developing recombinant forms of trail or trail receptor agonists for therapeutic purposes . in addition , the importance of trail in other clinical conditions , such as infectious diseases , autoimmunity , and cardiovascular diseases , is becoming more apparent . therefore , understanding the regulatory mechanisms of trail signaling will help in the future to control these health problems . neutrophils can protect against some tumors by secreting mmp-8 . in mice deficient in mmp-8 , an increase in skin tumors with an increase in neutrophil infiltrates to the tumors was reported . this protective effect is not clearly defined , but it involves the inhibition of neutrophil migration into the tumor site . antibodies directed to tumor cells can also bind to fc receptors on the membrane of immune cells . in many cases this antibody - dependent cell - mediated cytotoxicity ( adcc ) is capable of eliminating efficiently various types of tumors . nk cells are particularly efficient in this response via the fc receptors ( figure 4 ) . however , the mechanism of killing is not completely described , but it seems to be different from the classic adcc mechanism used by nk cells . it is worth noting that an important difference exists between murine and human neutrophils regarding fcr expression . in addition to fcriii , the only fc receptor on murine nk cells , murine neutrophils also express fcriv . in contrast , human neutrophils express two unique fc receptors not present in other species : fcriia ( cd32a ) ( homolog to murine fcriii ) and fcriiib ( cd16b ) ( a glycosylphosphatidylinositol- ( gpi- ) linked receptor ) . therefore special attention should be paid when interpreting data from murine models on adcc against tumors . human neutrophils present a more efficient adcc when they engage fcriia [ 184 , 185 ] . under stimulated conditions mainly with ifn- but also with g - csf , neutrophils can upregulate expression of fcri ( cd64 ) . this receptor seems also capable of promoting neutrophil adcc against tumors and in particular with squamous head and neck cancer . however , in other studies , it was shown that immature neutrophils with high expression of fcri had reduced adcc activity via this receptor . in fact , ample reports have demonstrated that the high affinity receptor for iga , fcri ( cd89 ) , is a more potent inducer of adcc by neutrophils [ 188 , 189 ] . the mechanism for tumor cytotoxicity from neutrophils is not completely known , and it seems to be multifactorial . both ros - dependent and ros - independent mechanisms have been suggested for neutrophil adcc . for the oxidative mechanism , close cell contact mediated by integrins however , studies with neutrophils from chronic granulomatous disease ( cgd ) patients and with ros scavengers suggest that ros are not as important for adcc as they are for antimicrobial functions . however , as mentioned before , expression of these enzymes in neutrophils remains controversial [ 166 , 167 ] . neutrophils invading tumors can modify t cell effector functions and in this way instruct t cells to reject tumors . cytotoxic cd8 t cells are key contributors in any immune response towards tumors . as mentioned before n2 neutrophils can be inhibitors of t cell functions [ 15 , 192 ] . however , the proinflammatory n1 neutrophils can recruit and activate cd8 t cells [ 15 , 193 ] ( figure 4 ) . also , after photodynamic therapy , there was a rapid neutrophil infiltration into the treated tumor bed . neutrophils were necessary for generation of tumor - specific primary and memory cd8 t cell responses . together , these reports indicate that neutrophils can influence the outcome of t cell functions depending on the type of cytokines they produce [ 4 , 194 ] . neutrophil extracellular traps ( nets ) constitute a recently described form of the antimicrobial arsenal of neutrophils . nets are fibers of chromatin released from neutrophils in an active process named netosis . in this process , neutrophils undergo dramatic changes starting with flattening of the cells . citrullination of histone h3 by peptidylarginine deiminase 4 ( pad4 ) is a major modification during netosis . dna fibers in nets are also decorated with various antimicrobial proteins from the neutrophil granules , including neutrophil elastase , mpo , cathepsin g , proteinase 3 , mmp-9 , and bactericidal / permeability increasing protein ( bpi ) . nets form a mesh - like structure where microorganisms get trapped and are either directly killed on some cases or more often subsequently phagocytosed by other neutrophils [ 198 , 199 ] . many microorganisms and various stimuli bacterial products such as lipopolysaccharide ( lps ) , formyl - methionyl - leucyl - phenylalanine ( fmlf ) , and also phorbol esters such as phorbol myristate acetate ( pma ) are efficient net inducers . recent reports also indicated that antigen - antibody complexes are capable of inducing net formation , thus suggesting a direct role for fc receptors in this function . in fact both fcri and fcriiib have been shown to induce net formation . this is interesting because various tumors are known to produce these cytokines and thus it is possible that tumors can enhance net formation . very little is known about the presence and effect of nets in different types of tumors . it is also not clear if distinct tans can make nets with different efficiency . in an initial study , tumor samples from eight patients with ewing sarcoma were evaluated for the presence of tans and nets , defined as extracellular staining for mop . in two ( 25% ) patients , intratumoral nets were found . thus , it was proposed that at least this type of tumor could induce tans to release nets . however , in cancer models of chronic myelogenous leukemia and mammary and lung carcinoma , peripheral neutrophils were more prone to net formation . neutrophils from tumor - bearing animals responded to platelet - activating factor ( paf ) forming more nets than neutrophils from tumor - free animals . in addition , higher amounts of circulating neutrophils and plasma cell - free dna were found in tumor - bearing animals . this free dna is probably in the form of nets , since a concomitant increase in neutrophils with hypercitrullinated histone h3 was also found . it seems then that some cancers may present a systemic effect on the host that predisposes neutrophils to form nets . as discussed earlier , many tumors presenting tans are associated with poor prognosis . in many of these tumors , thus the presence of nets in these tumors most certainly would be associated with tumor progression . supporting this idea , there are studies looking at the phenotype of tans during tumor development . in one study , neutrophil depletion at 14 days after implantation of lewis lung carcinoma ( llc ) and ab12 mesothelioma tumors resulted in reduced tumor growth . in contrast , neutrophil depletion at 7 days after implantation had no effect on tumor growth . tans from early tumors were more cytotoxic toward tumor cells , while tans from established tumors acquired a more protumorigenic phenotype . moreover , in initial tumors , tans were found in the periphery of the tumor , but in mature tumors tans and free dna were within the tumor . in another study , this defect caused an uneven compartmentalization of lymphoid and myeloid populations that led to aberrant interactions between nets and b cells . under these conditions , nets induced b cell proliferation and inhibition of apoptosis , resulting in malignant transformation . neutrophils would migrate to the new tumor and there tans would produce nets , which would promote tumor growth ( figure 5 ) . although evidence strongly indicates that nets within primary tumors can promote tumor progression , no mechanism for this effect has been revealed yet . however , because nets are made of chromatin fibers decorated with antimicrobial proteins such as neutrophil elastase , cathepsin g , and mpo , it is very likely that nets concentrate these factors to high local concentrations within the tumor microenvironment . as discussed above , these factors have all been implicated in tumor promotion . therefore , nets may be a way to enhance exposure of tumor cells to these bioactive proteins and in turn increase proliferation , inhibit apoptosis , and induce migration ( figure 5 ) . although in many instances the presence of neutrophils in tumors has a negative effect in cancer disease , these cells clearly have the capacity to destroy tumor cells . several novel therapeutic approaches are being considered to enhance the antitumor potential of neutrophils or to block the access of tans into growing tumors . n1 type murine neutrophils display an activated phenotype that leads to tumor control . in consequence , tumor cells modified to express activation of neutrophils with g - csf and ifn- can generate cells with an antitumor phenotype . due to the important role of neutrophils in antimicrobial responses , general activation of these cells is not good therapeutic approach since highly activated neutrophils without targeting specificity could cause excessive tissue damage . the two types of tans , n1 and n2 , suggest that the tumor microenvironment could be manipulated to generate more antitumor tans . this idea is supported by studies in murine cancer models where inhibition of tgf- induced the appearance of antitumor neutrophils . another therapeutic approach aims to block infiltration of neutrophils into tumors . as indicated before , the use of il-8 antagonists ( such as the fully humanized neutralizing monoclonal antibody abx - il8 ) to il-8 was shown to reduce tumor growth , metastasis , and angiogenesis of melanoma and lung cancer . because other chemokines also interact with the receptors cxcr1 and cxcr2 , a more effective way to block neutrophil migration may be the inhibition of these receptors . specific inhibitors for these receptors are now being developed with the idea of preventing neutrophil infiltration and retarding tumor progression . for example , the cxcr2 receptor antagonist , gsk135756 , is being considered to be used as an anti - inflammatory drug for chronic obstructive pulmonary disease . this inhibitor has shown to efficiently block neutrophil recruitment into tissues and to selectively target human breast cancer stem cells in xenograft models in mice . in addition to blocking neutrophil infiltration , inhibition of particular neutrophil - specific enzymes known to promote tumor progression is another therapeutic avenue being explored . for example , inhibition of ne was able to reduce significantly the growth of lung adenocarcinomas in a mouse model . also inhibition of mmp has been tried to prevent tumor angiogenesis . the bisphosphonate zoledronic acid , a strong mmp inhibitor , blocked mmp-9 expression and metalloprotease activity reducing angiogenesis and cervical cancer burden . however , in other models and clinical trials , inhibition of mmp-9 was not effective at reducing tumor growth [ 214 , 215 ] . a more promising approach is the use of antitumor monoclonal antibodies ( mabs ) to activate the adcc potential of neutrophils . upon fc receptor activation , today most mabs used in immunotherapy belong to the igg1 class , and they are effective at activating nk cells via fcriiia ( cd16a ) . in contrast , neutrophils activate adcc via fcriia ( cd32 ) by preferentially engaging igg2 class antibodies . this igg2-mediated adcc was influenced by the functional fcriia - r131h polymorphism and was induced more effectively by neutrophils from fcriia-131h homozygous donors than from fcriia-131r individuals . based on these findings , it has been proposed that fc receptor polymorphisms could be biomarkers for egfr antibodies such as panitumumab , the only human igg2 antibody approved for immunotherapy and inhibition of egfr . therefore , there is a big interest in developing new improved antibodies through fc engineering technologies in order to potentiate fcr - mediated functions . based on this methodology , it was possible to change the ability of an fcriii - optimized ( for nk cell ) anti - egfr antibody to efficiently activate neutrophil adcc against egfr - expressing tumors . however , it seems that fcri ( cd89 ) is a more potent inducer of adcc by neutrophils [ 188 , 189 ] . thus , it has been proposed that a new generation of cancer therapeutic mab should include iga class antibodies to fully take advantage of the cytotoxic potential of neutrophils . indeed , this idea is supported by a new iga2 anti - egfr antibody derived from the igg anti - egfr mab cetuximab . iga2 egfr was more effective than cetuximab in vivo against egfr - transfected ba / f3 target cells . very recently , it was also shown that the combination of igg and iga mabs to two different tumor targets ( egfr and her2 ) led to enhanced cytotoxicity compared with each isotype alone . the exact role for these tumor - associated neutrophils ( tans ) has yet to be completely elucidated . however , a tremendous body of clinical evidence has shown that neutrophils promote tumor progression in various ways . neutrophils can induce tumor proliferation and angiogenesis and can enhance tumor cell migration and metastasis . yet , a type of tans , named n1 , can indeed display antitumor functions . new therapeutic ways to recruit and activate these n1 type neutrophils are being investigated in order to turn protumorigenic neutrophils into antitumor effector cells . blocking neutrophil - derived components known to help tumor growth is a field of active research . also , very promising results have been found with the use of therapeutic antibodies , which induce neutrophils to perform adcc and to release cytokines that modulate the immune response against tumors . new antibodies are being designed so that they have better affinity for particular fc receptors and induce stronger antitumor responses . learning how to flip the neutrophil coin to the winning side , namely , functioning as antitumor effector cells , is a challenge for future research that will certainly provide us with new therapeutic options for cancer treatment .

neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections . in addition , neutrophils are also found infiltrating many types of tumors . tumor - associated neutrophils ( tans ) have relevant roles in malignant disease . indeed neutrophils may be potent antitumor effector cells . however , increasing clinical evidence shows tans correlate with poor prognosis . the tumor microenvironment controls neutrophil recruitment and in turn tans help tumor progression . hence , tans can be beneficial or detrimental to the host . it is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor , for neutrophils supporting tumor progression , and for neutrophil activation to enhance their antitumor functions .

1. Introduction 2. Neutrophils in Cancer 3. Recruitment 4. Protumor Function of Neutrophils 5. Antitumor Function of Neutrophils 6. Neutrophil Extracellular Traps 7. Therapeutic Approaches 8. Conclusion

neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections . invading microorganisms there , neutrophils destroy the microorganism by a series of mechanisms , mainly phagocytosis , release of antimicrobial substances , and the formation of neutrophil extracellular traps ( nets ) . activated neutrophils also release proteinases into the surrounding tissue , causing damage to the host . in addition , neutrophils are capable of producing many cytokines and chemokines , which can influence the inflammatory response , as well as the immune response [ 4 , 5 ] . besides this classical role in antimicrobial functions , neutrophils early studies suggested that these tumor - associated neutrophils ( tans ) were mere bystanders because it was hard to imagine that neutrophils , being short - lived cells , could have an effect on chronic and progressive diseases such as cancer . however , more recently it is becoming clear that tans have relevant roles in malignant disease . however , an increasing number of clinical observations and laboratory studies have shown that presence of neutrophils in tumors correlates with poor prognosis . in all these cases , tans are different from circulating neutrophils ( as discussed later ) , and , in untreated tumors of murine models , they can display a protumorigenic phenotype . the mechanisms for this phenotype are just beginning to be elucidated , but some of them involve genotoxicity , angiogenesis , and immunosuppression . these two types of tans described in mice have been named n1 and n2 in a similar manner as antitumor and protumor macrophages ( tams ) . it is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor , for neutrophils support to the tumor , and for neutrophil activation to enhance their antitumor functions and in the future improve cancer immunotherapy . however , this trend is now beginning to change with the realization that neutrophils are indeed important players in cancer development , as reflected by several recent reviews [ 1618 ] , and as we will see next . in addition , other cytokines such as granulocyte colony - stimulating factor ( g - csf ) , interleukin- ( il- ) 1 , and il-6 produced by tumors seem to contribute to elevated neutrophil numbers in blood . this neutrophilia is associated with poor prognosis in several types of cancers , such as lung , melanoma , and renal carcinomas [ 11 , 21 , 22 ] . reduced recurrence - free time and overall survival were reported for neutrophil - infiltrated tumors in renal carcinomas , hnscc , pancreatic adenocarcinomas , and liver carcinoma . due to its simplicity , nlr has shown to be a readily available and inexpensive biomarker for many types of tumors including non - small - cell lung cancer , hepatocellular carcinoma , nasopharyngeal carcinoma , colorectal cancer , melanoma , and breast cancer [ 29 , 30 ] . despite the clinical evidence from the many studies mentioned above , neutrophilia ( larger numbers of neutrophils in blood as a consequence of elevated egress of cells from the bone marrow ) is not always a bad indicator for cancer progression . in some types of tumors , for example in fact , the capacity of neutrophils to directly kill tumor cells both in vitro and in vivo was reported long time ago [ 3537 ] . also , neutrophils from tumor - bearing animals were reported to have enhanced cytotoxic activity [ 38 , 39 ] . in addition to the elevated number of neutrophils in blood , an increase in the frequency of immature myeloid cells at earlier stages of differentiation has also been detected in several types of tumors , including terminal patients with lung , breast , and gastrointestinal cancer . these mdscs can be divided phenotypically into granulocytic ( g - mdsc ) and monocytic ( mo - mdsc ) subgroups [ 45 , 46 ] and are found in great numbers in the spleens of tumor - bearing animals , where they display an immunosuppressive phenotype helping tumor progression [ 47 , 48 ] . the effect was found to be mediated by oxidation of the actin - remodeling protein cofilin . cofilin is activated through dephosphorylation at ser3 , and then it mediates severing and depolymerization of f - actin for formation of the immune synapse and t cell activation . in addition , exposure of ros to memory / effector cd45ro t cells results in inhibition of nf-b activation and reduction in th1 cytokines production . during mdsc - t cell contact , this process makes cd8-expressing t cells unable to bind peptide - mhc complexes and to respond to the specific peptide . because , g - mdscs share many properties with neutrophils but seem to be functionally different from mature neutrophils [ 13 , 62 ] , a transcriptomic analysis was conducted to compare in tumor - bearing animals circulating neutrophils with tans and with mdscs . thus , it seems that tgf- within the tumor microenvironment induces a population of tans with a protumor phenotype . in support of this idea , in two models of murine tumor cancer cell lines ( lewis lung carcinoma and ab12 mesothelioma ) , neutrophils were found primarily at the periphery of the tumor at early stages of tumor development . therefore , murine tans can have an antitumorigenic ( n1 ) phenotype but also a protumorigenic ( n2 ) phenotype capable of supporting tumor growth and suppressing the antitumor immune responses [ 14 , 41 ] , depending on the tumor microenvironment . in one study of surgically resected lung cancer patients , tans were isolated from digested human lung tumors and constituted 5%25% of the cells in the tumor . in addition , tans produced larger quantities of the proinflammatory factors mcp-1 , il-8 , mip-1 , and il-6 than blood neutrophils did . these activated dendritic cells induced t17 cells to secrete il-8 , tnf- , and gm - csf , thus leading to accumulation of neutrophils in the tumor . in early tumors , tans seem to be able to stimulate t cell responses , but later in established tumors tans are immunosuppressive . a very recent publication identifies several subpopulations of neutrophils in the blood of tumor - bearing mice and in human cancer patients and describes several relationships of these cells in connection to cancer progression . the hdns from cancer animals , which were previously reported as tumor - entrained neutrophils ( tens ) , displayed high cytotoxicity toward tumor cells in culture , whereas ldns were not cytotoxic . in addition , authors showed that hdns are capable of becoming ldns upon treatment with tgf- . it is interesting to note that tgf- was able to induce the change of hdns from tumor - bearing mice into ldns , but it had no effect on hdns from tumor - free mice . together , all these results support a model proposed by the authors , in which neutrophils are present in three subpopulations in cancer : normal high - density neutrophils , immature low - density neutrophils ( g - mdsc ) , and large mature low - density neutrophils . while the hdns are antitumor and the ldns are protumor , they can change under the influence of the different chemokines and cytokines in the tumor microenvironment . the tumor stroma contains fibroblasts , endothelial cells of blood vessels , and in many cases immune cells . these tumor - infiltrating immune cells highlight the inflammatory microenvironment that is commonly associated with tumor progression . in addition to lymphocytes and macrophages , neutrophils are found in great numbers in a wide variety of tumors [ 12 , 13 , 23 , 24 ] . clearly , neutrophils are recruited to the tumor by the action of neutrophil - attracting chemokines that can be produced not only by other immune cells but also directly by several tumor cells ( figure 1 ) . accordingly , increased il-8 levels were found in hnscc patients , and elimination of neutrophils in cancer murine models reduced tumor burden and metastasis . these findings support the notion that tumor - produced il-8 is important for neutrophil recruitment to help tumor progression . however , the il-8 receptors cxcr1 and cxcr2 are also expressed on other cell types including endothelial cells and tumor cells . using the same cxcr1 and cxcr2 receptors , neutrophils can also respond to other chemokines such as cxcl1 , cxcl2 , cxcl5 , cxcl6 , and cxcl7 ( figure 1 ) . supporting this idea , it was also found that in tans the expression of cxcl2 , and also cxcl1 , was upregulated more than 150-fold . therefore , it seems that neutrophils activate a positive feedback mechanism by releasing neutrophil chemokines that attract more neutrophils into the tumor , similarly to neutrophil recruitment into sites of infection . cxcl5 was found to be overexpressed in patients with recurrent tumors , and the levels of cxcl5 correlated with greater appearance of tans and with shorter overall survival . another chemokine that also participates in neutrophil recruitment to tumors is gcp-2/cxcl6 . in addition , migration inhibitory factor ( mif ) , another tumor - derived chemokine for neutrophils , was identified in hnscc tumors . many tumor cells can directly produce chemokines for neutrophils , but various other cells within the tumor may also be the source for these chemokines and other cytokines . although the influence of activated t cells in neutrophil recruitment to tumors is not known , regulatory t cells ( treg ) seem to be important for neutrophil infiltrating tumors . in one study , treg were found to inhibit neutrophil recruitment to a tumor site by reducing the expression of cxcl1 and cxcl2 . a large body of clinical evidence indicates that neutrophils are involved in cancer development and tumor progression . important molecules that can modify growth and invasiveness of tumors involve granule proteins , matrix - degrading proteinases , reactive oxygen species ( ros ) , chemokines , and cytokines . in addition to its roles in inflammation and bacteria destruction , ne has presented various protumor effects both in vivo and in vitro . because irs-1 binds to the regulatory unit of pi-3k , its degradation by ne led to more pi-3k available to enhance the proliferation pathway . similar results have been found with other types of tumor cells , including esophageal cancer , gastric cancer , and breast cancer . first cathepsin g binds to the tumor cell surface , independently of its catalytic site , and then induces cell aggregation , which is dependent on its enzymatic activity ( figure 2 ) . in addition , malignant keratinocyte transplantation resulted in tumors with neutrophils expressing predominantly mmp-9 and stromal cells expressing mainly mmp-2 and mmp-3 . moreover , in cancer patients , neutrophils expressing high levels of mmp-9 have also been found . in hnscc , expression of mmp-9 was larger by tans than by any other cell type in the tumor , and in hepatocellular carcinoma larger numbers of tans correlated with more angiogenesis [ 117 , 118 ] . direct proof for neutrophils being the major tumor - associated leukocyte type expressing mmp-9 was recently provided in a study employing human xenografts and syngeneic murine tumors . hence , neutrophil - derived mmp-9 is responsible for enhancing angiogenesis via release of vegf from the ecm in many types of tumors ( figure 2 ) . point mutations and dna strand breaks are induced in many different cell types when cocultured with neutrophils , and hocl has been reported to be mutagenic in lung epithelial a549 cells . thus , degranulation of neutrophils may exert an immunosuppressive effect in tumors by inhibiting t cells in a similar manner to the one described for g - mdsc . similarly , non - small cell lung cancer cells stimulated neutrophils through il-8 to release arg1 , and in tumors tans had reduced levels of arg1 . in several types of cancer these tumors include skin squamous cell carcinoma , melanoma , adenocarcinomas , hnscc , and breast cancer . in hnscc , tumor - derived mif not only recruits tans but also induced these cells to display promigratory effects on the tumor cells . in turn , neutrophils induce enhanced migration of the tumor cells . this process seems to involve at least in part the protease ne , which can induce severe tissue damage , and as mentioned before correlates with poor prognosis . in support of this once in circulation , neutrophils can also help tumor cells to survive by inducing tumor cell aggregation ( figure 3 ) . in patients with breast and prostate cancers , tumor cell clusters in blood have been associated with poor survival , and in animal models , injection of tumor cell clusters resulted in more metastases than injection of dispersed tumor cells . at least , for breast cancer mcf-7 cells , neutrophils can promote aggregation in vitro [ 99 , 101 ] . direct cell - cell interaction of neutrophils with breast carcinoma cells has been shown to involve the adhesion molecule icam-1 on the tumor cells and 2 integrins on neutrophils . neutrophils bound tumor cells engaging integrins and inducing icam-1 clustering on the tumor cell ( figure 3 ) . in addition , this enhanced migration was shown in vivo to result in increased metastasis to the liver . moreover , neutrophils seem to participate in facilitating metastasis even before the tumor cells arrive to the new site , the metastatic niche . in addition , coinjection of with 4t1 tumor cells with these gr-1cd11b cells , isolated from tumors and spleens of 4t1 mammary tumor - bearing mice , resulted in increased metastases to lungs . however , neutrophils are a good candidate because it has been reported that circulating neutrophils augment in number with increasing metastatic potential of various rat mammary adenocarcinomas , and tumors secreting il-8 also have an increased metastatic potential . despite the large amount of evidence for a negative role of neutrophils during tumor progression , there is also clear evidence for a positive role of neutrophils in carcinogenesis . in both models , neutrophils prevented metastasis to the lung . nevertheless , these findings underline the dual antitumor and protumor potential of neutrophils and suggest that neutrophils could be induced to enhance their antitumor responses . the mechanisms by which neutrophils accomplish this function are numerous and not completely understood , but they involve many of the same antimicrobial and immune regulatory functions of neutrophils ( figure 4 ) . because neutrophils require close contact mediated by integrins to induce killing , it is also possible that neutrophils may induce direct lysis of tumor cells by a mechanism similar to the one used by nk cells via the enzymes perforin and granzyme . in addition , the importance of trail in other clinical conditions , such as infectious diseases , autoimmunity , and cardiovascular diseases , is becoming more apparent . in many cases this antibody - dependent cell - mediated cytotoxicity ( adcc ) is capable of eliminating efficiently various types of tumors . the mechanism for tumor cytotoxicity from neutrophils is not completely known , and it seems to be multifactorial . in this process , neutrophils undergo dramatic changes starting with flattening of the cells . dna fibers in nets are also decorated with various antimicrobial proteins from the neutrophil granules , including neutrophil elastase , mpo , cathepsin g , proteinase 3 , mmp-9 , and bactericidal / permeability increasing protein ( bpi ) . very little is known about the presence and effect of nets in different types of tumors . it is also not clear if distinct tans can make nets with different efficiency . however , in cancer models of chronic myelogenous leukemia and mammary and lung carcinoma , peripheral neutrophils were more prone to net formation . in addition , higher amounts of circulating neutrophils and plasma cell - free dna were found in tumor - bearing animals . it seems then that some cancers may present a systemic effect on the host that predisposes neutrophils to form nets . as discussed earlier , many tumors presenting tans are associated with poor prognosis . moreover , in initial tumors , tans were found in the periphery of the tumor , but in mature tumors tans and free dna were within the tumor . although evidence strongly indicates that nets within primary tumors can promote tumor progression , no mechanism for this effect has been revealed yet . however , because nets are made of chromatin fibers decorated with antimicrobial proteins such as neutrophil elastase , cathepsin g , and mpo , it is very likely that nets concentrate these factors to high local concentrations within the tumor microenvironment . therefore , nets may be a way to enhance exposure of tumor cells to these bioactive proteins and in turn increase proliferation , inhibit apoptosis , and induce migration ( figure 5 ) . several novel therapeutic approaches are being considered to enhance the antitumor potential of neutrophils or to block the access of tans into growing tumors . the two types of tans , n1 and n2 , suggest that the tumor microenvironment could be manipulated to generate more antitumor tans . because other chemokines also interact with the receptors cxcr1 and cxcr2 , a more effective way to block neutrophil migration may be the inhibition of these receptors . for example , the cxcr2 receptor antagonist , gsk135756 , is being considered to be used as an anti - inflammatory drug for chronic obstructive pulmonary disease . this inhibitor has shown to efficiently block neutrophil recruitment into tissues and to selectively target human breast cancer stem cells in xenograft models in mice . in addition to blocking neutrophil infiltration , inhibition of particular neutrophil - specific enzymes known to promote tumor progression is another therapeutic avenue being explored . upon fc receptor activation , today most mabs used in immunotherapy belong to the igg1 class , and they are effective at activating nk cells via fcriiia ( cd16a ) . the exact role for these tumor - associated neutrophils ( tans ) has yet to be completely elucidated . however , a tremendous body of clinical evidence has shown that neutrophils promote tumor progression in various ways . new therapeutic ways to recruit and activate these n1 type neutrophils are being investigated in order to turn protumorigenic neutrophils into antitumor effector cells . learning how to flip the neutrophil coin to the winning side , namely , functioning as antitumor effector cells , is a challenge for future research that will certainly provide us with new therapeutic options for cancer treatment .

the institutional review board approved this study and did not require the approval of patients nor their informed consent for review of their images and records . from january 2002 to july 2004 , 303 women underwent breast mri examinations were initially considered for this study . the primary inclusion criterion of this retrospective analysis was a preoperative mri in patients with histologically confirmed breast cancer . among the 303 women , 213 patients had confirmed breast cancer . of these patients , 61 who underwent neoadjuvant chemotherapy were excluded because of a possible chemotherapeutic effect causing different kinetics and morphology of suspicious lesions ( 5 ) . the average age of the patients in the present study was 50 years ( range 23 to 79 years ) . indications for an mr examination included preoperative staging before planned breast conserving therapy to exclude multicentricity in patients who had one mammographic or sonographic suspicious lesion ( n = 123 ) , a search for an occult tumor with axillary metastases of suspected breast origin ( n = 5 ) and a postoperative examination to rule out residual disease ( n = 21 ) . a review of the mr images and final reports by an experienced radiologist specialized in breast imaging was conducted . we searched the cases with additional suspicious breast lesions that were initially detected by mri . by the definition of liberman ( 6 ) , mr lesions were considered to be additional sites if they were located in a different breast quadrant than the index cancer , if they were in the same quadrant but were separated from the index cancer by at least 1.0 cm of intervening normal - appearing tissue on mri , or if they were in the same quadrant and contiguous with the index cancer but extended at least 4.0 cm beyond the site of the index cancer . typically benign appearing enhancing lesions , i.e. those with a circumscribed margin or delayed enhancement were excluded . if mri revealed additional suspicious breast lesions other than the index cancer , a targeted us performed by the radiologist who interpreted the mr images was recommended to look for a us correlates amenable to further biopsy or localization . although there were no standardized protocols to follow , a targeted us examination by the radiologists who had knowledge of the mri findings was performed for the clinical and mammographical occult lesions . when targeted us was performed , the additional suspicious breast lesions were classified into two groups based on whether or not they had a us correlates . we compared the rate of identifying additional cancers between the lesions with and without a us correlate using the fisher 's exact test . the 31 patients that did not receive targeted us consisted of two groups ; one of the groups in whom the breast us was performed before the breast mr imaging and did not correlate with any additional detectable lesion by mri and the other group with mammographic microcalcifications , which were not detected initially and did correlate with the mri lesions . these lesions were confirmed by surgical excision or were followed with a subsequent examination . breast mri was performed with the patient prone in a 1.5-t commercially available imager ( signa ; ge medical system , milwaukee , wi ) with the use of a dedicated surface breast coil . the mri sequence used in this study included a fat suppressed axial fast spin echo t2-weighted sequence ( 4000/120 , repetition time msec / echo time msec ) and fat - suppressed unilateral sagittal dynamic imaging . dynamic imaging was performed with a t1-weighted three - dimensional , fat - suppressed fast spoiled gradient - echo sequence ( 17.3/1.3 ms ; flip angle , 30 ) three times ( one before and two after a rapid bolus injection of 0.1 mmol / kg gadopentetate dimeglumine [ magnevist ; berlex , wayne , nj ] ) ( 7 ) . section thickness was held between 1.0 - 2.0 mm without an intersection gap with a 256 192 matrix , an 18 - 24 cm field of view , and a scan time of 3 - 4 minutes . two sequential post - contrast scans were obtained without a break , beginning immediately after the saline flush . standard subtraction images were obtained by subtracting the precontrast images from the early peak ( or serial ) postcontrast image on a pixel - by - pixel basis . reverse subtraction images were obtained by subtracting the last postcontrast image from the early peak postcontrast image . if the lesion demonstrated a kinetic pattern showing an early rise and an early washout , the lesion would be observed with the remaining high signal intensity on reverse subtraction images . if any kinetic curves were equivocal , the morphologic features were considered together . if the lesion has a spiculate or an irregular margin , we regarded it as a morphological suspicious lesion . if a lesion had morphologic features that indicated it was probably benign but reverse subtraction images demonstrated a high signal intensity lesion , this was also considered to be suspicious ( 8) . criteria for the features of suspicious non - mass - like enhancements included linear , ductal , and segmental patterns . breast us examinations were performed by one of four breast imaging radiologists using a 7 - 10 mhz linear transducer ( logiq 700 ; general electric , milwaukee , wi , or hdi 5000 ; philips medical systems , bothell , wa ) . at the time of breast us thirty - eight lesions in 31 patients were performed with targeted us . of 21 patients with correlated suspicious enhancing lesions that were evident on us , eight underwent us - guided core biopsy for the suspicious lesion and 10 underwent us - guided localization , one with us - guided hook - wire localization and the remaining nine with us - guided carbon marking ( 9 ) . in the residual three patients , two patients had a scheduled mastectomy due to the extensive extent of an index cancer and one had breast conserving surgery because of the lesion localized 1 cm just inferior to the index cancer within the same quadrant . in the 10 patients without a us of the four that underwent a us - guided localization , us - guided needle localization was used in one and carbon marking in three . although lesions are not defined on us with certainty , localizations were performed in five lesions of four patients for subtle sonographic lesions likely consistent with the additional suspicious mr lesions . as the lesions without a us correlate could not be localized , careful histological examinations were requested at the area of any additional mr lesions . the institutional review board approved this study and did not require the approval of patients nor their informed consent for review of their images and records . from january 2002 to july 2004 , 303 women underwent breast mri examinations were initially considered for this study . the primary inclusion criterion of this retrospective analysis was a preoperative mri in patients with histologically confirmed breast cancer . among the 303 women , 213 patients had confirmed breast cancer . of these patients , 61 who underwent neoadjuvant chemotherapy were excluded because of a possible chemotherapeutic effect causing different kinetics and morphology of suspicious lesions ( 5 ) . the average age of the patients in the present study was 50 years ( range 23 to 79 years ) . indications for an mr examination included preoperative staging before planned breast conserving therapy to exclude multicentricity in patients who had one mammographic or sonographic suspicious lesion ( n = 123 ) , a search for an occult tumor with axillary metastases of suspected breast origin ( n = 5 ) and a postoperative examination to rule out residual disease ( n = 21 ) . a review of the mr images and final reports by an experienced radiologist specialized in breast imaging was conducted . we searched the cases with additional suspicious breast lesions that were initially detected by mri . by the definition of liberman ( 6 ) , mr lesions were considered to be additional sites if they were located in a different breast quadrant than the index cancer , if they were in the same quadrant but were separated from the index cancer by at least 1.0 cm of intervening normal - appearing tissue on mri , or if they were in the same quadrant and contiguous with the index cancer but extended at least 4.0 cm beyond the site of the index cancer . typically benign appearing enhancing lesions , i.e. those with a circumscribed margin or delayed enhancement were excluded . if mri revealed additional suspicious breast lesions other than the index cancer , a targeted us performed by the radiologist who interpreted the mr images was recommended to look for a us correlates amenable to further biopsy or localization . although there were no standardized protocols to follow , a targeted us examination by the radiologists who had knowledge of the mri findings was performed for the clinical and mammographical occult lesions . when targeted us was performed , the additional suspicious breast lesions were classified into two groups based on whether or not they had a us correlates . we compared the rate of identifying additional cancers between the lesions with and without a us correlate using the fisher 's exact test . the 31 patients that did not receive targeted us consisted of two groups ; one of the groups in whom the breast us was performed before the breast mr imaging and did not correlate with any additional detectable lesion by mri and the other group with mammographic microcalcifications , which were not detected initially and did correlate with the mri lesions . these lesions were confirmed by surgical excision or were followed with a subsequent examination . breast mri was performed with the patient prone in a 1.5-t commercially available imager ( signa ; ge medical system , milwaukee , wi ) with the use of a dedicated surface breast coil . the mri sequence used in this study included a fat suppressed axial fast spin echo t2-weighted sequence ( 4000/120 , repetition time msec / echo time msec ) and fat - suppressed unilateral sagittal dynamic imaging . dynamic imaging was performed with a t1-weighted three - dimensional , fat - suppressed fast spoiled gradient - echo sequence ( 17.3/1.3 ms ; flip angle , 30 ) three times ( one before and two after a rapid bolus injection of 0.1 mmol / kg gadopentetate dimeglumine [ magnevist ; berlex , wayne , nj ] ) ( 7 ) . section thickness was held between 1.0 - 2.0 mm without an intersection gap with a 256 192 matrix , an 18 - 24 cm field of view , and a scan time of 3 - 4 minutes . two sequential post - contrast scans were obtained without a break , beginning immediately after the saline flush . standard subtraction images were obtained by subtracting the precontrast images from the early peak ( or serial ) postcontrast image on a pixel - by - pixel basis . reverse subtraction images were obtained by subtracting the last postcontrast image from the early peak postcontrast image . if the lesion demonstrated a kinetic pattern showing an early rise and an early washout , the lesion would be observed with the remaining high signal intensity on reverse subtraction images . if any kinetic curves were equivocal , the morphologic features were considered together . if the lesion has a spiculate or an irregular margin , we regarded it as a morphological suspicious lesion . if a lesion had morphologic features that indicated it was probably benign but reverse subtraction images demonstrated a high signal intensity lesion , this was also considered to be suspicious ( 8) . criteria for the features of suspicious non - mass - like enhancements included linear , ductal , and segmental patterns . breast us examinations were performed by one of four breast imaging radiologists using a 7 - 10 mhz linear transducer ( logiq 700 ; general electric , milwaukee , wi , or hdi 5000 ; philips medical systems , bothell , wa ) . at the time of breast us examination , prior mr images were made available for direct correlation . of 21 patients with correlated suspicious enhancing lesions that were evident on us , eight underwent us - guided core biopsy for the suspicious lesion and 10 underwent us - guided localization , one with us - guided hook - wire localization and the remaining nine with us - guided carbon marking ( 9 ) . in the residual three patients , two patients had a scheduled mastectomy due to the extensive extent of an index cancer and one had breast conserving surgery because of the lesion localized 1 cm just inferior to the index cancer within the same quadrant . in the 10 patients without a us correlate , the lesions in six patients could not be localized . of the four that underwent a us - guided localization , us - guided needle localization was used in one and carbon marking in three . although lesions are not defined on us with certainty , localizations were performed in five lesions of four patients for subtle sonographic lesions likely consistent with the additional suspicious mr lesions . as the lesions without a us correlate could not be localized , careful histological examinations were requested at the area of any additional mr lesions . of the 149 total patients for whom mri was used for preoperative evaluation of breast cancer , 69 additional suspicious breast lesions were detected by mri in 62 patients ( 42% ) . of those 69 lesions , 26 ( 38% ) were confirmed as cancers by histology . by mri , 48 of 69 lesions ( 70% ) revealed a mass , and 21 ( 30% ) demonstrated nonmass - like enhancements and the average diameter of the 69 additional enhancing lesions in these 62 patients was 1.1 cm ( range , 0.4 - 5.0 cm ) . in addition , of these 62 patients with 69 lesions , 57 ( 92% ) had a single additional lesion , four ( 6% ) had two lesions , and one ( 2% ) had four lesions . among 69 lesions , contralateral enhancing lesions were detected in ten patients ( 16% ) , of which confirmed cancers in three patients ( 5% ) . of the 62 patients with additional lesions , 31 patients with 38 lesions underwent targeted us . histological findings of these 38 lesions in 31 patients performed with targeted us revealed cancers in 18 ( 47% ) lesions , high - risk lesions in two ( 5% ) , benign lesions in 15 ( 39% ) , and unidentified lesions in three ( 8% ) ( table 1 ) . the latter three lesions that were not confirmed by histological examination were removed by a mastectomy in one patient and breast conservative surgery with a wide extent in two patients . of the 38 lesions examined with targeted us , 27 ( 71% ) had a us correlate , which included 15 ( 56% , 15/27 ) cancers ( fig . 1 ) , two ( 7% , 2/27 ) high - risk lesions ( lobular carcinoma in situ and atypical ductal hyperplasia in each one ) and ten ( 37% , 10/27 ) benign lesions ( fig . 2 ) . of the 11 lesions without a us correlate , three ( 27% , 3/11 ) proved to be cancerous ( fig . 3 ) and five ( 45% , 5/11 ) were benign as determined by mastectomy or breast conserving surgery with additional excision ( fig . 4 ) , however , three ( 27% , 3/11 ) were not confirmed by histology . the cancer rate was statistically higher in lesions with a us correlate than in ones without a us correlate ( p = 0.028 ) ( table 2 ) . of 15 cancers with a us correlate us , ten ( 67% ) were confirmed as invasive ductal carcinoma , and five ( 33% ) as ductal carcinoma in situ . of the three cancers without a us correlate , two ( 67% ) were ductal carcinoma in situ , and one ( 33% ) as an invasive ductal carcinoma . the difference between invasive and in situ carcinoma was not statistically significant ( p = 0.528 ) . of us correlate lesions identified by histology , the high - risk lesions included lobular carcinoma in situ and atypical ductal hyperplasia for each one . the benign lesions were diagnosed as papilloma in three , fibroadenoma , stromal fibrosis , fibrocystic disease in two each , and columnar cell change in one . imaging findings and pathological results of us correlate lesions are summarized in table 3 . of 31 patients who underwent targeted us , the surgical plan was altered in 27 patients ( 87% ) and not in four patients . targeted us justified a change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) into an unnecessary surgical excision . of 21 patients with us correlates , the surgical treatment plan for 19 patients was altered preoperatively with the consent of the patient . these 19 patients included 14 with additional cancers , two with high - risk lesions and three with benign lesions ( one fibroadenoma and two intraductal papillomas ) . even though the us - guided core biopsy for three patients confirmed benign lesions , these patients underwent conserving surgery and a wider excision , because the surgeon did not want to defer the operation because of a personal concern of the patient or delayed pathological results with the consensus of the patient . of the ten patients without us correlates , surgical treatment was changed in eight patients . however , three patients for whom cancers were confirmed subsequently underwent mastectomy at the discretion of surgeon ( two ) , and conservative surgery with a wide excision during the operation ( one ) . five patients underwent unnecessary wide excision due to the difficulty in localizing the additional lesions . in 13 ( 76% ) of 17 patients ( 14 with us correlates and 3 without us correlates ) in whom mri detected additional cancers , the surgery was converted to mastectomy in ten or additional contralateral conserving surgery in three . of the 149 total patients for whom mri was used for preoperative evaluation of breast cancer , 69 additional suspicious breast lesions were detected by mri in 62 patients ( 42% ) . of those 69 lesions , 26 ( 38% ) were confirmed as cancers by histology . by mri , 48 of 69 lesions ( 70% ) revealed a mass , and 21 ( 30% ) demonstrated nonmass - like enhancements and the average diameter of the 69 additional enhancing lesions in these 62 patients was 1.1 cm ( range , 0.4 - 5.0 cm ) . in addition , of these 62 patients with 69 lesions , 57 ( 92% ) had a single additional lesion , four ( 6% ) had two lesions , and one ( 2% ) had four lesions . among 69 lesions , contralateral enhancing lesions were detected in ten patients ( 16% ) , of which confirmed cancers in three patients ( 5% ) . of the 62 patients with additional lesions , 31 patients with 38 lesions underwent targeted us . histological findings of these 38 lesions in 31 patients performed with targeted us revealed cancers in 18 ( 47% ) lesions , high - risk lesions in two ( 5% ) , benign lesions in 15 ( 39% ) , and unidentified lesions in three ( 8% ) ( table 1 ) . the latter three lesions that were not confirmed by histological examination were removed by a mastectomy in one patient and breast conservative surgery with a wide extent in two patients . of the 38 lesions examined with targeted us , 27 ( 71% ) had a us correlate , which included 15 ( 56% , 15/27 ) cancers ( fig . 1 ) , two ( 7% , 2/27 ) high - risk lesions ( lobular carcinoma in situ and atypical ductal hyperplasia in each one ) and ten ( 37% , 10/27 ) benign lesions ( fig . 2 ) . of the 11 lesions without a us correlate , three ( 27% , 3/11 ) proved to be cancerous ( fig . 3 ) and five ( 45% , 5/11 ) were benign as determined by mastectomy or breast conserving surgery with additional excision ( fig . 4 ) , however , three ( 27% , 3/11 ) were not confirmed by histology . the cancer rate was statistically higher in lesions with a us correlate than in ones without a us correlate ( p = 0.028 ) ( table 2 ) . of 15 cancers with a us correlate us , ten ( 67% ) were confirmed as invasive ductal carcinoma , and five ( 33% ) as ductal carcinoma in situ . of the three cancers without a us correlate , two ( 67% ) were ductal carcinoma in situ , and one ( 33% ) as an invasive ductal carcinoma . the difference between invasive and in situ carcinoma was not statistically significant ( p = 0.528 ) . of us correlate lesions identified by histology , the high - risk lesions included lobular carcinoma in situ and atypical ductal hyperplasia for each one . the benign lesions were diagnosed as papilloma in three , fibroadenoma , stromal fibrosis , fibrocystic disease in two each , and columnar cell change in one . of 31 patients who underwent targeted us , the surgical plan was altered in 27 patients ( 87% ) and not in four patients . targeted us justified a change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) into an unnecessary surgical excision . of 21 patients with us correlates , the surgical treatment plan for 19 patients was altered preoperatively with the consent of the patient . these 19 patients included 14 with additional cancers , two with high - risk lesions and three with benign lesions ( one fibroadenoma and two intraductal papillomas ) . even though the us - guided core biopsy for three patients confirmed benign lesions , these patients underwent conserving surgery and a wider excision , because the surgeon did not want to defer the operation because of a personal concern of the patient or delayed pathological results with the consensus of the patient . of the ten patients without us correlates , surgical treatment was changed in eight patients . however , three patients for whom cancers were confirmed subsequently underwent mastectomy at the discretion of surgeon ( two ) , and conservative surgery with a wide excision during the operation ( one ) . five patients underwent unnecessary wide excision due to the difficulty in localizing the additional lesions . in 13 ( 76% ) of 17 patients ( 14 with us correlates and 3 without us correlates ) in whom mri detected additional cancers , the surgery was converted to mastectomy in ten or additional contralateral conserving surgery in three . breast mri is generally accepted as the preferred diagnostic method for patients with breast cancer because of its high sensitivity ( 10 - 12 ) . in contrast , the specificity of breast mr images remains highly variable ( 37 - 100% ) ( 10 , 11 ) . in situations where there is a high probability of cancer such as preoperative staging , use of mri the detection of additional lesions by mri in breast cancer patients was frequent ( 42% ) . among the 69 additional lesions detected by mri , 26 ( 38% ) were shown to be cancers following a histological examination . because of the need to biopsy and localize these lesions initially seen only on mri , and until recently , the lack of biopsy systems that were mr compatible and commercially available , we designed an alternative method . if a lesion that is detected only on mri can be found on us through careful re - examination , the visualization of the lesion on us will enable a us - guided intervention by a skilled radiologist and allow the patients to possibly avoid a multi - step surgery . some investigators have previously reported on the reliability of targeted ultrasound for suspicious mri - detected lesions ( 13 - 15 ) . because targeted us might be complementary to a subjective us by referencing mr imaging , this technical procedure is more helpful in reducing the number of missed cancers than using us alone or using us before mri . in our study , targeted us found 71% of the additional lesions seen only on mr in breast cancer patients . in comparison to a previous report where 23% of the cases were detected , we suggest the basis for these differing results include the selection of patients that had a high probability of cancer , the more frequent use of bilateral whole breast ultrasound due to unfamiliarity with a skillful mr - guided biopsy technique , and an advantage in lesion detection due to the relatively smaller - sized breasts of asian women included in the study that allowed routine use of a smaller film size ( eg . biopsy or localization was successfully completed in all patients with a us correlate , and 15 ( 71% ) of these patients had additional sites of cancer subsequently identified by mri . of these 31 patients who underwent targeted us , carcinoma was found in 56% of patients that had a us correlate ; in comparison , cancer was identified in 27% of patients lacking a us correlate . similar to a previous study , a us correlate was more often observed for cancers in the present study ( 3 ) . twenty lesions ( 74% ) out of 27 with a us correlate were detected as a mass ( or small nodule ) on mri and were observed more often for invasive forms ( 67% ) than for in situ forms ( 33% ) ( p = 0.528 ) . additional lesions that are verified by mri may lead to either a justified surgical excision or over - treatment . in a previous study on preoperative mri , planned surgical management was altered in 26% of the cases ( 16 ) . in our study , we demonstrated that the targeted us results altered subsequent surgical management from what had been planned initially for 27 ( 87% ) of all 31 patients who underwent targeted us . targeted us in addition to mri seems to contribute to an alteration in the surgical plan . targeted us justified the change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) to unnecessary surgical excision . the five over - treated patients who underwent additional treatment had lesions without a us correlate . a retrospective study such as this has deficiencies . first , histological confirmation was absent or unavailable in some instances , because of the inability to conduct mri - guided percutaneous biopsy . thus , excision of lesions without a us correlate sometimes resulted in sacrificing large amounts of normal tissue to examine an mr - suspicious area . therefore , we speculate that an mr - guided biopsy provides the unique ability to identify these lesions that are not visualized by us ( 17 , 18 ) . second , although us in this study was performed by four skilled breast imaging radiologists and carefully correlated with additional imaging studies , us interpretation is highly dependent upon both the operator and the equipment . as for the us findings in our study , radiologists tend to search for a sonographic hypoechoic lesion in order for it to be considered as suspicious . there may be a chance of missing the additional lesion that would be seen as a hyperechoic or isoechoic lesion by targeted us . it has been our experience that if a suspicious lesion in patients with breast cancer is additionally depicted only on mri , re - examination with another modality , specifically ultrasound , can improve the accuracy of identification and characterization of the lesion , thereby increasing confidence in the application of an intervention . however , our results also highlighted the need of mr - guided biopsies in some cases . in conclusion , targeted us can play a useful role in the evaluation of additional suspicious mr lesions in breast cancer patients , but is limited in lesions without a us correlate .

objectiveto investigate the usefulness of targeted ultrasound ( us ) in the identification of additional suspicious lesions found by magnetic resonance ( mr ) imaging in breast cancer patients and the changes in treatment based on the identification of the lesions by the use of targeted us.materials and methodsone - hundred forty nine patients who underwent breast mr imaging for a preoperative evaluation of breast cancer between january 2002 and july 2004 were included in the study . we searched all cases for any additional lesions that were found initially by mr imaging and investigated the performance of targeted us in identifying the lesions . we also investigated their pathological outcomes and changes in treatment as a result of lesion identification.resultsof the 149 patients with breast cancer , additional suspicious lesions were detected with mr imaging in 62 patients ( 42% ) . of the 69 additional lesions found in those 62 patients , 26 ( 38% ) were confirmed as cancers by histology . thirty - eight lesions in 31 patients were examined with targeted us and were histologically revealed as cancers in 18 ( 47% ) , high risk lesions in two ( 5% ) , benign lesions in 15 ( 39% ) , and unidentified lesions in three ( 8% ) . the cancer rate was statistically higher in lesions with a us correlate than in lesions without a us correlate ( p = 0.028 ) . of 31 patients , the surgical plan was altered in 27 ( 87% ) . the use of targeted us justified a change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) into having an unnecessary surgical excision.conclusiontargeted us can play a useful role in the evaluation of additional suspicious lesions detected by mr imaging in breast cancer patients , but is limited in lesions without a us correlate .

MATERIALS AND METHODS Patients and Lesions Breast MRI Technique Breast US and US-Guided Intervention Technique RESULTS Patients and Lesions Pathological Outcomes of Additional Lesions Evaluated with Targeted US Changes in Surgical Management as a Result of Targeted US DISCUSSION

from january 2002 to july 2004 , 303 women underwent breast mri examinations were initially considered for this study . the primary inclusion criterion of this retrospective analysis was a preoperative mri in patients with histologically confirmed breast cancer . of these patients , 61 who underwent neoadjuvant chemotherapy were excluded because of a possible chemotherapeutic effect causing different kinetics and morphology of suspicious lesions ( 5 ) . the average age of the patients in the present study was 50 years ( range 23 to 79 years ) . indications for an mr examination included preoperative staging before planned breast conserving therapy to exclude multicentricity in patients who had one mammographic or sonographic suspicious lesion ( n = 123 ) , a search for an occult tumor with axillary metastases of suspected breast origin ( n = 5 ) and a postoperative examination to rule out residual disease ( n = 21 ) . a review of the mr images and final reports by an experienced radiologist specialized in breast imaging was conducted . we searched the cases with additional suspicious breast lesions that were initially detected by mri . by the definition of liberman ( 6 ) , mr lesions were considered to be additional sites if they were located in a different breast quadrant than the index cancer , if they were in the same quadrant but were separated from the index cancer by at least 1.0 cm of intervening normal - appearing tissue on mri , or if they were in the same quadrant and contiguous with the index cancer but extended at least 4.0 cm beyond the site of the index cancer . if mri revealed additional suspicious breast lesions other than the index cancer , a targeted us performed by the radiologist who interpreted the mr images was recommended to look for a us correlates amenable to further biopsy or localization . although there were no standardized protocols to follow , a targeted us examination by the radiologists who had knowledge of the mri findings was performed for the clinical and mammographical occult lesions . when targeted us was performed , the additional suspicious breast lesions were classified into two groups based on whether or not they had a us correlates . we compared the rate of identifying additional cancers between the lesions with and without a us correlate using the fisher 's exact test . the 31 patients that did not receive targeted us consisted of two groups ; one of the groups in whom the breast us was performed before the breast mr imaging and did not correlate with any additional detectable lesion by mri and the other group with mammographic microcalcifications , which were not detected initially and did correlate with the mri lesions . these lesions were confirmed by surgical excision or were followed with a subsequent examination . section thickness was held between 1.0 - 2.0 mm without an intersection gap with a 256 192 matrix , an 18 - 24 cm field of view , and a scan time of 3 - 4 minutes . at the time of breast us thirty - eight lesions in 31 patients were performed with targeted us . of 21 patients with correlated suspicious enhancing lesions that were evident on us , eight underwent us - guided core biopsy for the suspicious lesion and 10 underwent us - guided localization , one with us - guided hook - wire localization and the remaining nine with us - guided carbon marking ( 9 ) . in the residual three patients , two patients had a scheduled mastectomy due to the extensive extent of an index cancer and one had breast conserving surgery because of the lesion localized 1 cm just inferior to the index cancer within the same quadrant . in the 10 patients without a us of the four that underwent a us - guided localization , us - guided needle localization was used in one and carbon marking in three . as the lesions without a us correlate could not be localized , careful histological examinations were requested at the area of any additional mr lesions . from january 2002 to july 2004 , 303 women underwent breast mri examinations were initially considered for this study . the primary inclusion criterion of this retrospective analysis was a preoperative mri in patients with histologically confirmed breast cancer . of these patients , 61 who underwent neoadjuvant chemotherapy were excluded because of a possible chemotherapeutic effect causing different kinetics and morphology of suspicious lesions ( 5 ) . the average age of the patients in the present study was 50 years ( range 23 to 79 years ) . indications for an mr examination included preoperative staging before planned breast conserving therapy to exclude multicentricity in patients who had one mammographic or sonographic suspicious lesion ( n = 123 ) , a search for an occult tumor with axillary metastases of suspected breast origin ( n = 5 ) and a postoperative examination to rule out residual disease ( n = 21 ) . a review of the mr images and final reports by an experienced radiologist specialized in breast imaging was conducted . we searched the cases with additional suspicious breast lesions that were initially detected by mri . by the definition of liberman ( 6 ) , mr lesions were considered to be additional sites if they were located in a different breast quadrant than the index cancer , if they were in the same quadrant but were separated from the index cancer by at least 1.0 cm of intervening normal - appearing tissue on mri , or if they were in the same quadrant and contiguous with the index cancer but extended at least 4.0 cm beyond the site of the index cancer . if mri revealed additional suspicious breast lesions other than the index cancer , a targeted us performed by the radiologist who interpreted the mr images was recommended to look for a us correlates amenable to further biopsy or localization . although there were no standardized protocols to follow , a targeted us examination by the radiologists who had knowledge of the mri findings was performed for the clinical and mammographical occult lesions . when targeted us was performed , the additional suspicious breast lesions were classified into two groups based on whether or not they had a us correlates . we compared the rate of identifying additional cancers between the lesions with and without a us correlate using the fisher 's exact test . the 31 patients that did not receive targeted us consisted of two groups ; one of the groups in whom the breast us was performed before the breast mr imaging and did not correlate with any additional detectable lesion by mri and the other group with mammographic microcalcifications , which were not detected initially and did correlate with the mri lesions . these lesions were confirmed by surgical excision or were followed with a subsequent examination . breast mri was performed with the patient prone in a 1.5-t commercially available imager ( signa ; ge medical system , milwaukee , wi ) with the use of a dedicated surface breast coil . of 21 patients with correlated suspicious enhancing lesions that were evident on us , eight underwent us - guided core biopsy for the suspicious lesion and 10 underwent us - guided localization , one with us - guided hook - wire localization and the remaining nine with us - guided carbon marking ( 9 ) . in the residual three patients , two patients had a scheduled mastectomy due to the extensive extent of an index cancer and one had breast conserving surgery because of the lesion localized 1 cm just inferior to the index cancer within the same quadrant . in the 10 patients without a us correlate , the lesions in six patients could not be localized . of the four that underwent a us - guided localization , us - guided needle localization was used in one and carbon marking in three . as the lesions without a us correlate could not be localized , careful histological examinations were requested at the area of any additional mr lesions . of the 149 total patients for whom mri was used for preoperative evaluation of breast cancer , 69 additional suspicious breast lesions were detected by mri in 62 patients ( 42% ) . of those 69 lesions , 26 ( 38% ) were confirmed as cancers by histology . by mri , 48 of 69 lesions ( 70% ) revealed a mass , and 21 ( 30% ) demonstrated nonmass - like enhancements and the average diameter of the 69 additional enhancing lesions in these 62 patients was 1.1 cm ( range , 0.4 - 5.0 cm ) . in addition , of these 62 patients with 69 lesions , 57 ( 92% ) had a single additional lesion , four ( 6% ) had two lesions , and one ( 2% ) had four lesions . among 69 lesions , contralateral enhancing lesions were detected in ten patients ( 16% ) , of which confirmed cancers in three patients ( 5% ) . of the 62 patients with additional lesions , 31 patients with 38 lesions underwent targeted us . histological findings of these 38 lesions in 31 patients performed with targeted us revealed cancers in 18 ( 47% ) lesions , high - risk lesions in two ( 5% ) , benign lesions in 15 ( 39% ) , and unidentified lesions in three ( 8% ) ( table 1 ) . the latter three lesions that were not confirmed by histological examination were removed by a mastectomy in one patient and breast conservative surgery with a wide extent in two patients . of the 38 lesions examined with targeted us , 27 ( 71% ) had a us correlate , which included 15 ( 56% , 15/27 ) cancers ( fig . 1 ) , two ( 7% , 2/27 ) high - risk lesions ( lobular carcinoma in situ and atypical ductal hyperplasia in each one ) and ten ( 37% , 10/27 ) benign lesions ( fig . of the 11 lesions without a us correlate , three ( 27% , 3/11 ) proved to be cancerous ( fig . 4 ) , however , three ( 27% , 3/11 ) were not confirmed by histology . the cancer rate was statistically higher in lesions with a us correlate than in ones without a us correlate ( p = 0.028 ) ( table 2 ) . of 15 cancers with a us correlate us , ten ( 67% ) were confirmed as invasive ductal carcinoma , and five ( 33% ) as ductal carcinoma in situ . of the three cancers without a us correlate , two ( 67% ) were ductal carcinoma in situ , and one ( 33% ) as an invasive ductal carcinoma . the difference between invasive and in situ carcinoma was not statistically significant ( p = 0.528 ) . of us correlate lesions identified by histology , the high - risk lesions included lobular carcinoma in situ and atypical ductal hyperplasia for each one . the benign lesions were diagnosed as papilloma in three , fibroadenoma , stromal fibrosis , fibrocystic disease in two each , and columnar cell change in one . of 31 patients who underwent targeted us , the surgical plan was altered in 27 patients ( 87% ) and not in four patients . targeted us justified a change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) into an unnecessary surgical excision . of 21 patients with us correlates , the surgical treatment plan for 19 patients was altered preoperatively with the consent of the patient . however , three patients for whom cancers were confirmed subsequently underwent mastectomy at the discretion of surgeon ( two ) , and conservative surgery with a wide excision during the operation ( one ) . five patients underwent unnecessary wide excision due to the difficulty in localizing the additional lesions . in 13 ( 76% ) of 17 patients ( 14 with us correlates and 3 without us correlates ) in whom mri detected additional cancers , the surgery was converted to mastectomy in ten or additional contralateral conserving surgery in three . of the 149 total patients for whom mri was used for preoperative evaluation of breast cancer , 69 additional suspicious breast lesions were detected by mri in 62 patients ( 42% ) . of those 69 lesions , 26 ( 38% ) were confirmed as cancers by histology . by mri , 48 of 69 lesions ( 70% ) revealed a mass , and 21 ( 30% ) demonstrated nonmass - like enhancements and the average diameter of the 69 additional enhancing lesions in these 62 patients was 1.1 cm ( range , 0.4 - 5.0 cm ) . in addition , of these 62 patients with 69 lesions , 57 ( 92% ) had a single additional lesion , four ( 6% ) had two lesions , and one ( 2% ) had four lesions . among 69 lesions , contralateral enhancing lesions were detected in ten patients ( 16% ) , of which confirmed cancers in three patients ( 5% ) . of the 62 patients with additional lesions , 31 patients with 38 lesions underwent targeted us . histological findings of these 38 lesions in 31 patients performed with targeted us revealed cancers in 18 ( 47% ) lesions , high - risk lesions in two ( 5% ) , benign lesions in 15 ( 39% ) , and unidentified lesions in three ( 8% ) ( table 1 ) . the latter three lesions that were not confirmed by histological examination were removed by a mastectomy in one patient and breast conservative surgery with a wide extent in two patients . of the 38 lesions examined with targeted us , 27 ( 71% ) had a us correlate , which included 15 ( 56% , 15/27 ) cancers ( fig . 1 ) , two ( 7% , 2/27 ) high - risk lesions ( lobular carcinoma in situ and atypical ductal hyperplasia in each one ) and ten ( 37% , 10/27 ) benign lesions ( fig . of the 11 lesions without a us correlate , three ( 27% , 3/11 ) proved to be cancerous ( fig . 3 ) and five ( 45% , 5/11 ) were benign as determined by mastectomy or breast conserving surgery with additional excision ( fig . 4 ) , however , three ( 27% , 3/11 ) were not confirmed by histology . the cancer rate was statistically higher in lesions with a us correlate than in ones without a us correlate ( p = 0.028 ) ( table 2 ) . of 15 cancers with a us correlate us , ten ( 67% ) were confirmed as invasive ductal carcinoma , and five ( 33% ) as ductal carcinoma in situ . of the three cancers without a us correlate , two ( 67% ) were ductal carcinoma in situ , and one ( 33% ) as an invasive ductal carcinoma . the difference between invasive and in situ carcinoma was not statistically significant ( p = 0.528 ) . of us correlate lesions identified by histology , the high - risk lesions included lobular carcinoma in situ and atypical ductal hyperplasia for each one . the benign lesions were diagnosed as papilloma in three , fibroadenoma , stromal fibrosis , fibrocystic disease in two each , and columnar cell change in one . of 31 patients who underwent targeted us , the surgical plan was altered in 27 patients ( 87% ) and not in four patients . targeted us justified a change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) into an unnecessary surgical excision . of 21 patients with us correlates , the surgical treatment plan for 19 patients was altered preoperatively with the consent of the patient . even though the us - guided core biopsy for three patients confirmed benign lesions , these patients underwent conserving surgery and a wider excision , because the surgeon did not want to defer the operation because of a personal concern of the patient or delayed pathological results with the consensus of the patient . however , three patients for whom cancers were confirmed subsequently underwent mastectomy at the discretion of surgeon ( two ) , and conservative surgery with a wide excision during the operation ( one ) . in 13 ( 76% ) of 17 patients ( 14 with us correlates and 3 without us correlates ) in whom mri detected additional cancers , the surgery was converted to mastectomy in ten or additional contralateral conserving surgery in three . breast mri is generally accepted as the preferred diagnostic method for patients with breast cancer because of its high sensitivity ( 10 - 12 ) . in contrast , the specificity of breast mr images remains highly variable ( 37 - 100% ) ( 10 , 11 ) . in situations where there is a high probability of cancer such as preoperative staging , use of mri the detection of additional lesions by mri in breast cancer patients was frequent ( 42% ) . among the 69 additional lesions detected by mri , 26 ( 38% ) were shown to be cancers following a histological examination . because of the need to biopsy and localize these lesions initially seen only on mri , and until recently , the lack of biopsy systems that were mr compatible and commercially available , we designed an alternative method . if a lesion that is detected only on mri can be found on us through careful re - examination , the visualization of the lesion on us will enable a us - guided intervention by a skilled radiologist and allow the patients to possibly avoid a multi - step surgery . some investigators have previously reported on the reliability of targeted ultrasound for suspicious mri - detected lesions ( 13 - 15 ) . in our study , targeted us found 71% of the additional lesions seen only on mr in breast cancer patients . in comparison to a previous report where 23% of the cases were detected , we suggest the basis for these differing results include the selection of patients that had a high probability of cancer , the more frequent use of bilateral whole breast ultrasound due to unfamiliarity with a skillful mr - guided biopsy technique , and an advantage in lesion detection due to the relatively smaller - sized breasts of asian women included in the study that allowed routine use of a smaller film size ( eg . biopsy or localization was successfully completed in all patients with a us correlate , and 15 ( 71% ) of these patients had additional sites of cancer subsequently identified by mri . of these 31 patients who underwent targeted us , carcinoma was found in 56% of patients that had a us correlate ; in comparison , cancer was identified in 27% of patients lacking a us correlate . similar to a previous study , a us correlate was more often observed for cancers in the present study ( 3 ) . twenty lesions ( 74% ) out of 27 with a us correlate were detected as a mass ( or small nodule ) on mri and were observed more often for invasive forms ( 67% ) than for in situ forms ( 33% ) ( p = 0.528 ) . additional lesions that are verified by mri may lead to either a justified surgical excision or over - treatment . in a previous study on preoperative mri , planned surgical management was altered in 26% of the cases ( 16 ) . in our study , we demonstrated that the targeted us results altered subsequent surgical management from what had been planned initially for 27 ( 87% ) of all 31 patients who underwent targeted us . targeted us in addition to mri seems to contribute to an alteration in the surgical plan . targeted us justified the change in treatment for 22 patients ( 81% ) and misled five patients ( 19% ) to unnecessary surgical excision . the five over - treated patients who underwent additional treatment had lesions without a us correlate . thus , excision of lesions without a us correlate sometimes resulted in sacrificing large amounts of normal tissue to examine an mr - suspicious area . second , although us in this study was performed by four skilled breast imaging radiologists and carefully correlated with additional imaging studies , us interpretation is highly dependent upon both the operator and the equipment . there may be a chance of missing the additional lesion that would be seen as a hyperechoic or isoechoic lesion by targeted us . it has been our experience that if a suspicious lesion in patients with breast cancer is additionally depicted only on mri , re - examination with another modality , specifically ultrasound , can improve the accuracy of identification and characterization of the lesion , thereby increasing confidence in the application of an intervention . in conclusion , targeted us can play a useful role in the evaluation of additional suspicious mr lesions in breast cancer patients , but is limited in lesions without a us correlate .

dendritic cells ( dcs ) are professional antigen - presenting cells that possess the unique capacity to activate and prime naive cd4 and cd8 t cells . they form a heterogeneous population consisting of specialized dc subsets that differ in their surface marker expression , molecular phenotype , and antigen - processing and antigen - presentation capacity [ 24 ] . in peripheral blood , at least two major types of dcs can be distinguished , namely , myeloid dcs ( mdcs ) and plasmacytoid dcs ( pdcs ) [ 5 , 6 ] . myeloid dcs express high levels of cd11c and can further be subdivided based on the differential expression of either cd1c ( blood dendritic cell antigen 1 = bdca1 ) or cd141 ( bdca3 ) . each dc subset has its own repertoire of toll - like receptors ( tlrs ) , underlining their functional specialization [ 3 , 7 ] . both mdc subsets express tlr3 and tlr8 among others , although expression levels of tlr3 are much higher in cd141 mdcs . plasmacytoid dcs are key effectors of innate immune responses due to their capacity to produce large amounts of type i ifns in response to bacterial or viral infections ; this production can also be induced by tlr agonists such as r848 and oligodeoxynucleotides class c ( cpg ) [ 8 , 9 ] . besides their role in the innate immune system , pdcs also participate in priming t helper ( th ) cells , depending on the stimulus they receive ( summarized in ) . myeloid dcs , on the other hand , have the capacity to produce the th1 skewing cytokine interleukin- ( il- ) 12 . for both pdcs and mdcs , it has been shown that they induce proliferation in an allogeneic setting and that they can cross - present exogenous antigens to prime cd8 t cells [ 1016 ] . as a result of their unique capacity to orchestrate adaptive immune responses , recently , more advanced examination of primary blood dcs has come within reach through the availability of efficient isolation techniques . primary dcs are hypothesized to be stronger inducers of anticancer responses than monocyte derived dcs in cell - based vaccination strategies since they differentiate in vivo and require only short ex vivo handling . the first clinical studies utilizing primary blood dcs have recently been conducted by our group , demonstrating the safety and efficacy of cd1c mdcs and pdcs in cancer immunotherapy [ 17 , 18 ] . in order for dc - based immunotherapy to elicit potent antitumor t cell responses , the administered dcs need to raise an immune - stimulatory rather than tolerogenic t cell response . naive t cells will proliferate upon encounter with antigen - presenting cells presenting their specific antigen in the presence of costimulatory signals . the nature of costimulation and cytokines from the dc will influence the polarization of the t cells into different t helper phenotypes such as th1 , th2 , and th17 or regulatory t cells ( tregs ) . for example , the presence of il-12 promotes the induction of th1 cells , whereas il-10 inhibits induction of th1 cells and promotes the differentiation of tregs [ 20 , 21 ] . in antiviral responses , th1 cells and antigen - specific cytotoxic cd8 t cells this type of immune response is also highly desirable in antitumor strategies , in which the aim is to eradicate tumor cells . toll - like receptor ligands such as polyinosinic : polycytidylic acid ( polyi : c ) , r848 , and cpg have been shown to possess th1 polarizing capacity when used as adjuvants or maturation agents for dcs [ 2226 ] . to be able to successfully manipulate t cell responses for therapeutic strategies , a better understanding of the functional specialization of human dc subsets is needed . in this study , we compared the cd4 t cell stimulatory and polarizing capacity of human blood mdcs and pdcs side by side especially the capacity to induce th1 responses upon differential stimulation . human blood dcs were isolated from buffy coats ( sanquin ) obtained from healthy volunteers after written informed consent and according to institutional guidelines . pbmcs were purified via ficoll density gradient centrifugation ( lymphoprep by axis - shield ) . for this , lineage positive cells were depleted from pbmcs either with dynabeads human dc enrichment kit ( invitrogen by life technologies ) or with anti - fitc microbeads ( miltenyi biotec ) after incubation with fitc - conjugated anti - lin1 antibody cocktail ( cd3cd14cd16cd19cd20cd56 ) ( bd biosciences ) . the remaining cells were labeled with fitc - conjugated anti - lin1 antibody cocktail ( bd biosciences ) , pe - cy7-conjugated anti - hla - dr ( bd biosciences ) , bv421-conjugated anti - cd1c ( biolegend ) , apc - conjugated anti - cd141 ( miltenyi biotec ) , and pe - conjugated anti - bdca4 ( miltenyi biotec ) . subsets were sorted to obtain cd1c mdcs , cd141 mdcs , or pdcs , respectively ( purity 98100% ) ( see suppl . in some experiments , cd1c mdcs were isolated from pbmcs with a cd1c dc isolation kit ( miltenyi biotec ) . cd141 mdcs and pdcs were isolated from pbls by positive selection with anti - cd141 ( cd141 ) and anti - bdca4 magnetic microbeads , respectively ( miltenyi biotec ) . naive cd4 t cells were isolated from pbls by depleting cd4 cells with macs multisort beads and additional use of pe - conjugated anti - cd45ro ( dako ) and anti - pe beads ( miltenyi biotec ) for the depletion of cd45ro memory t cells ( purity > 95% ) . all cells were cultured in x - vivo 15 medium ( lonza ) supplemented with 2% human serum ( sigma - aldrich ) . the dcs were stimulated with the following tlr ligands : 4 g / ml r848 ( axxora ) , 2 g / ml polyi : c ( sigma ) ( figures 1 and 2 ) or 20 g / ml polyi : c ( enzo life sciences ) ( figures 3 and 4 ) , 450 u / ml gm - csf ( cellgenix ) , or 5 g / ml cpg - c ( designated cpg throughout text ; enzo life sciences ) . for the control condition of pdcs , the medium was supplemented with 10 ng / ml recombinant human il-3 ( cellgenix ) to ensure pdc survival . cell sorting was performed on a bd aria and flow cytometry on a bd calibur or bd verse . the dc subsets were incubated overnight at 37c with different stimuli in triplicate ( cd1c mdcs , pdcs ) or in duplicate ( cd141 mdcs ) . the next day , supernatants were taken and cells were labeled with pe - conjugated anti - mhc class i and fitc - conjugated anti - mhc class ii ( bd ) , pe - conjugated anti - cd80 ( bd biosciences ) , and pe - conjugated anti - cd86 ( bd biosciences ) . supernatants were analyzed for il-10 ( ebioscience ) and il-12p70 ( m122 and m121b by pierce endogen , standard by bd biosciences ) by standard sandwich elisa . depicted in figure 2 is the cytokine production by 50,000 dcs in a volume of 100 l . for cd141 mdcs , in some instances cd1c mdcs , cd141 mdcs , or pdcs ( 1 10 cells ) were incubated overnight at 37c with different stimuli in triplicate . the next day , allogeneic naive cd4 t cells were added to the dcs at a ratio of 1 : 5 ( dc : t cell ) . proliferative responses were determined by adding 1 ci [ 0.037 mbq]/well of tritiated thymidine ( h ) ( mp biomedicals ) to the cells after three days of coculture . h incorporation over a time course of 16 hours was measured with a scintillation counter . dendritic cells ( 1 10 ) were stimulated overnight with the different stimuli in 100 l medium . next , allogeneic naive cd4 t cells ( 4 10 ) were added at a ratio of 1 : 4 ( dc : t cell ) in a final volume of 200 l medium containing 10 pg / ml superantigen staphylococcus aureus enterotoxin b ( seb ) ( sigma ) . at day 5 , human ril-2 ( 20 iu / ml , novartis ) was added and the cultures expanded for the next 68 days . on days 1113 , resting t cells were counted and analyzed by flow cytometry with three panels . panel 1 includes anti - cd25-apc ( bd bioscience ) , anti - cd127-pe ( ebioscience ) , and anti - foxp3-a488 ( ebioscience ) . panel 2 includes anti - t - bet - a488 , anti - gata-3-pe , and anti - rort - apc ( all ebioscience ) . panel 3 includes anti - cd45ro - apc ( bd bioscience ) , anti - cd197 ( r&d systems ) with goat - anti - mouse igg2a - a488 ( life technologies ) , and anti - cd62-l ( ebioscience ) with rat - anti - mouse igg1-pe ( bd pharmingen ) . the population of tregs was determined by selecting cd25 cd127 cells and subsequently gating on the foxp3 population ( suppl . tcm were determined by further gating on cd197/cd62-l and tem were determined by further gating on cd197 cells . both populations are shown as percentage of live cells ( forward - sideward scatter ) ( suppl . furthermore , 5 10 of the t cells of each condition were restimulated with 5 10 anti - cd3/anti - cd28 beads ( dynabeads gibco by life technologies ) in triplicate and supernatants from 24-hour cultures were analyzed for levels of ifn- ( pierce endogen ) , il-5 , and il-10 ( ebioscience ) by standard sandwich elisa . data were analyzed by kruskal - wallis test followed by dunn 's testing , by a 1-way anova followed by tukey testing or with paired student 's t - test using prism5 ( graphpad prism5 ) . statistical significance was defined as < 0.05 ( p < .05 ; p < .01 ; p < .001 ) . high expression of mhc molecules is a hallmark of dcs , underlining their antigen - presenting capacities . accordingly , we found high levels of both mhc class i and mhc class ii molecules on all three dc subsets ( figure 1(a ) ) . the levels of both mhc class i and mhc class ii molecules were highest for cd141 mdcs and comparable for cd1c mdcs and pdcs , both on freshly isolated cells and after tlr activation . the expression of costimulatory molecules by dcs is essential to activate t cells and can be induced by tlr ligands . throughout the study , cd1c and cd141 mdc maturation cd1c mdcs were also stimulated with granulocyte - macrophage colony - stimulating factor ( gm - csf ) . plasmacytoid dcs were stimulated with r848 or cpg and il-3 used for the control to secure pdc survival . on cd1c mdcs , the costimulatory molecule cd86 was already highly expressed after overnight culture in medium alone ; on cd141 mdcs , this holds true for the expression of both cd80 and cd86 ( figure 1(b ) ) . in comparison , cd141 mdcs showed the highest expression of cd80 and cd86 , both after culturing in medium alone or after tlr ligation ( figure 1(b ) ) . although cd80 and cd86 molecules were expressed already at high levels on immature dcs , expression of both molecules was significantly increased upon culture with tlr ligands on all dcs . dendritic cell - derived il-10 is known to inhibit th1 cells and induce type 1 tregs ( tregs producing il-10 ) , whereas il-12 is a th1-inducing cytokine and therefore desirable in the context of anticancer therapy [ 20 , 21 ] . plasmacytoid dcs did not secrete il-10 or il-12 at detectable levels , whereas cd1c and cd141 mdcs secreted both il-10 and il-12 at differential levels depending on the stimulus ( figure 2 ) . r848 and polyi : c , alone or in combination , induced il-10 production in cd1c mdcs , while only the combination of both tlr ligands induced a significant increase in the secretion of il-12 . cd141 mdcs secreted low amounts of il-10 , irrespective of the stimulus and at lower levels than cd1c mdcs . we observed a significant increase in il-12 production by cd141 mdcs after stimulation with both polyi : c and r848 , which is higher than the production by cd1c mdcs . we can therefore conclude that cd141 mdcs produce less il-10 and more il-12 than cd1c mdcs . a primary immune response constitutes the activation of naive t cells in response to antigen and their subsequent proliferation and differentiation . besides recognition of their cognate antigen , naive t cells depend on costimulation by the antigen - presenting cell to start such a primary response . the ability of blood dcs to induce proliferation of naive t cells was directly compared by coculturing overnight stimulated pdcs and cd1c and cd141 mdcs of the same donors with allogeneic naive cd4 t cells . all primary blood dc subsets showed the ability to induce proliferation of naive cd4 t cells ( figure 3 ) . even so , r848-matured mdcs induced the highest levels of proliferation , while polyi : c maturation did not further increase proliferation as compared to unstimulated mdcs . for pdcs , r848 and il-3 ( control ) treatment stimulate similar levels of naive cd4 t cell proliferation , while the levels for cpg - treated pdcs tend to be lower than for r848 or il-3 . besides providing effector t cells , a primary immune response can generate immunological memory in the form of memory t cells . while central memory t cells ( tcm ) are responsible for rapid clonal expansion after reexposure to antigen and localize in lymphoid organs , effector memory t cells ( tem ) localize in mucosal tissue and mediate rapid effector functions there . although the formation and longevity of such memory cells can only be accurately measured in vivo , we wanted to get an idea of the individual capacities of the different dc subsets to induce them . for this , we cocultured naive , allogeneic cd4 t cells with the differentially activated blood dc subsets until the t cells had ceased to proliferate . at this resting state ( after ~12 days ) , we analyzed their cd45ro , ccr7 ( cd197 ) , and l - selectin ( cd62l ) expression . the percentages of cd45ro ccr7 ( tem ) and cd45ro l - selectin ccr7 ( tcm ) among the t cells did not differ significantly between the subsets or different stimuli ( suppl . at the time point measured , the t cells comprise a larger proportion of tem ( mean 47.14%71.51% ) than tcm ( mean 13.47%24% ) . taken together , all subsets can effectively induce proliferation of naive t cells and are probably able to induce memory t cells . however , mdcs induce significantly higher proliferation when matured with r848 in comparison to polyi : c maturation or culturing alone . dendritic cells play a critical role in the polarization of naive cd4 t cells into different t helper phenotypes or tregs . in a th1 response , cytotoxic cd8 t cells that are able to kill cells bearing their specific antigen are elicited . therefore , this type of immune response is highly desirable in antitumor strategies . to compare the differential t cell stimulatory and polarizing capacity especially the capacity to induce th1 responses upon differential stimulation with polyi : c , r848 , and cpg and possible treg induction by human blood mdcs and pdcs , naive cd4 t cells were cocultured with the dc subsets until they reached resting state . importantly , analysis of the resting t cells did not show a large fraction of tregs for any dc subset or condition ( mean 3%10% ) ( figure 4(a ) ; suppl . 2a ) . although the differences are small , it is interesting to note that the percentages of these cells were lowest for polyi : c and r848-matured mdcs and were highest for gm - csf - stimulated cd1c mdcs ( mean 3% and 10% , resp . ) . for the pdc cocultures , there is a tendency of a higher proportion of tregs being induced after r848 or cpg stimulation compared to the control ( il-3-treated cells ) ( mean 7% , 7% , and 4% , resp . ) . furthermore , we analyzed the induction of th subset - specifying transcription factors t - bet , gata-3 , and rort ( figure 4(b ) ) . we found pronounced populations expressing t - bet across the subsets and stimuli ( cd1c mdcs : 9%35% , cd141 mdcs : 23%35% , and pdcs : 32%42% ) , indicating th1 polarization by all subsets . cd141 mdcs showed the most pronounced gata-3 expression for control and r848 stimulation ( mean 5.1% and 4.25% , resp . ) , which was significantly reduced with polyi : c or combined r848 and polyi : c stimulation of cd141 mdcs ( mean 1.87% for both ) . furthermore , rort expression was only detected in a very small population of cd4 t cells across the subsets ( 0.08%0.72% ) , indicating th1 rather than th17 polarization of these cells . resting cd4 t cells were also restimulated with anti - cd3/anti - cd28 beads and their supernatants analyzed for il-5 , il-10 , il-17 , and ifn- production to determine the th1 polarization capacity of the dc subsets . interleukin-5 is a th2 cytokine , while il-10 inhibits th1 polarization and ifn- is a strong th1 inducer [ 20 , 21 , 28 ] . notably , coculture with all three blood dc subsets induced t cells with prominent ifn- production after restimulation even without tlr maturation ( figure 4(c ) ) . t cells primed by cd141 or cd1c mdcs induced prominent populations of t - bet expressing cells and secreted high levels of ifn- , indicating th1 skewing ( figure 4(c ) , lower panel ) . gm - csf - stimulated cd1c mdcs induced smaller populations of t - bet expressing cells and lower levels of ifn- and similar levels of both il-5 and il-10 as the medium control or tlr - matured dcs ; therefore , gm - csf maturation of cd1c mdcs does not induce the most potent th1 response . also pdcs induce a prominent population of t - bet expressing cells and ifn- release from restimulated cd4 t cells , although the levels of ifn- are lower than for optimally stimulated mdcs . plasmacytoid dcs induce similar levels of il-5 in cocultured t cells as mdcs . however , the levels of il-10 are higher , especially for r848 and cpg stimulated pdcs , which coincides with a tendency for a bigger proportion of tregs ( cd25 cd127 foxp3 ) induced in these conditions . we measured no il-17 for pdcs and modest levels for mdcs stimulated with r848 or cd1c mdcs stimulated with the combination of r848 and polyi : c ( suppl . 4 ) . together with the rort expression data we conclude a th1 rather than th17 polarization of the nave cd4 t cells . in sum , all subsets polarize naive cd4 t cells mainly towards th1 cells with a strong t - bet signature producing mainly ifn- after restimulation . in order to manipulate t cell responses for dc - based cancer immunotherapy , a better understanding of the functional specialization of human dc subsets is needed . in this study , we compared the capacities of primary human blood mdcs and pdcs to activate and polarize cd4 t cells side by side . we report that cd1c mdcs , cd141 mdcs , and pdcs all induce proliferation of naive cd4 t cells . importantly , naive cd4 t cells are not skewed towards a regulatory phenotype by coculture with either mature mdcs or pdcs . despite differences in activation and cytokine profile , both cd141 and cd1c mdcs polarize naive cd4 t cells towards t cells with a strong ifn- signature ; also pdcs induce ifn- , although at lower levels and accompanied by a higher il-10 production . while all dc subsets mature upon tlr ligation , we observed distinct cytokine responses for different subsets and stimuli . cd1c mdcs produced only a limited amount of il-12 after maturation with either r848 or polyi : c alone , but production was significantly increased with a combination of these tlr ligands . even higher levels of il-12 are produced by cd141 mdcs when stimulated with the combination of polyi : c and r848 . in contrast to our findings , nizzoli et al . did not find il-12 production for cd141 mdcs after combined polyi : c and r848 stimulation . other studies have shown that , in order to induce strong il-12 responses in human and mouse dcs , both an innate trigger such as tlr ligation and a second trigger like ligation of cd40 by cd40l on t cells are needed [ 7 , 30 ] . more recently , it has been shown for cd1c mdcs that the combination of the tlr ligands r848 and lps can trigger significant il-12 production . in the case of cd141 mdcs , a cocktail of polyi : c together with the cytokines ifn- , tnf- , ifn- , and il-1 was shown to induce significant levels of il-12 . showed that cd141 mdcs produced less il-12 as compared to cd1c mdcs for single tlr ligation but that a higher proportion produced il-12 after tlr1/2 or tlr3 ligation in a whole blood assay . our data supports the notion that a single stimulus is not sufficient to induce high il-12 production and with polyi : c and r848 we describe a new combination that can trigger substantial il-12 secretion by both human mdc subsets . all dc subsets induced proliferation of naive cd4 t cells regardless of the stimulus . the level of t cell proliferation induced by polyi : c - matured mdcs is similar to nonstimulated mdcs . strikingly , gm - csf - stimulated cd1c mdcs and r848-matured cd1c and cd141 mdcs cause an extra boost in proliferation of naive cd4 t cells . this is in accordance with an earlier study by jongbloed et al . , which described equally high induction of proliferation of naive cd4 t cells for nonstimulated or polyi : c stimulated cd1c and cd141 mdcs after six days . because of the upregulation of the expression of costimulatory molecules with both stimuli compared to untreated dcs , one would expect a higher proliferation rate than with untreated dcs . certainly , other cytokines and immunostimulatory , but also immunoinhibitory , molecules expressed by cultured dcs are integrated into a single response by the t cells and possible differences in these factors might cause the observed differences in t cell proliferation . only a minor percentage of cd4 t cells that grew out of cocultures with the different dc subsets displayed a treg phenotype . earlier studies suggest that pdcs can act as th1 , th2 , th17 , or even treg inducers in t cell priming , depending on the stimulus they receive ( summarized in ) . one stimulus that can induce this regulatory t cell phenotype is cpg and the proposed mechanism is via the expression of inducible costimulator ligand ( icos - l ) . icos - l upon cpg maturation , which triggers il-10 production of t cells but no production of il-4 , il-5 , or il-13 . this is in accordance with our findings , where we observed higher levels of il-10 and a tendency of a higher proportion of tregs for pdcs matured with cpg or r848 compared to matured mdcs but no elevated levels of il-5 . however , regardless of the stimulus we also found a strong t - bet expression and ifn- production by cd4 t cells that had grown out of cocultures . plasmacytoid dcs secrete large amounts of type i ifns in response to bacterial or viral stimuli , including r848 and cpg . type i ifns not only are important in innate responses , but can also help to skew t cells towards a th1 phenotype . type i ifns secreted by pdcs might play a role in the observed ifn- induction . regulatory t cell induction with functional effects on t cells has been described in one study for tissue mdcs of the skin . we show here that primary blood mdcs induce only a low proportion of tregs and , importantly , the overall cd4 t cell population displays a th1 phenotype after coculture ( pronounced t - bet expression and high ifn- production ) and no th2 or th17 phenotype . this is in line with the ability of mdcs to produce il-12 after combined polyi : c and r848 stimulation . for the other conditions , one can speculate whether the addition of the cd4 t cells and therefore ligation of cd40 on the dcs give the needed second signal for il-12 production and help the th1 skewing . different groups have shown that blood mdcs can induce ifn- production in naive cd4 and cd8 t cells [ 7 , 35 , 36 ] . found that cd141 mdcs were more potent than cd1c dcs at inducing ifn- responses in total cd4 t cells , especially after polyi : c stimulation . we found that although cd1c mdcs show a less mature phenotype than cd141 mdcs including higher il-10 and lower il-12 production , cd1c and cd141 mdcs induce similar ifn- responses after coculture with naive cd4 t cells . however , there is a tendency for cd141 mdcs to induce less il-5 and il-10 than cd1c , also arguing for an overall stronger th1 skewing by this subset . for cd141 mdcs , high tlr3 expression and the ability to produce ifn- and cxcl10 , both known to induce antiviral responses , all suggest their capability to induce th1 skewing in t cells [ 7 , 37 , 38 ] . certainly , r848 and polyi : c can trigger distinct pathways as tlr3 signals through a trif - to - irf3 pathway , rather than an myd88-to - irf7 pathway that is used by tlr8 . it is interesting to note that both mdc subsets react strongly and in a similar way to polyi : c , although the expression levels of tlr3 are much higher in cd141 mdcs than in cd1c mdcs . likely , other receptors for polyi : c contribute to the response in one or both of the mdc subsets . the synthetic dsrna analog is a ligand for multiple pathogen recognition receptors , and besides tlr3 also triggers cytosolic rig - i - like receptors that are expressed by mdcs [ 39 , 40 ] . suggest in a study on mdcs and nk cells that both tlr3 stimulation and rig - i - like receptor ligation are needed for ifn- induction by mdcs . in addition to their cd4 t cell activating capacities , all three subsets can cross - present exogenous antigens for cognate restimulation of previously activated cd8 t cells [ 1014 ] , making them promising candidates for dc - based vaccination strategies against cancer . both cd1c mdcs and pdcs have generated promising results in first clinical studies utilizing these primary blood dc subsets as vaccines [ 17 , 18 ] . these studies support their excellent in vivo functioning and mark them as the next generation of cancer vaccines . in this context , we have learned from the current work that gm - csf is not the optimal stimulus for cd1c mdcs , since gm - csf stimulation showed an overall lower th1 skewing capacity and induced more tregs than other stimuli . while maturation with polyi : c or the combination of polyi : c and r848 induces the most pronounced th1 skewing , the number of t cells that grow out with these stimuli is lower than , for example , with r848 stimulation . considering the proliferation data and the similar polarization capacity by all subsets and with all stimuli including control dcs , one can only speculate about a recommendation for a suited stimulation of dcs for dc - based vaccination strategies . for example , tlr activation of dcs can lead to the upregulation of otherwise unexpressed tlr ligands in the dcs , making them sensitive to a broader range of danger signals . furthermore , in an in vivo situation also other cell types might play a crucial role for the overall outcome of a therapy . such cells include nk and cd8 t cells , for which type i ifns and il-12secreted at higher levels upon tlr maturation are important [ 4346 ] . as discussed above , cd141 mdcs certainly display promising properties for dc - based anticancer vaccination strategies . besides their th1-inducing capacity , human cd141 mdcs are also excellent cross - presenters of exogenous antigens to cd8 t cells . while some publications show superior cross - presentation capacity of cd141 mdcs [ 31 , 4749 ] and put them forward as the human counterparts of mouse cd8 dcs [ 31 , 4750 ] , other studies suggest that the different human dcs subsets bear similar cross - presentation capacities at least for soluble antigens delivered through early endosomes [ 14 , 15 , 51 ] . the type and size of the antigen as well as the compartments they are targeted to probably underlie these differing outcomes ( reviewed in [ 52 , 53 ] ) . in addition to using single subsets for therapeutic approaches , we hypothesize that using a combination of several dc subsets could further increase t cell activating properties , since earlier studies have shown that cell - cell interactions as well as soluble factors can act to cross - activate the different dcs ( summarized in ) . with this comparative study , we have reinforced the establishment of human circulating cd1c mdcs , cd141 mdcs , and pdcs as promising candidates for dc - based immunotherapy in the context of cancer .

dendritic cells ( dcs ) are central players of immune responses ; they become activated upon infection or inflammation and migrate to lymph nodes , where they can initiate an antigen - specific immune response by activating naive t cells . two major types of naturally occurring dcs circulate in peripheral blood , namely , myeloid and plasmacytoid dcs ( pdcs ) . myeloid dcs ( mdcs ) can be subdivided based on the expression of either cd1c or cd141 . these human dc subsets differ in surface marker expression , toll - like receptor ( tlr ) repertoire , and transcriptional profile , suggesting functional differences between them . here , we directly compared the capacity of human blood mdcs and pdcs to activate and polarize cd4 + t cells . cd141 + mdcs show an overall more mature phenotype over cd1c+ mdc and pdcs ; they produce less il-10 and more il-12 than cd1c+ mdcs . despite these differences , all subsets can induce the production of ifn- in naive cd4 + t cells . cd1c+ and cd141 + mdcs especially induce a strong t helper 1 profile . importantly , naive cd4 + t cells are not polarized towards regulatory t cells by any subset . these findings further establish all three human blood dcs despite their differences as promising candidates for immunostimulatory effectors in cancer immunotherapy .

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

dendritic cells ( dcs ) are professional antigen - presenting cells that possess the unique capacity to activate and prime naive cd4 and cd8 t cells . they form a heterogeneous population consisting of specialized dc subsets that differ in their surface marker expression , molecular phenotype , and antigen - processing and antigen - presentation capacity [ 24 ] . in peripheral blood , at least two major types of dcs can be distinguished , namely , myeloid dcs ( mdcs ) and plasmacytoid dcs ( pdcs ) [ 5 , 6 ] . myeloid dcs express high levels of cd11c and can further be subdivided based on the differential expression of either cd1c ( blood dendritic cell antigen 1 = bdca1 ) or cd141 ( bdca3 ) . each dc subset has its own repertoire of toll - like receptors ( tlrs ) , underlining their functional specialization [ 3 , 7 ] . plasmacytoid dcs are key effectors of innate immune responses due to their capacity to produce large amounts of type i ifns in response to bacterial or viral infections ; this production can also be induced by tlr agonists such as r848 and oligodeoxynucleotides class c ( cpg ) [ 8 , 9 ] . besides their role in the innate immune system , pdcs also participate in priming t helper ( th ) cells , depending on the stimulus they receive ( summarized in ) . myeloid dcs , on the other hand , have the capacity to produce the th1 skewing cytokine interleukin- ( il- ) 12 . for both pdcs and mdcs , it has been shown that they induce proliferation in an allogeneic setting and that they can cross - present exogenous antigens to prime cd8 t cells [ 1016 ] . as a result of their unique capacity to orchestrate adaptive immune responses , recently , more advanced examination of primary blood dcs has come within reach through the availability of efficient isolation techniques . the first clinical studies utilizing primary blood dcs have recently been conducted by our group , demonstrating the safety and efficacy of cd1c mdcs and pdcs in cancer immunotherapy [ 17 , 18 ] . naive t cells will proliferate upon encounter with antigen - presenting cells presenting their specific antigen in the presence of costimulatory signals . the nature of costimulation and cytokines from the dc will influence the polarization of the t cells into different t helper phenotypes such as th1 , th2 , and th17 or regulatory t cells ( tregs ) . in antiviral responses , th1 cells and antigen - specific cytotoxic cd8 t cells this type of immune response is also highly desirable in antitumor strategies , in which the aim is to eradicate tumor cells . toll - like receptor ligands such as polyinosinic : polycytidylic acid ( polyi : c ) , r848 , and cpg have been shown to possess th1 polarizing capacity when used as adjuvants or maturation agents for dcs [ 2226 ] . to be able to successfully manipulate t cell responses for therapeutic strategies , a better understanding of the functional specialization of human dc subsets is needed . in this study , we compared the cd4 t cell stimulatory and polarizing capacity of human blood mdcs and pdcs side by side especially the capacity to induce th1 responses upon differential stimulation . human blood dcs were isolated from buffy coats ( sanquin ) obtained from healthy volunteers after written informed consent and according to institutional guidelines . cd141 mdcs and pdcs were isolated from pbls by positive selection with anti - cd141 ( cd141 ) and anti - bdca4 magnetic microbeads , respectively ( miltenyi biotec ) . naive cd4 t cells were isolated from pbls by depleting cd4 cells with macs multisort beads and additional use of pe - conjugated anti - cd45ro ( dako ) and anti - pe beads ( miltenyi biotec ) for the depletion of cd45ro memory t cells ( purity > 95% ) . the dc subsets were incubated overnight at 37c with different stimuli in triplicate ( cd1c mdcs , pdcs ) or in duplicate ( cd141 mdcs ) . the next day , allogeneic naive cd4 t cells were added to the dcs at a ratio of 1 : 5 ( dc : t cell ) . dendritic cells ( 1 10 ) were stimulated overnight with the different stimuli in 100 l medium . next , allogeneic naive cd4 t cells ( 4 10 ) were added at a ratio of 1 : 4 ( dc : t cell ) in a final volume of 200 l medium containing 10 pg / ml superantigen staphylococcus aureus enterotoxin b ( seb ) ( sigma ) . panel 3 includes anti - cd45ro - apc ( bd bioscience ) , anti - cd197 ( r&d systems ) with goat - anti - mouse igg2a - a488 ( life technologies ) , and anti - cd62-l ( ebioscience ) with rat - anti - mouse igg1-pe ( bd pharmingen ) . both populations are shown as percentage of live cells ( forward - sideward scatter ) ( suppl . furthermore , 5 10 of the t cells of each condition were restimulated with 5 10 anti - cd3/anti - cd28 beads ( dynabeads gibco by life technologies ) in triplicate and supernatants from 24-hour cultures were analyzed for levels of ifn- ( pierce endogen ) , il-5 , and il-10 ( ebioscience ) by standard sandwich elisa . high expression of mhc molecules is a hallmark of dcs , underlining their antigen - presenting capacities . accordingly , we found high levels of both mhc class i and mhc class ii molecules on all three dc subsets ( figure 1(a ) ) . the levels of both mhc class i and mhc class ii molecules were highest for cd141 mdcs and comparable for cd1c mdcs and pdcs , both on freshly isolated cells and after tlr activation . the expression of costimulatory molecules by dcs is essential to activate t cells and can be induced by tlr ligands . on cd1c mdcs , the costimulatory molecule cd86 was already highly expressed after overnight culture in medium alone ; on cd141 mdcs , this holds true for the expression of both cd80 and cd86 ( figure 1(b ) ) . although cd80 and cd86 molecules were expressed already at high levels on immature dcs , expression of both molecules was significantly increased upon culture with tlr ligands on all dcs . plasmacytoid dcs did not secrete il-10 or il-12 at detectable levels , whereas cd1c and cd141 mdcs secreted both il-10 and il-12 at differential levels depending on the stimulus ( figure 2 ) . we observed a significant increase in il-12 production by cd141 mdcs after stimulation with both polyi : c and r848 , which is higher than the production by cd1c mdcs . we can therefore conclude that cd141 mdcs produce less il-10 and more il-12 than cd1c mdcs . a primary immune response constitutes the activation of naive t cells in response to antigen and their subsequent proliferation and differentiation . besides recognition of their cognate antigen , naive t cells depend on costimulation by the antigen - presenting cell to start such a primary response . the ability of blood dcs to induce proliferation of naive t cells was directly compared by coculturing overnight stimulated pdcs and cd1c and cd141 mdcs of the same donors with allogeneic naive cd4 t cells . all primary blood dc subsets showed the ability to induce proliferation of naive cd4 t cells ( figure 3 ) . besides providing effector t cells , a primary immune response can generate immunological memory in the form of memory t cells . while central memory t cells ( tcm ) are responsible for rapid clonal expansion after reexposure to antigen and localize in lymphoid organs , effector memory t cells ( tem ) localize in mucosal tissue and mediate rapid effector functions there . although the formation and longevity of such memory cells can only be accurately measured in vivo , we wanted to get an idea of the individual capacities of the different dc subsets to induce them . for this , we cocultured naive , allogeneic cd4 t cells with the differentially activated blood dc subsets until the t cells had ceased to proliferate . at this resting state ( after ~12 days ) , we analyzed their cd45ro , ccr7 ( cd197 ) , and l - selectin ( cd62l ) expression . taken together , all subsets can effectively induce proliferation of naive t cells and are probably able to induce memory t cells . dendritic cells play a critical role in the polarization of naive cd4 t cells into different t helper phenotypes or tregs . therefore , this type of immune response is highly desirable in antitumor strategies . to compare the differential t cell stimulatory and polarizing capacity especially the capacity to induce th1 responses upon differential stimulation with polyi : c , r848 , and cpg and possible treg induction by human blood mdcs and pdcs , naive cd4 t cells were cocultured with the dc subsets until they reached resting state . importantly , analysis of the resting t cells did not show a large fraction of tregs for any dc subset or condition ( mean 3%10% ) ( figure 4(a ) ; suppl . although the differences are small , it is interesting to note that the percentages of these cells were lowest for polyi : c and r848-matured mdcs and were highest for gm - csf - stimulated cd1c mdcs ( mean 3% and 10% , resp . ) furthermore , we analyzed the induction of th subset - specifying transcription factors t - bet , gata-3 , and rort ( figure 4(b ) ) . we found pronounced populations expressing t - bet across the subsets and stimuli ( cd1c mdcs : 9%35% , cd141 mdcs : 23%35% , and pdcs : 32%42% ) , indicating th1 polarization by all subsets . resting cd4 t cells were also restimulated with anti - cd3/anti - cd28 beads and their supernatants analyzed for il-5 , il-10 , il-17 , and ifn- production to determine the th1 polarization capacity of the dc subsets . notably , coculture with all three blood dc subsets induced t cells with prominent ifn- production after restimulation even without tlr maturation ( figure 4(c ) ) . t cells primed by cd141 or cd1c mdcs induced prominent populations of t - bet expressing cells and secreted high levels of ifn- , indicating th1 skewing ( figure 4(c ) , lower panel ) . gm - csf - stimulated cd1c mdcs induced smaller populations of t - bet expressing cells and lower levels of ifn- and similar levels of both il-5 and il-10 as the medium control or tlr - matured dcs ; therefore , gm - csf maturation of cd1c mdcs does not induce the most potent th1 response . also pdcs induce a prominent population of t - bet expressing cells and ifn- release from restimulated cd4 t cells , although the levels of ifn- are lower than for optimally stimulated mdcs . plasmacytoid dcs induce similar levels of il-5 in cocultured t cells as mdcs . together with the rort expression data we conclude a th1 rather than th17 polarization of the nave cd4 t cells . in sum , all subsets polarize naive cd4 t cells mainly towards th1 cells with a strong t - bet signature producing mainly ifn- after restimulation . in order to manipulate t cell responses for dc - based cancer immunotherapy , a better understanding of the functional specialization of human dc subsets is needed . in this study , we compared the capacities of primary human blood mdcs and pdcs to activate and polarize cd4 t cells side by side . we report that cd1c mdcs , cd141 mdcs , and pdcs all induce proliferation of naive cd4 t cells . importantly , naive cd4 t cells are not skewed towards a regulatory phenotype by coculture with either mature mdcs or pdcs . despite differences in activation and cytokine profile , both cd141 and cd1c mdcs polarize naive cd4 t cells towards t cells with a strong ifn- signature ; also pdcs induce ifn- , although at lower levels and accompanied by a higher il-10 production . while all dc subsets mature upon tlr ligation , we observed distinct cytokine responses for different subsets and stimuli . other studies have shown that , in order to induce strong il-12 responses in human and mouse dcs , both an innate trigger such as tlr ligation and a second trigger like ligation of cd40 by cd40l on t cells are needed [ 7 , 30 ] . in the case of cd141 mdcs , a cocktail of polyi : c together with the cytokines ifn- , tnf- , ifn- , and il-1 was shown to induce significant levels of il-12 . all dc subsets induced proliferation of naive cd4 t cells regardless of the stimulus . strikingly , gm - csf - stimulated cd1c mdcs and r848-matured cd1c and cd141 mdcs cause an extra boost in proliferation of naive cd4 t cells . , which described equally high induction of proliferation of naive cd4 t cells for nonstimulated or polyi : c stimulated cd1c and cd141 mdcs after six days . because of the upregulation of the expression of costimulatory molecules with both stimuli compared to untreated dcs , one would expect a higher proliferation rate than with untreated dcs . certainly , other cytokines and immunostimulatory , but also immunoinhibitory , molecules expressed by cultured dcs are integrated into a single response by the t cells and possible differences in these factors might cause the observed differences in t cell proliferation . only a minor percentage of cd4 t cells that grew out of cocultures with the different dc subsets displayed a treg phenotype . earlier studies suggest that pdcs can act as th1 , th2 , th17 , or even treg inducers in t cell priming , depending on the stimulus they receive ( summarized in ) . one stimulus that can induce this regulatory t cell phenotype is cpg and the proposed mechanism is via the expression of inducible costimulator ligand ( icos - l ) . icos - l upon cpg maturation , which triggers il-10 production of t cells but no production of il-4 , il-5 , or il-13 . this is in accordance with our findings , where we observed higher levels of il-10 and a tendency of a higher proportion of tregs for pdcs matured with cpg or r848 compared to matured mdcs but no elevated levels of il-5 . however , regardless of the stimulus we also found a strong t - bet expression and ifn- production by cd4 t cells that had grown out of cocultures . plasmacytoid dcs secrete large amounts of type i ifns in response to bacterial or viral stimuli , including r848 and cpg . type i ifns not only are important in innate responses , but can also help to skew t cells towards a th1 phenotype . regulatory t cell induction with functional effects on t cells has been described in one study for tissue mdcs of the skin . we show here that primary blood mdcs induce only a low proportion of tregs and , importantly , the overall cd4 t cell population displays a th1 phenotype after coculture ( pronounced t - bet expression and high ifn- production ) and no th2 or th17 phenotype . for the other conditions , one can speculate whether the addition of the cd4 t cells and therefore ligation of cd40 on the dcs give the needed second signal for il-12 production and help the th1 skewing . different groups have shown that blood mdcs can induce ifn- production in naive cd4 and cd8 t cells [ 7 , 35 , 36 ] . found that cd141 mdcs were more potent than cd1c dcs at inducing ifn- responses in total cd4 t cells , especially after polyi : c stimulation . we found that although cd1c mdcs show a less mature phenotype than cd141 mdcs including higher il-10 and lower il-12 production , cd1c and cd141 mdcs induce similar ifn- responses after coculture with naive cd4 t cells . however , there is a tendency for cd141 mdcs to induce less il-5 and il-10 than cd1c , also arguing for an overall stronger th1 skewing by this subset . for cd141 mdcs , high tlr3 expression and the ability to produce ifn- and cxcl10 , both known to induce antiviral responses , all suggest their capability to induce th1 skewing in t cells [ 7 , 37 , 38 ] . the synthetic dsrna analog is a ligand for multiple pathogen recognition receptors , and besides tlr3 also triggers cytosolic rig - i - like receptors that are expressed by mdcs [ 39 , 40 ] . suggest in a study on mdcs and nk cells that both tlr3 stimulation and rig - i - like receptor ligation are needed for ifn- induction by mdcs . in addition to their cd4 t cell activating capacities , all three subsets can cross - present exogenous antigens for cognate restimulation of previously activated cd8 t cells [ 1014 ] , making them promising candidates for dc - based vaccination strategies against cancer . both cd1c mdcs and pdcs have generated promising results in first clinical studies utilizing these primary blood dc subsets as vaccines [ 17 , 18 ] . in this context , we have learned from the current work that gm - csf is not the optimal stimulus for cd1c mdcs , since gm - csf stimulation showed an overall lower th1 skewing capacity and induced more tregs than other stimuli . besides their th1-inducing capacity , human cd141 mdcs are also excellent cross - presenters of exogenous antigens to cd8 t cells . while some publications show superior cross - presentation capacity of cd141 mdcs [ 31 , 4749 ] and put them forward as the human counterparts of mouse cd8 dcs [ 31 , 4750 ] , other studies suggest that the different human dcs subsets bear similar cross - presentation capacities at least for soluble antigens delivered through early endosomes [ 14 , 15 , 51 ] . in addition to using single subsets for therapeutic approaches , we hypothesize that using a combination of several dc subsets could further increase t cell activating properties , since earlier studies have shown that cell - cell interactions as well as soluble factors can act to cross - activate the different dcs ( summarized in ) . with this comparative study , we have reinforced the establishment of human circulating cd1c mdcs , cd141 mdcs , and pdcs as promising candidates for dc - based immunotherapy in the context of cancer .

the synthetic ligands for lxr ( gw3965 and t1317 ) , ppar ( gw9544 ) , ppar ( gw742 ) , and ppar ( gw7845 ) were provided by glaxosmithkline . the lxr ligand was used at a concentration of 1 m , whereas the ppar ligands were all used at 100 nm . lps from salmonella typhimurium and poly(i : c ) were purchased from sigma and used at 100 ng / ml and 2.5 mg / ml , respectively . bm - derived macrophages were differentiated in dmem containing 20% fbs and 30% l929-conditioned media for 7 days . cells were harvested from wild - type , ppar , ppar , or ppar mice . the mx cre , pparfl / fl , and ppar mice are all on the c57bl/6 background ( more than nine generations backcrossed ) . the recipient wild - type mice used for the bmt studies were irradiated with 900 rads the night prior to reconstitution . bm cells from recipient mice were harvested and injected into tail veins of the recipient mice . the irradiated mice were kept in sterile cages with autoclaved food and trimethoprim - sulfamethoxazole - treated water for 2 weeks . mice were challenged with a 60% caloric fat diet ( research diets ) for 16 weeks . mice were fasted the night prior to glucose tolerance tests , and glucose levels were monitored after intraperitoneal injections of glucose ( 2 g / kg ; sigma ) . for gavage experiments , mice were gavaged with either vehicle or rosiglitazone ( 30 mg / kg ; cayman chemicals ) for 8 days . wild - type , ppar , and ppar bmt mice were fasted overnight , and blood was collected in heparin tubes . samples were then spun at 8,000 rpm for 5 min to isolate serum . an ultrasensitive mouse insulin elisa kit ( crystal chem , inc . ) total rna was extracted from cells using trizol reagent ( invitrogen ) and was reverse transcribed to obtain cdna ( applied biosystems ) . real - time quantitative pcr assays were performed using an applied biosystems 7900 sequence detector as previously described ( 23 ) . statistical significance was determined using student 's t - test for in vitro assays . a one - way or two - way anova test was used to determine statistical significance for all glucose tolerance tests . the synthetic ligands for lxr ( gw3965 and t1317 ) , ppar ( gw9544 ) , ppar ( gw742 ) , and ppar ( gw7845 ) were provided by glaxosmithkline . the lxr ligand was used at a concentration of 1 m , whereas the ppar ligands were all used at 100 nm . lps from salmonella typhimurium and poly(i : c ) were purchased from sigma and used at 100 ng / ml and 2.5 mg / ml , respectively . bm - derived macrophages were differentiated in dmem containing 20% fbs and 30% l929-conditioned media for 7 days . cells were harvested from wild - type , ppar , ppar , or ppar mice . the mx cre , pparfl / fl , and ppar mice are all on the c57bl/6 background ( more than nine generations backcrossed ) . the recipient wild - type mice used for the bmt studies were irradiated with 900 rads the night prior to reconstitution . bm cells from recipient mice were harvested and injected into tail veins of the recipient mice . the irradiated mice were kept in sterile cages with autoclaved food and trimethoprim - sulfamethoxazole - treated water for 2 weeks . mice were challenged with a 60% caloric fat diet ( research diets ) for 16 weeks . mice were fasted the night prior to glucose tolerance tests , and glucose levels were monitored after intraperitoneal injections of glucose ( 2 g / kg ; sigma ) . for gavage experiments , mice were gavaged with either vehicle or rosiglitazone ( 30 mg / kg ; cayman chemicals ) for 8 days . wild - type , ppar , and ppar bmt mice were fasted overnight , and blood was collected in heparin tubes . samples were then spun at 8,000 rpm for 5 min to isolate serum . an ultrasensitive mouse insulin elisa kit ( crystal chem , inc . ) total rna was extracted from cells using trizol reagent ( invitrogen ) and was reverse transcribed to obtain cdna ( applied biosystems ) . real - time quantitative pcr assays were performed using an applied biosystems 7900 sequence detector as previously described ( 23 ) . statistical significance was determined using student 's t - test for in vitro assays . a one - way or two - way anova test was used to determine statistical significance for all glucose tolerance tests . to investigate the function of macrophage ppar , we used a cre recombinase system to disrupt the ppar gene . specifically , we crossed c57bl/6 mice with loxp sites flanking the second exon of ppar ( pparfl / fl ) with c57bl/6 mice expressing cre recombinase downstream of the / interferon - inducible ( mx ) promoter . cre recombinase activity was initiated by intraperitoneal injection of the mx activator poly ic 3 to 4 weeks after birth . interestingly , injection of poly ic at weaning age caused a permanent disruption of ppar in myeloid precursor cells ; new myeloid cells generated postinjection remain ppar-null for the life of the mouse . typically , we analyzed macrophages from these mice at least 4 weeks after inducing recombination to reduce any potential effects of the poly ic . quantitative real - time pcr confirmed that the mx cre system was extremely efficient , yielding nearly complete ( > 95% ) deletion of ppar exon 2 in vivo ( fig . as expected , induction of ppar target genes ( ap2 , adrp , pgar ) by ppar but not ppar agonists was lost in mx cre ppar macrophages ( fig . induction of the mx promoter effectively disrupts ppar , whereas lysm cre can not efficiently delete ppar exon 2 . the presented data are representative of at least three independent experiments . a : thioglycollate - elicited wild - type and mx cre ppar peritoneal macrophages were treated with ppar or ppar ligands ( gw7845 or gd742 , respectively ; 100 nm each ) overnight . ppar exon 2 could not be detected in ppar cells ( p < 0.005 ) , whereas ppar exon 6 expression was unaffected . known ppar target genes ( ap2 , adrp , and pgar ) were tested to confirm loss of ligand response in ppar macrophages . b : thioglycollate - elicited wild - type and lysm cre ppar peritoneal macrophages were treated with ppar ligand ( gw7845 ; 100 nm ) overnight . ppar exon 2 could still be detected in ppar macrophages , and pgar , a known ppar target gene , could still be induced by ppar ligand in these cells ( p < 0.02 ) . in addition to using the mx cre system , we also generated mice with macrophage - specific ppar deletion using the lysm cre system . unlike mx cre , lysm cre is constitutively active . however , compared with the mx cre system , the lysm cre system was not as efficient in recombining ppar to create a disrupted gene in our hands . after harvesting peritoneal macrophages from wild - type and lysm cre ppar mice , we treated the cells with ppar ligand to test whether disruption of ppar was able to prevent ppar target gene regulation . we found that ppar exon 2 could still be detected by quantitative real - time pcr and that pgar , a ppar target gene , could be regulated by ligand even in ppar macrophages ( fig . in contrast , the mx cre system yields cell populations with complete deletion of ppar exon 2 expression , and more importantly , these cells are unable to respond to ppar ligand and do not regulate target genes . recent work suggests that ppars mediate inflammatory signaling pathways in macrophages and may affect inflammation associated with insulin resistance ( 18 , 22 ) . to address this issue in our genetic loss - of - function system , thioglycollate - elicited ppar , ppar , or ppar peritoneal macrophages cells were then stimulated with lps ( 10 ng / ml ) for 6 h , and inflammatory and receptor target gene was measured by real - time pcr . as shown in fig . 2a , the ppar target gene ap2 , and the lxr target gene abca1 , were upregulated in wild - type cells by ligand , confirming proper ligand response . as expected , lxr agonist ( gw3965 ) efficiently suppressed inflammatory gene expression ( inos ) . furthermore , ppar ligand ( gw7845 ) repressed il-6 , mcp-1 , and inos expression in wild - type cells but not in ppar-null cells ( fig . ppar agonist ( gw742 ) had no significant effect on expression of these genes in any genotype . ppar , ppar , and ppar macrophages exhibited heightened inflammatory gene expression compared with wild - type cells in vitro . a : thioglycollate - elicited wild - type , ppar , ppar , and ppar peritoneal macrophages were harvested and pretreated with gw7845 ( 100 nm ) , gw742 ( 100 nm ) , or gw3965 ( 1 m ) overnight . the cells were then stimulated with lps ( 10 ng / ml ) for 6 h. ppar and lxr target genes ( ap2 and abca1 , respectively ) were tested to confirm ligand response . inos was markedly upregulated in macrophages with deleted ppars ; in wild - type cells , treatment with lxr ligands consistently suppressed inflammatory genes , whereas treatment only with ppar and not ppar ligand resulted in repression . c : additional inflammatory genes were tested in response to lps and ppar ligand treatments . however , il-1 has a different expression profile compared with the other genes and highlights inconsistencies in ppar signaling in inflammation . error bars represent sem . we also noted a significant effect of loss of ppar expression on lps - inducible inflammatory gene expression in the absence of receptor ligands . loss of either ppar or ppar alone led to enhanced induction of inos and il-6 by lps ( fig . moreover , the combined deletion of both receptors had an additive effect , i.e. , inflammatory gene expression was most responsive in the double knockouts . interestingly , the combined loss of ppar and ppar affected the expression of some inflammatory markers more than others . for example , in contrast to il-6 , mcp-1 , and inos , expression of il-1 was not altered in the wild - type cells ( fig . 2c ) . to investigate the impact of bm ppar expression on systemic glucose metabolism in c57bl/6 mice , we used wild - type , ppar , ppar , or ppar bm ( from mice more than 10 generations backcrossed to c57/bl6 ) to reconstitute irradiated wild - type mice . after recovery for 4 weeks , the mice were challenged with a 60% fat diet for an additional 14 weeks to cause obesity . all four groups of mice gained weight at a similar rate and had equivalent food intake during the course of the feeding ( fig . 3a ; data not shown ) . to confirm the degree of bm reconstitution after 14 weeks of high - fat diet , we harvested thioglycollate - elicted peritoneal macrophages from ppar and wild - type bmt - recipient mice . real - time pcr verified that virtually all of the macrophages from ppar bmt mice were derived from the donor cells , because ppar exon 2 was not expressed ( fig . we also tested whether the macrophages were responsive to ligand by examining ppar target gene expression . 3c , expression of adrp was induced by ppar ligand ( gw7845 ) in cells from wild - type but not ppar bmt mice . all ppar mice had equivalent weight gain and exhibited reconstitution with myeloid cells derived from donor mice . a : irradiated mice transplanted with wild - type , ppar , ppar , or ppar myeloid cells were challenged with a 60% fat diet . all groups of mice gained weight at similar rates . b : thioglycollate - elicted wild - type and ppar peritoneal macrophages were harvested from recipient mice to determine degree of reconstitution . ppar exon 2 could not be detected in ppar macrophages ( p < 0.05 ) , whereas ppar exon 6 could be detected . cells were harvested and pooled from four wild - type and three ppar bone marrow transplant ( bmt ) mice . ppar target genes ( ap2 and adrp ) did not increase in expression as a result of ligand treatment in ppar cells . cells were harvested and pooled from four wild - type and three ppar bmt mice . after mice were on the diet for 14 weeks , we performed glucose tolerance tests to determine whether reconstitution with differing ppar knockout macrophages would alter systemic metabolism . mice clearly became glucose intolerant as a result of high - fat feeding ; however , high - fat - fed mice lacking bm expression of ppar , ppar , or both on the c57bl/6 background showed no significant difference from controls ( fig . a second set of glucose tolerance tests performed 4 weeks later yielded similar results ( fig . 4b ) . fasting serum insulin levels were also not significantly different between groups , as determined by elisa ( p > 0.05 ; data not shown ) . a : after 14 weeks on the diet , the four groups of ppar mice were equally diabetic and similarly insulin resistant ( p > 0.05 ) . after the mice were fasted overnight , basal blood glucose levels were measured ( 0 min ) before intraperitoneal administration of 2 g / kg glucose . b : a second glucose tolerance test performed after an additional 4 weeks yielded similar results ( p > 0.05 ) . c : wild - type and ppar mice were treated with rosiglitazone ( 30 mg / kg ) for 8 days . rosiglitazone improved insulin sensitivity in both wild - type and ppar mice , whereas vehicle treatment did not demonstrate a decrease in blood glucose levels in either group ( p < 0.02 ) . we also assessed the influence of bm ppar expression for the anti - diabetic effects of ppar agonists on a c57bl/6 background . we treated wild - type and ppar bmt - recipient mice with rosiglitazone for 8 days . 4c , both groups treated with rosiglitazone showed markedly and equivalently improved glucose tolerance , compared with vehicle controls . this observation suggests that tissues other than bm are the primary mediators of the anti - diabetic effects of ppar ligand in mice on a c57bl/6 background under the experimental conditions employed here . macrophage infiltration into adipose tissue is a well - characterized feature in obese mouse models ( 6 ) . furthermore , administration of thiazoladinediones reverses macrophage infiltration , presumably by activating ppar. we therefore investigated whether the lack of macrophage ppar would affect macrophage infiltration into adipose tissue . to our surprise , we found that there was no change in the numbers of macrophages present in adipose tissue when we compared wild - type and ppar bmt mice ( data not shown ) . additionally , the size of adipocytes was similar in wild - type and ppar bmt mice and we did not observe any obvious differences in body composition ( data not shown ) . previous work has established that both lxr and lxr are expressed at high levels in macrophages , and both isotypes have strong anti - inflammatory effects ( 19 ) . as shown in fig . we hypothesized that loss of lxr receptor expression in bm cells might exacerbate inflammatory signaling in vivo and impact glucose metabolism . we therefore transplanted bm cells from wild - type and lxr mice into irradiated wild - type recipients . as with the mice used for ppar studies , the animals used for these experiments were also more than 10 generations backcrossed to c57bl/6 mice . after 4 weeks of recovery , the bmt mice were challenged with a 60% fat diet to induce obesity and insulin resistance . all groups consumed similar amounts of food and gained weight at similar rates ( fig . we confirmed that the reconstituted animals had been repopulated with lxr bm by harvesting thioglycollate - elicted peritoneal macrophages and performing real - time pcr to examine the expression of lxrs and their downstream target genes . as expected , lxr ligands ( gw3965 and t1317 ) induced expression of abca1 in wild - type but not in lxr bmt cells ( fig . we also verified that lxrs had maintained their anti - inflammatory abilities after reconstitution by stimulating macrophages with lps in the presence or absence of lxr ligand . indeed , lxr ligand repressed expression of tnf in wild - type but not lxr - null bm recipients ( fig . recipient mice showed equivalent weight gain and exhibited efficient reconstitution with wild - type or lxr - null donor bone marrow ( bm ) . a : wild - type and lxr mice gained weight at similar rates on a 60% fat diet . b : thioglycollate - elicted wild - type and lxr peritoneal macrophages were harvested from recipient mice to determine degree of reconstitution . wild - type and lxr macrophages were pretreated with gw3965 or t1317 ( 1 m ) and then stimulated with lps ( 10 ng / ml ) for 6 h. the lxr target gene , abca1 , did not increase in expression as a result of ligand treatment in lxr cells . furthermore , lxr cells failed to repress tnf gene expression . wild - type cells treated with lxr ligand were able to exert anti - inflammatory control . after mice were on on a high - fat diet for 14 weeks , a glucose tolerance test was performed . similar to the results obtained with the ppar bm transplants , we observed no difference in glucose tolerance or insulin levels between mice transplanted with wild - type or lxr bm ( fig . these observations indicate that despite their anti - inflammatory effects , loss of lxr expression in the bm does not exert a major influence on glucose tolerance in high - fat - fed mice . glucose tolerance tests in wild - type and lxr mice revealed no difference in blood glucose levels . a two - way anova test was used to determine significance . a : wild - type and lxr mice were challenged with a 60% fat diet for 14 weeks . glucose ( 2 g / kg ) was administered , then blood glucose was assessed every 30 min for 2 h. both groups of mice were equally insulin resistant ( p > 0.05 ) . b : a second glucose tolerance test was performed after an additional 4 weeks as previously described . it has been previously reported that alternative activation of balb / c macrophages relies significantly on appropriate ppar signaling ( 22 , 24 ) . studies have demonstrated that ppar-deficient macrophages express low levels of m2 marker genes and are not effectively alternatively activated ( 22 ) . as a result , these cells may be more inflammatory and may act as contributors to worsened insulin sensitivity . to test whether ppar or ppar macrophages from c57bl/6 mice also expressed reduced levels of alternative activation marker genes , we stimulated bm - derived macrophages with cytokines that upregulate th-2 response , such as il-4 or il-13 ( 25 ) . we found that the absence of ppars did not compromise m2 inflammatory responses under our experimental conditions . expression of classical m2 marker genes such as chi3l3 , pdcd1lg2 , and clec7a ( 26 , 27 ) in response to il-4 or il-13 in ppar , ppar , or ppar macrophages was not significantly different from wild - type macrophages ( fig . 7 ) . however , the loss of ppar in macrophages resulted in lower expression of arginase i ( argi ) , a primary m2 marker gene ( 28 ) , in response to il-4 or il-13 stimulation . argi has been previously described as a direct ppar and ppar target gene , and this may explain the reduced argi expression in ppar-deficient macrophages ( 22 , 29 ) . interestingly , argi expression was unique in its dependence on ppar for induction ; the expression of the remaining m2 marker genes we tested was not altered by the loss of ppars . classical m2 gene expression is not absolutely dependent on ppar expression in bm - derived macrophages . wild - type , ppar , ppar , and ppar bm cells were harvested , differentiated into macrophages , and treated with il-4 or il-13 ( 10 ng / ml each ) overnight . expression of m2 genes was analyzed to determine impact of ppar , ppar , or both ppar and ppar deficiencies . in the present study , we report that loss of bm expression of anti - inflammatory nuclear receptors is not a dominant contributor to the development of diet - induced obesity or glucose intolerance in high - fat - fed c57bl/6 mice . this was true not only for ppar , which has been reported to play a role in insulin resistance in balb / c mice , but also for ppar , lxr , and lxr , which have not been previously studied in this context . using a bmt approach , we found that loss of ppar , ppar , both ppar and ppar , or both lxr and lxr did not alter weight gain in mice challenged with a 60% fat diet or have a significant effect on glucose tolerance . we also found that administration of rosiglitazone to ppar bmt mice improved glucose tolerance , suggesting that bm expression of this receptor is not absolutely required for the therapeutic effects of thiazolidinediones in c57bl/6 mice . previous work has reported that macrophage ppar expression affects the development of obesity and insulin resistance ( 18 , 22 ) . it is important to point out that our studies were conducted in parallel to those of glass and chawla ( 18 , 22 ) and were not designed to replicate the experimental conditions used in either of these reports . therefore , our results are not necessarily in conflict with those of prior work , and there are a number of factors that may have contributed to the differences in experimental outcomes . the macrophages in prior work were derived from either balb / c or mixed - background mice . genetic background in mice contributes to the dominance of m1 or m2 activation of macrophages and can bias how mouse models respond to specific stimuli . for example , the balb / c mouse strain is th2-permissive and is more susceptible to diseases that require an aggressive immune response ( 1 ) . conversely , c57bl/6 mice are dominant for th1 signaling and display more - robust inflammatory signaling and the upregulation of pro - inflammatory genes . the c57bl/6 strain is most widely used for the study of metabolic disorders because of its susceptibility to diet - induced insulin resistance and atherosclerosis . another plausible explanation for the difference between our results and those of the chawla and glass groups ( 18 , 22 ) is environment and the immune status of the mice . all animal facilities are different , and mice at different sites may therefore be exposed to a range of different pathogens and commensal bacteria . it is conceivable that such differences alter the immune systems of experimental mice in ways that may affect macrophage responses . hevener et al . ( 18 ) reported more - significant differences in blood glucose levels between wild - type and ppar bmt mice fed a chow diet rather than a high - fat diet . we chose to use a 60% fat diet because our aim was to study ppar / lxr function in obese models . however , it is possible that our high - fat diet may have been too robust a diabetic stimulus and therefore masked differences otherwise seen on a chow diet . finally , the bmt approach employed in our study may have contributed to differences in results . we chose to use mx cre - mediated deletion coupled with bmt because of incomplete deletion by lysm cre in our hands ( fig . 1 ) . however , it is possible that the bmt procedure itself has a confounding effect on systemic metabolism . furthermore , although we confirmed reconstitution of recipient mice with the appropriate donor bm cells , there is a possibility that the tissue macrophage reconstitution was not complete at the time we performed our studies . prior work has shown that adipose tissue macrophages and hepatic kupffer cells are effectively replaced by bm cells in the time frame employed in our study ( more than 4 months ) . however , the possibility that some wild - type macrophages may have remained and affected our results can not be excluded . while this paper was under review , two additional studies reported an impact of loss of ppar expression on glucose tolerance and alternative macrophage activation ( 30 , 31 ) . some of the findings reported in these studies also differ from the results we report here . as outlined above for ppar , it is likely that a number of factors may contribute to the prominence of ppar signaling in different experimental contexts . additional studies will be required to more precisely define the role of both ppar and ppar in macrophage inflammatory signaling in the context of metabolic disease . in conclusion , our studies suggest that the in vitro anti - inflammatory activities of nuclear receptors alone are not predictive of in vivo metabolic effects . macrophage lxrs show profound anti - inflammatory effects in vitro but do not dramatically influence systemic glucose metabolism under the conditions used here . furthermore , our results suggest that careful consideration of the immunological differences between mouse strains is warranted in the context of metabolic disease studies . inherent biases in mouse strains can steer immune responses and affect the outcomes of various disease models in different ways . finally , our data indicate that the influence of bm nuclear expression on systemic metabolism may differ between experimental context and genetic background and that these factors may not be obligatory components of disease pathogenesis or thiazolidinedione therapeutic action in all contexts .

macrophage lipid metabolism and inflammatory responses are both regulated by the nuclear receptors ppar and lxr . emerging links between inflammation and metabolic disease progression suggest that ppar and lxr signaling may alter macrophage function and thereby impact systemic metabolism . in this study , the function of macrophage ppar and lxr in th1-biased c57bl/6 mice was tested using a bone marrow transplantation approach with ppar/ , ppar/ , ppar/ , and lxr/ cells . despite their inhibitory effects on inflammatory gene expression , loss of ppars or lxrs in macrophages did not exert major effects on obesity or glucose tolerance induced by a high - fat diet . treatment with rosiglitazone effectively improved glucose tolerance in mice lacking macrophage ppar , suggesting that cell types other than macrophages are the primary mediators of the anti - diabetic effects of ppar agonists in our model system . c57bl/6 macrophages lacking ppars or lxrs exhibited normal expression of most alternative activation gene markers , indicating that macrophage alternative activation is not absolutely dependent on these receptors in the c57bl/6 background under the conditions used here . these studies suggest that genetic background may be an important modifier of nuclear receptor effects in macrophages . our results do not exclude a contribution of macrophage ppar and lxr expression to systemic metabolism in certain contexts , but these factors do not appear to be dominant contributors to glucose tolerance in a high - fat - fed th1-biased bone marrow transplant model .

METHODS Reagents and cell culture Animal studies Insulin ELISA RNA and quantitative PCR Statistical analysis RESULTS DISCUSSION

the synthetic ligands for lxr ( gw3965 and t1317 ) , ppar ( gw9544 ) , ppar ( gw742 ) , and ppar ( gw7845 ) were provided by glaxosmithkline . the mx cre , pparfl / fl , and ppar mice are all on the c57bl/6 background ( more than nine generations backcrossed ) . bm cells from recipient mice were harvested and injected into tail veins of the recipient mice . mice were challenged with a 60% caloric fat diet ( research diets ) for 16 weeks . mice were fasted the night prior to glucose tolerance tests , and glucose levels were monitored after intraperitoneal injections of glucose ( 2 g / kg ; sigma ) . wild - type , ppar , and ppar bmt mice were fasted overnight , and blood was collected in heparin tubes . a one - way or two - way anova test was used to determine statistical significance for all glucose tolerance tests . cells were harvested from wild - type , ppar , ppar , or ppar mice . the mx cre , pparfl / fl , and ppar mice are all on the c57bl/6 background ( more than nine generations backcrossed ) . bm cells from recipient mice were harvested and injected into tail veins of the recipient mice . mice were fasted the night prior to glucose tolerance tests , and glucose levels were monitored after intraperitoneal injections of glucose ( 2 g / kg ; sigma ) . wild - type , ppar , and ppar bmt mice were fasted overnight , and blood was collected in heparin tubes . a one - way or two - way anova test was used to determine statistical significance for all glucose tolerance tests . to investigate the function of macrophage ppar , we used a cre recombinase system to disrupt the ppar gene . specifically , we crossed c57bl/6 mice with loxp sites flanking the second exon of ppar ( pparfl / fl ) with c57bl/6 mice expressing cre recombinase downstream of the / interferon - inducible ( mx ) promoter . cre recombinase activity was initiated by intraperitoneal injection of the mx activator poly ic 3 to 4 weeks after birth . interestingly , injection of poly ic at weaning age caused a permanent disruption of ppar in myeloid precursor cells ; new myeloid cells generated postinjection remain ppar-null for the life of the mouse . typically , we analyzed macrophages from these mice at least 4 weeks after inducing recombination to reduce any potential effects of the poly ic . quantitative real - time pcr confirmed that the mx cre system was extremely efficient , yielding nearly complete ( > 95% ) deletion of ppar exon 2 in vivo ( fig . as expected , induction of ppar target genes ( ap2 , adrp , pgar ) by ppar but not ppar agonists was lost in mx cre ppar macrophages ( fig . induction of the mx promoter effectively disrupts ppar , whereas lysm cre can not efficiently delete ppar exon 2 . known ppar target genes ( ap2 , adrp , and pgar ) were tested to confirm loss of ligand response in ppar macrophages . ppar exon 2 could still be detected in ppar macrophages , and pgar , a known ppar target gene , could still be induced by ppar ligand in these cells ( p < 0.02 ) . however , compared with the mx cre system , the lysm cre system was not as efficient in recombining ppar to create a disrupted gene in our hands . after harvesting peritoneal macrophages from wild - type and lysm cre ppar mice , we treated the cells with ppar ligand to test whether disruption of ppar was able to prevent ppar target gene regulation . we found that ppar exon 2 could still be detected by quantitative real - time pcr and that pgar , a ppar target gene , could be regulated by ligand even in ppar macrophages ( fig . in contrast , the mx cre system yields cell populations with complete deletion of ppar exon 2 expression , and more importantly , these cells are unable to respond to ppar ligand and do not regulate target genes . recent work suggests that ppars mediate inflammatory signaling pathways in macrophages and may affect inflammation associated with insulin resistance ( 18 , 22 ) . to address this issue in our genetic loss - of - function system , thioglycollate - elicited ppar , ppar , or ppar peritoneal macrophages cells were then stimulated with lps ( 10 ng / ml ) for 6 h , and inflammatory and receptor target gene was measured by real - time pcr . 2a , the ppar target gene ap2 , and the lxr target gene abca1 , were upregulated in wild - type cells by ligand , confirming proper ligand response . as expected , lxr agonist ( gw3965 ) efficiently suppressed inflammatory gene expression ( inos ) . furthermore , ppar ligand ( gw7845 ) repressed il-6 , mcp-1 , and inos expression in wild - type cells but not in ppar-null cells ( fig . ppar agonist ( gw742 ) had no significant effect on expression of these genes in any genotype . ppar , ppar , and ppar macrophages exhibited heightened inflammatory gene expression compared with wild - type cells in vitro . a : thioglycollate - elicited wild - type , ppar , ppar , and ppar peritoneal macrophages were harvested and pretreated with gw7845 ( 100 nm ) , gw742 ( 100 nm ) , or gw3965 ( 1 m ) overnight . the cells were then stimulated with lps ( 10 ng / ml ) for 6 h. ppar and lxr target genes ( ap2 and abca1 , respectively ) were tested to confirm ligand response . inos was markedly upregulated in macrophages with deleted ppars ; in wild - type cells , treatment with lxr ligands consistently suppressed inflammatory genes , whereas treatment only with ppar and not ppar ligand resulted in repression . we also noted a significant effect of loss of ppar expression on lps - inducible inflammatory gene expression in the absence of receptor ligands . loss of either ppar or ppar alone led to enhanced induction of inos and il-6 by lps ( fig . moreover , the combined deletion of both receptors had an additive effect , i.e. , inflammatory gene expression was most responsive in the double knockouts . interestingly , the combined loss of ppar and ppar affected the expression of some inflammatory markers more than others . for example , in contrast to il-6 , mcp-1 , and inos , expression of il-1 was not altered in the wild - type cells ( fig . to investigate the impact of bm ppar expression on systemic glucose metabolism in c57bl/6 mice , we used wild - type , ppar , ppar , or ppar bm ( from mice more than 10 generations backcrossed to c57/bl6 ) to reconstitute irradiated wild - type mice . after recovery for 4 weeks , the mice were challenged with a 60% fat diet for an additional 14 weeks to cause obesity . all four groups of mice gained weight at a similar rate and had equivalent food intake during the course of the feeding ( fig . to confirm the degree of bm reconstitution after 14 weeks of high - fat diet , we harvested thioglycollate - elicted peritoneal macrophages from ppar and wild - type bmt - recipient mice . we also tested whether the macrophages were responsive to ligand by examining ppar target gene expression . 3c , expression of adrp was induced by ppar ligand ( gw7845 ) in cells from wild - type but not ppar bmt mice . a : irradiated mice transplanted with wild - type , ppar , ppar , or ppar myeloid cells were challenged with a 60% fat diet . cells were harvested and pooled from four wild - type and three ppar bone marrow transplant ( bmt ) mice . ppar target genes ( ap2 and adrp ) did not increase in expression as a result of ligand treatment in ppar cells . after mice were on the diet for 14 weeks , we performed glucose tolerance tests to determine whether reconstitution with differing ppar knockout macrophages would alter systemic metabolism . mice clearly became glucose intolerant as a result of high - fat feeding ; however , high - fat - fed mice lacking bm expression of ppar , ppar , or both on the c57bl/6 background showed no significant difference from controls ( fig . a second set of glucose tolerance tests performed 4 weeks later yielded similar results ( fig . a : after 14 weeks on the diet , the four groups of ppar mice were equally diabetic and similarly insulin resistant ( p > 0.05 ) . b : a second glucose tolerance test performed after an additional 4 weeks yielded similar results ( p > 0.05 ) . c : wild - type and ppar mice were treated with rosiglitazone ( 30 mg / kg ) for 8 days . rosiglitazone improved insulin sensitivity in both wild - type and ppar mice , whereas vehicle treatment did not demonstrate a decrease in blood glucose levels in either group ( p < 0.02 ) . we also assessed the influence of bm ppar expression for the anti - diabetic effects of ppar agonists on a c57bl/6 background . we treated wild - type and ppar bmt - recipient mice with rosiglitazone for 8 days . 4c , both groups treated with rosiglitazone showed markedly and equivalently improved glucose tolerance , compared with vehicle controls . this observation suggests that tissues other than bm are the primary mediators of the anti - diabetic effects of ppar ligand in mice on a c57bl/6 background under the experimental conditions employed here . we therefore investigated whether the lack of macrophage ppar would affect macrophage infiltration into adipose tissue . additionally , the size of adipocytes was similar in wild - type and ppar bmt mice and we did not observe any obvious differences in body composition ( data not shown ) . previous work has established that both lxr and lxr are expressed at high levels in macrophages , and both isotypes have strong anti - inflammatory effects ( 19 ) . we hypothesized that loss of lxr receptor expression in bm cells might exacerbate inflammatory signaling in vivo and impact glucose metabolism . we therefore transplanted bm cells from wild - type and lxr mice into irradiated wild - type recipients . as with the mice used for ppar studies , the animals used for these experiments were also more than 10 generations backcrossed to c57bl/6 mice . after 4 weeks of recovery , the bmt mice were challenged with a 60% fat diet to induce obesity and insulin resistance . we confirmed that the reconstituted animals had been repopulated with lxr bm by harvesting thioglycollate - elicted peritoneal macrophages and performing real - time pcr to examine the expression of lxrs and their downstream target genes . as expected , lxr ligands ( gw3965 and t1317 ) induced expression of abca1 in wild - type but not in lxr bmt cells ( fig . we also verified that lxrs had maintained their anti - inflammatory abilities after reconstitution by stimulating macrophages with lps in the presence or absence of lxr ligand . indeed , lxr ligand repressed expression of tnf in wild - type but not lxr - null bm recipients ( fig . recipient mice showed equivalent weight gain and exhibited efficient reconstitution with wild - type or lxr - null donor bone marrow ( bm ) . a : wild - type and lxr mice gained weight at similar rates on a 60% fat diet . wild - type and lxr macrophages were pretreated with gw3965 or t1317 ( 1 m ) and then stimulated with lps ( 10 ng / ml ) for 6 h. the lxr target gene , abca1 , did not increase in expression as a result of ligand treatment in lxr cells . furthermore , lxr cells failed to repress tnf gene expression . wild - type cells treated with lxr ligand were able to exert anti - inflammatory control . after mice were on on a high - fat diet for 14 weeks , a glucose tolerance test was performed . similar to the results obtained with the ppar bm transplants , we observed no difference in glucose tolerance or insulin levels between mice transplanted with wild - type or lxr bm ( fig . these observations indicate that despite their anti - inflammatory effects , loss of lxr expression in the bm does not exert a major influence on glucose tolerance in high - fat - fed mice . glucose tolerance tests in wild - type and lxr mice revealed no difference in blood glucose levels . a : wild - type and lxr mice were challenged with a 60% fat diet for 14 weeks . it has been previously reported that alternative activation of balb / c macrophages relies significantly on appropriate ppar signaling ( 22 , 24 ) . as a result , these cells may be more inflammatory and may act as contributors to worsened insulin sensitivity . to test whether ppar or ppar macrophages from c57bl/6 mice also expressed reduced levels of alternative activation marker genes , we stimulated bm - derived macrophages with cytokines that upregulate th-2 response , such as il-4 or il-13 ( 25 ) . we found that the absence of ppars did not compromise m2 inflammatory responses under our experimental conditions . expression of classical m2 marker genes such as chi3l3 , pdcd1lg2 , and clec7a ( 26 , 27 ) in response to il-4 or il-13 in ppar , ppar , or ppar macrophages was not significantly different from wild - type macrophages ( fig . however , the loss of ppar in macrophages resulted in lower expression of arginase i ( argi ) , a primary m2 marker gene ( 28 ) , in response to il-4 or il-13 stimulation . argi has been previously described as a direct ppar and ppar target gene , and this may explain the reduced argi expression in ppar-deficient macrophages ( 22 , 29 ) . interestingly , argi expression was unique in its dependence on ppar for induction ; the expression of the remaining m2 marker genes we tested was not altered by the loss of ppars . classical m2 gene expression is not absolutely dependent on ppar expression in bm - derived macrophages . wild - type , ppar , ppar , and ppar bm cells were harvested , differentiated into macrophages , and treated with il-4 or il-13 ( 10 ng / ml each ) overnight . expression of m2 genes was analyzed to determine impact of ppar , ppar , or both ppar and ppar deficiencies . in the present study , we report that loss of bm expression of anti - inflammatory nuclear receptors is not a dominant contributor to the development of diet - induced obesity or glucose intolerance in high - fat - fed c57bl/6 mice . this was true not only for ppar , which has been reported to play a role in insulin resistance in balb / c mice , but also for ppar , lxr , and lxr , which have not been previously studied in this context . using a bmt approach , we found that loss of ppar , ppar , both ppar and ppar , or both lxr and lxr did not alter weight gain in mice challenged with a 60% fat diet or have a significant effect on glucose tolerance . we also found that administration of rosiglitazone to ppar bmt mice improved glucose tolerance , suggesting that bm expression of this receptor is not absolutely required for the therapeutic effects of thiazolidinediones in c57bl/6 mice . previous work has reported that macrophage ppar expression affects the development of obesity and insulin resistance ( 18 , 22 ) . therefore , our results are not necessarily in conflict with those of prior work , and there are a number of factors that may have contributed to the differences in experimental outcomes . genetic background in mice contributes to the dominance of m1 or m2 activation of macrophages and can bias how mouse models respond to specific stimuli . for example , the balb / c mouse strain is th2-permissive and is more susceptible to diseases that require an aggressive immune response ( 1 ) . the c57bl/6 strain is most widely used for the study of metabolic disorders because of its susceptibility to diet - induced insulin resistance and atherosclerosis . another plausible explanation for the difference between our results and those of the chawla and glass groups ( 18 , 22 ) is environment and the immune status of the mice . ( 18 ) reported more - significant differences in blood glucose levels between wild - type and ppar bmt mice fed a chow diet rather than a high - fat diet . we chose to use a 60% fat diet because our aim was to study ppar / lxr function in obese models . however , it is possible that our high - fat diet may have been too robust a diabetic stimulus and therefore masked differences otherwise seen on a chow diet . finally , the bmt approach employed in our study may have contributed to differences in results . we chose to use mx cre - mediated deletion coupled with bmt because of incomplete deletion by lysm cre in our hands ( fig . however , it is possible that the bmt procedure itself has a confounding effect on systemic metabolism . prior work has shown that adipose tissue macrophages and hepatic kupffer cells are effectively replaced by bm cells in the time frame employed in our study ( more than 4 months ) . however , the possibility that some wild - type macrophages may have remained and affected our results can not be excluded . while this paper was under review , two additional studies reported an impact of loss of ppar expression on glucose tolerance and alternative macrophage activation ( 30 , 31 ) . some of the findings reported in these studies also differ from the results we report here . as outlined above for ppar , it is likely that a number of factors may contribute to the prominence of ppar signaling in different experimental contexts . additional studies will be required to more precisely define the role of both ppar and ppar in macrophage inflammatory signaling in the context of metabolic disease . in conclusion , our studies suggest that the in vitro anti - inflammatory activities of nuclear receptors alone are not predictive of in vivo metabolic effects . macrophage lxrs show profound anti - inflammatory effects in vitro but do not dramatically influence systemic glucose metabolism under the conditions used here . furthermore , our results suggest that careful consideration of the immunological differences between mouse strains is warranted in the context of metabolic disease studies . finally , our data indicate that the influence of bm nuclear expression on systemic metabolism may differ between experimental context and genetic background and that these factors may not be obligatory components of disease pathogenesis or thiazolidinedione therapeutic action in all contexts .

the inflammasomes are signaling platforms , which are assembled in response to pathogen - associated and damage - associated molecular pattern molecules and environmental irritants . currently , inflammasomes are distinguished into two families : the nod - like receptor ( nlr ) family and the pyrin and hin200 ( haematopoietic interferon - inducible nuclear antigens with 200 amino - acid repeats ) domain - containing protein ( pyhin ) family . the nlr family consists of nlrp1 , nlrp2 , nlrp3 , nlrp6 , nlrc4 , and nlrp12 . the nlrp3 inflammasome is a multiprotein , large cytoplasmic complex ( > 700 kda ) , composed of a specific member of the nod - like receptor protein ( nlrp ) subfamily , the adaptor protein named apoptosis - associated speck - like protein containing a card ( asc ) , and procaspase-1 , which are preferentially expressed in adipose tissue macrophages ( atms ) . unlike the typical signaling cascades downstream of many innate receptors such as other nlrp members , nlrp3 contains an n - terminal pyrin domain ( pyd ) , which is used to physically interact with the pyd domain of asc , thus facilitating the subsequent recruitment and activation of procaspase-1 . , once activated , caspase-1 , as far as we are currently aware , cleaves the proforms of two potent proinflammatory cytokines interleukin- ( il- ) 1 and il-18 in the cytoplasm . this has two main effects ; firstly it activates the two cytokines and secondly in this mature form these cytokines can be released from the cell . the active form of caspase-1 also has the ability to induce the release of il-1 and hmgb-1 ( high mobility group box 1 ) , as well as initiate a lytic form of cell death called pyroptosis [ 24 ] ( figure 1 ) . the primary role of the inflammasome and its products seems to be as part of the body 's innate immune system , in that they can be triggered to assist in the defense against invading pathogens . indeed much of the data published on the inflammasome / caspase 1 is on its role in the body 's response to microbial molecules ( bacterial , fungal , or viral ) with conserved molecular structures known as pathogen associated molecular patterns ( pamps ) [ 5 , 6 ] . in addition to pamps , the nlrp3 inflammasome is also proficient in sensing stress to endogenous ( nonmicrobial ) danger signals ( danger associated molecular patterns , damps ) from damaged cells . damps can include molecules such as reactive oxygen species ( ros ) , adenosine triphosphate ( atp ) , hypotonic stress , uric acid crystals , or noxious exogenous factors such as environmental insults , asbestos , and uv radiation . there are a number of potential mechanisms for the assembly of the nlrp3 inflammasome , as described earlier . according to one hypothesis , mitochondria are the principal source of reactive oxygen species ( ros ) required for inflammasome activation ; several recent studies have implicated ros produced by mitochondria , rather than phagosomes , in nlrp3 activation exerting an indirect effect on pathways of metabolism [ 8 , 9 ] . a second mechanism involves the disruption of lysosomal membrane integrity by crystalline materials and peptide aggregates [ 10 , 11 ] . upon uptake of such substances , lysosomal rupture leads to the leakage of lysosomal proteases , specifically cathepsins b and l , into the cytosol where they could possibly mediate nlrp3 inflammasome activation by an as - yet - undefined cleavage event . in addition , type-2 diabetic patients and mice fed a high - fat diet demonstrate il-1 production following inflammasome activation from obesity - induced danger signals . mice have also been shown to become glucose intolerant following activation of the inflammasome in hematopoietic cells by the saturated fatty acid palmitate . very recently , vajjhala and colleagues have shed light on the molecular details of the complex mechanisms of nlrp3 inflammasome assembly and activation , identifying multiple binding sites on the pyd domain of the adaptor protein asc which allow self - association and interaction with binding partners . several in vitro , in vivo studies and clinical trials provide evidence that supports a causative role of il-1 in the pathogenesis of type 2 diabetes , and elevation in circulating levels of il-1 predicts type 2 diabetes when combined with serum il-6 levels . prolonged il-1 treatment has been demonstrated to reduce the insulin - induced glucose uptake in murine adipocytes . in contrast , addition of the il-1 receptor antagonist to adipocytes resulted in increased insulin sensitivity as reflected by increased levels of phosphorylated akt in response to insulin . these results were confirmed by showing that il-1 knockout mice were more insulin sensitive as compared to wild - type control animals . in humans , elevated plasma levels of il-1 have been found to be predictive of type 2 diabetes , and clinical studies have suggested that treatment with the il-1 receptor antagonist anakinra has beneficial effects in type 2 diabetic patients . a number of recent landmark studies have pointed out a key role for an excessive nlrp3 inflammasome activation in the il-1-related development of type 2 diabetes . the association between the nlrp3 inflammasome and both insulin resistance and obesity has been suggested by animal studies showing that genetic ablation of nlrp3 improved insulin sensitivity and glucose homeostasis . specifically , adipocytes isolated from nlrp3-deficient mice showed an increase in insulin sensitivity as determined by phosphorylation of akt . in line with the rise in insulin sensitivity , il-1 production of adipose tissue isolated from nlrp3 knockout mice other studies [ 12 , 13 ] have shown that improvement in insulin sensitivity ( increased phosphorylation of the insulin receptor subtrate-1 and akt ) can also be detected in liver and muscle of nlrp3 knockout mice on a high - fat diet for 12 weeks . this effect was associated with a significant reduction in the tissue mrna expression of inflammatory cytokines compared to wild - type control . ablation of the nlrp3 in mice has been also reported to protect from obesity - associated macrophage activation in adipose tissue , reducing m1-like macrophage gene expression ( tumor necrosis factor- , chemokine ligand 20 , and chemokine ligand 11 ) and increasing the expression of m2-like cytokines ( interleukin-10 ) . this effect was associated with an increase in the number of m2 macrophages in nlrp3-deficient obese mice , without affecting the m1 macrophage frequency . to confirm the clinical relevance of these data generated from mouse models , the same authors have demonstrated that weight loss reduced nlrp3 expression in abdominal subcutaneous adipose tissue in obese patients with type 2 diabetes , which was accompanied by improved glucose homeostasis . furthermore , strong correlations between the expression of nlrp3 inflammasome - related genes and insulin resistance have been recently reported in obese male subjects with impaired glucose tolerance . additionally , type 2 diabetic patients showed elevated levels of nlrp3 , asc , il-1 , and il-18 mrna and protein expression in monocyte - derived macrophages , compared with those in healthy control subjects . besides , the cleavage of caspase-1 and release of mature il-1 were significantly elevated in monocyte - derived macrophages from type 2 diabetic patients compared with controls . inflammatory cytokines are known to contribute crucially to the development of insulin resistance by activating different kinases that disrupt insulin signaling . the endoplasmatic reticulum ( er ) is an extensive membrane network which has been recently demonstrated to be involved in the transduction of cytokines effects into activation of different kinases . the early steps of insulin biosynthesis occur in the er of pancreatic cells , thus further suggesting the key role of er load and folding activity in the insulin biosynthesis . a major role of the er is to ensure the synthesis and folding of membrane and secreted proteins , and any disturbance in this function ( e.g. , excessive protein synthesis or accumulation of unfolded or misfolded proteins in the er lumen ) leads to an er stress the recent literature suggests that er stress may act directly as a negative modulator of the insulin biosynthesis and insulin signaling pathways but also indirectly by promoting lipid accumulation [ 25 , 26 ] . er stress also plays a role in the dysregulation of adipokine secretion by adipose tissue , frequently observed in obesity and insulin resistance [ 27 , 28 ] , and cd14 + monocytes isolated from diabetic patients showed evidence of er stress , which may underlie the functional defects in these cells . interestingly , er stress has been recently demonstrated to activate the nlrp3 inflammasome , resulting in the subsequent release of il-1 by human macrophages , with an activation mechanism similar to that of other known nlrp3 activators , requiring ros generation and potassium efflux . the thioredoxin - interacting protein ( txnip ) , a critical node in the development of er stress leading to programmed cell death of pancreatic cells , activates the nlrp3 inflammasome , causing procaspase-1 cleavage and il-1 secretion in human monocytic cells . the role of er stress in promoting nlrp3 inflammasome activation is consistent with the subcellular localization of nlrp3 . in resting cells , nlrp3 is associated with er membranes , and then upon activation nlrp3 is redistribute to the perinuclear space where it colocalizes with endoplasmic reticulum and mitochondria organelle clusters . nlrp3 inflammasome plays a substantial role in sensing obesity - associated inducers of caspase-1 activation and therefore regulates the magnitude of the inflammation and its downstream effects on insulin signaling in different organs , as reported here later . nlrp3 expression is detected mainly in the cytosol of granulocytes , monocytes , dendritic cells , t and b cells , and osteoblasts . thus , most of the first studies characterizing the role of nlrp3 signaling have been conducted in cells of the immune system . several studies on innate immune cells have demonstrated that the myeloid - derived nlrp3 inflammasome complex may contribute to promote inflammatory cytokine production and insulin resistance through reduction of insulin signaling . in vitro experiments have shown that elevated concentration of saturated fatty acids ( sfas ) , caused by a high - fat diet , may activate the nlrp3 inflammasome in macrophages through a newly identified amp - activated protein kinase and unc-51-like kinase-1 autophagy signaling cascade . besides , both ex vivo and in vivo exposure of bone marrow derived dendritic cells to dietary sfa resulted in increased nlrp3 inflammasome activation and reduced adipocyte insulin sensitivity . more specifically , dietary sfa may act as a primer of the nlrp3 inflammasome protein complex enhancing nlrp3 , caspase-1 , and pro - il-1 mrna expression . a second signal is then required to induce maturation of il-1 from inactive pro - il-1. this second step can be triggered by exposure to atp , ros , or ceramide . overall , these data suggest that exposure to dietary sfa represents the key metabolic stressor relevant to both priming and processing of il-1 in both adipocytes and innate immune cells . however , it must be stressed that the high expression of nlrp3 in primary adipocyte fractions of enzymatically digested adipose tissue may be attributable in large part to lipid - laden macrophages that contaminate enriched adipocyte fractions , as also suggested by immunofluorescence and qrt - pcr data , showing that nlrp3 is highly expressed in adipose tissue macrophages with low expression in adipocytes . besides , standard isolation procedures for isolating primary adipose cells often involve collagenase digestion , which have been shown to be a potent inducer of cytokine gene transcription and protein secretion . these findings highlight a new model of organ crosstalk , in which leukocyte and macrophage recruitment in key insulin target tissues , such as liver , adipose , and muscle , may promote insulin resistance by enhancing inflammasome activation . this is in keeping with recent studies showing that il-1 's role in regulating the endocrine function of adipose tissue is mediated by its own ability to evoke local macrophage recruitment and lipid accumulation in an autocrine / paracrine manner . as in diabetic patients pancreas , adipose tissue , liver , and kidney , with infiltrated macrophages , are major sites of origin of inflammation , it might be intriguing to investigate the specific contribution of nlrp3 inflammasome activation in these different insulin target tissues and to identify the specific inducers that selectivity participate in the mechanism of tissue nlrp3 inflammasome activation . pancreatic islets of type 2 diabetic patients have amyloid deposition and increased production of proinflammatory cytokines and chemokines . the unique , primary component of islet amyloid deposits is the islet amyloid polypeptide ( iapp ; also known as amylin ) . mice overexpressing iapp produce higher amounts of il-1 , and exposure to high levels of il-1 has been demonstrated to induce beta cell death in cell culture , interfering with signaling to nf-b through ikk or the ib super - repressor . in keeping with these results , neutralizing il-1 on isolated beta cells using il-1 receptor antagonist however , the precise mechanism(s ) by which il-1 affects pancreatic -cell failure is still debated . were the first to identify a possible signaling pathway involved in nlrp3 inflammasome activation under conditions of metabolic stress . they showed that thioredoxin - interacting protein ( txnip ) , also known as vitamin d3 upregulated protein 1 ( vdup1 ) , is an upstream and highly selective activating ligand for nlrp3 , with no effect on the activity of other inflammasomes ( e.g. , nlrc4 and aim2 ) . txnip - dependent nlrp3 inflammasome activation drives il-1 secretion from pancreatic islets in response to chronic elevated glucose , thus suggesting , for the first time , that nlrp3 , activated under conditions of metabolic stress , mediates il-1-driven islet failure . other authors have identified oligomers of iapp , as a key trigger for nlrp3 inflammasome activation and the following processing of il-1 . obesity - induced pancreatic -cell death is regulated , at least in part , by the nlrp3 inflammasome , as demonstrated in nlrp3-deficient mice in late - stage obesity , where the ablation of nlrp3 is associated with reduced cell death and increase in pancreatic islet size and local insulin levels . as noted by vandanmagsar et al . , the nlrp3 inflammasome is activated in adipose tissue in mouse models of obesity and attenuated by calorie restriction . nlrp3 inflammasome levels also correlate with glycaemia in type 2 diabetes patients after weight loss interventions . besides , mice deficient in inflammasome components are protected from body - weight gain and adipocyte hypertrophy , induced by chronic exposure to a high - fat diet . nlrp3 inflammasome components have been reported to be abundantly represented in adipocytes of patients with metabolic syndrome , mainly in adipocytes from samples of visceral adipose tissue . in contrast , the inflammasome in subcutaneous adipose tissue adipocytes did not seem to be grossly influenced by the presence of the metabolic syndrome . interestingly , caspase-1 activation in adipose tissue of obese animals takes place partly independent of macrophage infiltration . partial depletion of macrophages from adipose tissue of obese animals decreased the expression of the macrophage marker cd68 , with no significant alteration in the expression of caspase-1 , thus suggesting that the effects of the nlrp3-asc - caspase-1 protein complex on adipose tissue are not only exerted though infiltrating macrophages . this observation was also confirmed in in vitro experiments showing caspase-1 activation in adipocytes in settings free of inflammatory cells and increased insulin sensitivity in adipocytes lacking of caspase-1 or the inflammasome component nlrp3 . in addition , adipocyte upregulation of il-1 expression and secretion in response to inflammatory stimuli has been shown to induce hepatic insulin resistance , thus suggesting a further intriguing role for nlrp3 inflammasome activation in the dysfunctional communication between adipocytes and hepatocytes [ 35 , 43 ] . overall these data reveal a novel metabolic function of the nlrp3 inflammasome in adipose tissue , suggesting that its pharmacological modulation in obese and/or patients with type 2 diabetes may restore the metabolic function of adipose tissue and subsequently improve insulin sensitivity . the involvement of the inflammasome in nonalcoholic fatty liver disease ( nafld ) and non - alcoholic steatohepatitis ( nash ) is slowly being elucidated . the presence of nlrp3 inflammasome and/or inflammasome activation has been shown in sinusoidal endothelial cells , stellate cells , and hepatocytes . recently , inflammasome activation has been associated with nash , and long - term high - fat diet administration resulted in reduced hepatic steatosis in nlrp3 knockout mice . selective deficiency in il-1 in liver parenchymal cells , but not in bone - marrow - derived cells , protected mice from diet - induced steatohepatitis and fibrosis . increased mrna expression of nlrp3 inflammasome components was found in human livers of nash patients where nlrp3 levels were decreased after weight loss . these observations suggest that inflammasome activation by different cell types may contribute to different aspects of steatohepatitis . in contrast , to date , there are no data suggesting a potential role of nlrp3 inflammasome activation on impaired glycogen synthesis and/or augmented glycogenolysis . there is evidence that the inflammasome components are important in the maintenance of the integrity of the intestinal epithelium and the defense against pathogenic organisms that can invade the gastrointestinal tract . for instance , mice lacking the inflammasome components nlrp3 and caspase-1 are hypersusceptible to gastrointestinal inflammation induced by citrobacter rodentium , an enteric bacterial pathogen of the mouse intestinal tract that triggers inflammatory responses resembling those of humans infected with enteropathogenic and enterohemorrhagic escherichia coli . the increased host susceptibility to c. rodentium is due to the failure to produce normal levels of il-1 and il-18 in the presence of nlrp3 and caspase-1 deficiency . nlrp3-deficient mice had been reported to show increased susceptibility to dextran - sulfate - sodium- ( dss- ) induced colitis with increased mortality and weight loss in three different studies [ 4951 ] . however , other authors did not show a negative regulatory effect of nlrp3 on colitis , showing that nlrp3-null mice or mice pretreated with the caspase-1 inhibitor pralnacasan had less severe colitis when treated with dss , which was related to decreased il-1 secretion of dss - exposed nlrp3-deficient macrophages in vitro [ 52 , 53 ] . this discrepancy could be due not only to differences in protocols but also to baseline differences in the gut microbiota that might account for the dissimilar phenotypes . the crucial role of inflammasome components in the impairments of the gut microbiota composition is also suggested by recent studies demonstrating that nlrp3 inflammasome regulates the gastrointestinal microbiome and can thereby affect host susceptibility to diseases beyond the gastrointestinal tract , including obesity and diabetes . in particular , modulation of the intestinal microbiota through multiple inflammasome components has been recently demonstrated to be a critical determinant of nafld / nash progression as well as multiple other aspects of metabolic syndrome such as weight gain and glucose homeostasis . inflammasome - deficiency - associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of tlr4 and tlr9 agonists into the portal circulation , leading to enhanced hepatic tumour - necrosis factor- ( tnf- ) expression that drives nash progression . little is known of the role of the nlrp3 inflammasome complex in the development of renal metabolic damage . in humans , il-18 and caspase-1 are expressed in renal tubular epithelium , and patients with chronic kidney disease or the nephrotic syndrome exhibit elevated levels of il-18 [ 5557 ] . in a cohort of renal biopsies from patients with nondiabetic kidney disease , levels of mrna encoding nlrp3 correlate with renal function , strongly suggesting that nlrp3 contributes to the pathogenesis of chronic kidney disease . this is supported by experimental data showing that inflammasome - regulated cytokines such as il-1 and il-18 are implicated in animal models of chronic kidney disease , including glomerulonephritis and renal ischemic injury . in an animal study aimed to evaluate the renal consequences of the chronic administration of high - fructose corn syrup ( hfcs-55 ) , the major sweetener in foods and soft - drinks , we have recently demonstrated that hfcs-55 feeding caused a significant increase in body weight and more importantly dyslipidemia , hyperinsulinemia , and an increase in insulin resistance due to impaired insulin signaling . most notably , the hfcs-55 diet evoked upregulation of renal nlrp3 expression , resulting in activation of caspase-1 and the subsequent cleavage of pro - il1 to the biologically active secreted form il-1. these effects were due , at least in part , to the marked hyperuricemia afforded by the dietary manipulation , as also confirmed by a previous study demonstrating that increased levels of uric acid directly activate the nlrp3 inflammasome . similarly , rats fed with fructose , which is known to raise uric acid levels , showed a significant increase in renal protein levels of nlrp3 . however , one important question that remains to be answered regards the specific cell types involved in renal nlrp3 activation . as several studies have shown that monocyte / macrophage recruitment to the kidney significantly contributes to the renal injury , we can not rule out that the increased nlrp3 activation in the kidney is due to an increase in the infiltrating macrophages or , more likely , to a crosstalk between macrophages and tubular / glomerular cells . although it has been recently proposed that sarcopenia ( loss of muscle mass ) and myosteatosis ( fat infiltration in skeletal muscle ) exert a key role in triggering insulin resistance in obese patients , so far the potential role of nlrp3 complex activation in muscle activity and muscle production of inflammatory mediators has not yet been investigated . however , there is evidence that components of the inflammasome complex are upregulated in dysferlin - deficient human muscle , thus suggesting that skeletal muscle cells can actively participate in inflammasome activation . this is a crucial point as recent studies have demonstrated that skeletal muscle cells produce and release cytokines ( myokines ) that act in an autocrine , paracrine , and/or endocrine manner to modulate metabolic and inflammatory process . for example , it has been demonstrated very recently that muscular expression of pgc-1 alpha stimulates the secretion of a newly identified myokine , irisin , which improves glucose homeostasis and causes weight loss . however , the interactions between local nlrp3 expression / activity and myokines production as well as the effects of these interactions on muscle structure , function , and insulin - sensitivity in animals and humans have never been investigated . interestingly , both il-1 and il-18 seem to exert a crucial role also in the initiation and progression of the idiopathic inflammatory myopathies , a heterogeneous group of chronic disorders with predominant inflammation in muscle tissue , including dermatomyositis , polymyositis , and myositis [ 6567 ] . studies elucidating the detailed involvement of muscular inflammasome protein complex may thus provide promising targets for new therapies for this heterogeneous group of inflammatory muscle diseases . individuals with obesity and insulin resistance have an increased burden of cardiovascular disease ( cvd ) . in the kuopio ischemic heart disease study , lakka et al . reported a 4.26-fold relative risk for mortality due to heart disease and a 1.77 relative risk for all - cause mortality in obese , insulin - resistant patients . similarly , in the botnia study the risk for coronary heart disease ( chd ) and stroke was shown to be increased threefold and the risk for cardiovascular mortality was increased six fold . the hoorn study examined 615 men and 749 women aged 50 to 75 years without diabetes or a history of cvd at baseline and reported that the development of insulin resistance and/or obesity was associated with about a twofold increase in age - adjusted risk of fatal cvd in men and nonfatal cvd in women . studies published during the past decade have convincingly demonstrated a pathophysiological role for the inflammatory response in the development of both insulin resistance and related cvd . the finding a little over a decade ago that the secretion of il-1 and il-18 was increased in an ischemia / reperfusion ( i / r ) model of suprafused human atrial myocardium provided the first clear link between inflammasome activation and cvd development . experimental studies in mice with genetic deletion of caspase-1 have identified caspase-1 inhibition as a potential target for pharmacological intervention in the setting of cvd [ 7274 ] . a recent report described formation of the inflammasome in a mouse model of myocardial i / r , mainly in cardiac fibroblasts and infiltrating cells , and reported that asc knockout mice were protected , with a significant decline in cardiac infiltration of phagocytes , inflammatory cytokine levels , infarct size , and myocardial fibrosis and dysfunction . the inflammasome was also detected in cardiomyocytes bordering the infarct zone during the infarct process , and prevention of inflammasome activation limited infarct size and cardiac enlargement after acute myocardial i / r injury in the mouse . nlrp3 deficiency protects mice also from renal i / r injury [ 77 , 78 ] . both studies showed that the absence of nlrp3 protected kidneys against renal i / r injury to a greater extent than the absence of asc , suggesting that nlrp3 may play an additional role in renal i / r injury independently of asc and caspase-1 . overall , the ability of members of the nlrp3 inflammasome protein complex to target molecular and cellular pathways involved in both metabolic and cardiovascular diseases suggest that selective pharmacological modulation of nlrp3 inflammasome has the potential to exert synergistic effects in the control of metabolic disorders and its cardiovascular complications . thus , this unique therapeutic strategy could decrease the burden of cardiovascular morbidity and mortality in the presence of obesity and insulin resistance , although , to date , there are no clinical data to support this concept . in conclusion , nlrp3 inflammasome is a novel protein complex that integrates multiple exogenous and endogenous danger signals into the immediate secretion of il-1 and il-18 . most recent data suggest that activation of the nlrp3 inflammasome complex contributes to the pathophysiological mechanisms that explain the development of visceral obesity and insulin resistance . thanks to its wide distribution in different tissues and organs , the nlrp3 inflammasome protein complex may represent a crucial signaling pathway that facilitates organ crosstalk and local injury in tissues target of metabolic damage . a better understanding of this novel pathway could help to clarify the crucial role of the molecular mechanisms of interorgan crosstalk during obesity and insulin resistance development . studies using animal models and human biopsies will be useful to determine the spatial and temporal expression of inflammasome components inside the organs and to correlate these findings with disease activity or prognosis . gene polymorphism studies on suitable patient cohorts could help to determine the functional significance of the protein expression data . finally , the identification of selective pharmacological tools able to affect expression and/or activity of this novel pathway could represent the ultimate proof of significance of the inflammasome - caspase-1-il-1/18 axis in the development of metabolic inflammation . the effects evoked by these novel pharmacological tools should be compared with effects obtained by targeting selective cytokine receptor activities in order to better elucidate the potential crucial role of nlrp3 inflammasome protein complex in mediating inflammatory diseases . this approach may not only offer a potentially fruitful area of research , but it will also hopefully lead to novel and specific therapies for obesity - related conditions such as insulin resistance and its associated cardiovascular complications .

the combination of obesity and type 2 diabetes is a serious health problem , which is projected to afflict 300 million people worldwide by 2020 . both clinical and translational laboratory studies have demonstrated that chronic inflammation is associated with obesity and obesity - related conditions such as insulin resistance . however , the precise etiopathogenetic mechanisms linking obesity to diabetes remain to be elucidated , and the pathways that mediate this phenomenon are not fully characterized . one of the most recently identified signaling pathways , whose activation seems to affect many metabolic disorders , is the inflammasome , a multiprotein complex composed of nlrp3 ( nucleotide - binding domain and leucine - rich repeat protein 3 ) , asc ( apoptosis - associated speck - like protein containing a card ) , and procaspase-1 . nlrp3 inflammasome activation leads to the processing and secretion of the proinflammatory cytokines interleukin- ( il- ) 1 and il-18 . the goal of this paper is to review new insights on the effects of the nlrp3 inflammasome activation in the complex mechanisms of crosstalk between different organs , for a better understanding of the role of chronic inflammation in metabolic disease pathogenesis . we will provide here a perspective on the current research on nlrp3 inflammasome , which may represent an innovative therapeutic target to reverse the detrimental metabolic consequences of the metabolic inflammation .

1. The NLRP3 Inflammasome: An Overview 2. The NLRP3 Inflammasome in Obesity and Type 2 Diabetes 3. The NLRP3 Inflammasome and the Organ Crosstalk in the Metabolic Inflammation 4. NLRP3 Activation and Cardiovascular Complications of Metabolic Disorders 5. Conclusions

the inflammasomes are signaling platforms , which are assembled in response to pathogen - associated and damage - associated molecular pattern molecules and environmental irritants . the nlrp3 inflammasome is a multiprotein , large cytoplasmic complex ( > 700 kda ) , composed of a specific member of the nod - like receptor protein ( nlrp ) subfamily , the adaptor protein named apoptosis - associated speck - like protein containing a card ( asc ) , and procaspase-1 , which are preferentially expressed in adipose tissue macrophages ( atms ) . unlike the typical signaling cascades downstream of many innate receptors such as other nlrp members , nlrp3 contains an n - terminal pyrin domain ( pyd ) , which is used to physically interact with the pyd domain of asc , thus facilitating the subsequent recruitment and activation of procaspase-1 . , once activated , caspase-1 , as far as we are currently aware , cleaves the proforms of two potent proinflammatory cytokines interleukin- ( il- ) 1 and il-18 in the cytoplasm . the primary role of the inflammasome and its products seems to be as part of the body 's innate immune system , in that they can be triggered to assist in the defense against invading pathogens . indeed much of the data published on the inflammasome / caspase 1 is on its role in the body 's response to microbial molecules ( bacterial , fungal , or viral ) with conserved molecular structures known as pathogen associated molecular patterns ( pamps ) [ 5 , 6 ] . in addition to pamps , the nlrp3 inflammasome is also proficient in sensing stress to endogenous ( nonmicrobial ) danger signals ( danger associated molecular patterns , damps ) from damaged cells . damps can include molecules such as reactive oxygen species ( ros ) , adenosine triphosphate ( atp ) , hypotonic stress , uric acid crystals , or noxious exogenous factors such as environmental insults , asbestos , and uv radiation . there are a number of potential mechanisms for the assembly of the nlrp3 inflammasome , as described earlier . upon uptake of such substances , lysosomal rupture leads to the leakage of lysosomal proteases , specifically cathepsins b and l , into the cytosol where they could possibly mediate nlrp3 inflammasome activation by an as - yet - undefined cleavage event . very recently , vajjhala and colleagues have shed light on the molecular details of the complex mechanisms of nlrp3 inflammasome assembly and activation , identifying multiple binding sites on the pyd domain of the adaptor protein asc which allow self - association and interaction with binding partners . several in vitro , in vivo studies and clinical trials provide evidence that supports a causative role of il-1 in the pathogenesis of type 2 diabetes , and elevation in circulating levels of il-1 predicts type 2 diabetes when combined with serum il-6 levels . in humans , elevated plasma levels of il-1 have been found to be predictive of type 2 diabetes , and clinical studies have suggested that treatment with the il-1 receptor antagonist anakinra has beneficial effects in type 2 diabetic patients . a number of recent landmark studies have pointed out a key role for an excessive nlrp3 inflammasome activation in the il-1-related development of type 2 diabetes . the association between the nlrp3 inflammasome and both insulin resistance and obesity has been suggested by animal studies showing that genetic ablation of nlrp3 improved insulin sensitivity and glucose homeostasis . in line with the rise in insulin sensitivity , il-1 production of adipose tissue isolated from nlrp3 knockout mice other studies [ 12 , 13 ] have shown that improvement in insulin sensitivity ( increased phosphorylation of the insulin receptor subtrate-1 and akt ) can also be detected in liver and muscle of nlrp3 knockout mice on a high - fat diet for 12 weeks . ablation of the nlrp3 in mice has been also reported to protect from obesity - associated macrophage activation in adipose tissue , reducing m1-like macrophage gene expression ( tumor necrosis factor- , chemokine ligand 20 , and chemokine ligand 11 ) and increasing the expression of m2-like cytokines ( interleukin-10 ) . to confirm the clinical relevance of these data generated from mouse models , the same authors have demonstrated that weight loss reduced nlrp3 expression in abdominal subcutaneous adipose tissue in obese patients with type 2 diabetes , which was accompanied by improved glucose homeostasis . furthermore , strong correlations between the expression of nlrp3 inflammasome - related genes and insulin resistance have been recently reported in obese male subjects with impaired glucose tolerance . additionally , type 2 diabetic patients showed elevated levels of nlrp3 , asc , il-1 , and il-18 mrna and protein expression in monocyte - derived macrophages , compared with those in healthy control subjects . a major role of the er is to ensure the synthesis and folding of membrane and secreted proteins , and any disturbance in this function ( e.g. , excessive protein synthesis or accumulation of unfolded or misfolded proteins in the er lumen ) leads to an er stress the recent literature suggests that er stress may act directly as a negative modulator of the insulin biosynthesis and insulin signaling pathways but also indirectly by promoting lipid accumulation [ 25 , 26 ] . er stress also plays a role in the dysregulation of adipokine secretion by adipose tissue , frequently observed in obesity and insulin resistance [ 27 , 28 ] , and cd14 + monocytes isolated from diabetic patients showed evidence of er stress , which may underlie the functional defects in these cells . interestingly , er stress has been recently demonstrated to activate the nlrp3 inflammasome , resulting in the subsequent release of il-1 by human macrophages , with an activation mechanism similar to that of other known nlrp3 activators , requiring ros generation and potassium efflux . the thioredoxin - interacting protein ( txnip ) , a critical node in the development of er stress leading to programmed cell death of pancreatic cells , activates the nlrp3 inflammasome , causing procaspase-1 cleavage and il-1 secretion in human monocytic cells . the role of er stress in promoting nlrp3 inflammasome activation is consistent with the subcellular localization of nlrp3 . in resting cells , nlrp3 is associated with er membranes , and then upon activation nlrp3 is redistribute to the perinuclear space where it colocalizes with endoplasmic reticulum and mitochondria organelle clusters . nlrp3 inflammasome plays a substantial role in sensing obesity - associated inducers of caspase-1 activation and therefore regulates the magnitude of the inflammation and its downstream effects on insulin signaling in different organs , as reported here later . thus , most of the first studies characterizing the role of nlrp3 signaling have been conducted in cells of the immune system . several studies on innate immune cells have demonstrated that the myeloid - derived nlrp3 inflammasome complex may contribute to promote inflammatory cytokine production and insulin resistance through reduction of insulin signaling . in vitro experiments have shown that elevated concentration of saturated fatty acids ( sfas ) , caused by a high - fat diet , may activate the nlrp3 inflammasome in macrophages through a newly identified amp - activated protein kinase and unc-51-like kinase-1 autophagy signaling cascade . more specifically , dietary sfa may act as a primer of the nlrp3 inflammasome protein complex enhancing nlrp3 , caspase-1 , and pro - il-1 mrna expression . these findings highlight a new model of organ crosstalk , in which leukocyte and macrophage recruitment in key insulin target tissues , such as liver , adipose , and muscle , may promote insulin resistance by enhancing inflammasome activation . as in diabetic patients pancreas , adipose tissue , liver , and kidney , with infiltrated macrophages , are major sites of origin of inflammation , it might be intriguing to investigate the specific contribution of nlrp3 inflammasome activation in these different insulin target tissues and to identify the specific inducers that selectivity participate in the mechanism of tissue nlrp3 inflammasome activation . in keeping with these results , neutralizing il-1 on isolated beta cells using il-1 receptor antagonist however , the precise mechanism(s ) by which il-1 affects pancreatic -cell failure is still debated . they showed that thioredoxin - interacting protein ( txnip ) , also known as vitamin d3 upregulated protein 1 ( vdup1 ) , is an upstream and highly selective activating ligand for nlrp3 , with no effect on the activity of other inflammasomes ( e.g. txnip - dependent nlrp3 inflammasome activation drives il-1 secretion from pancreatic islets in response to chronic elevated glucose , thus suggesting , for the first time , that nlrp3 , activated under conditions of metabolic stress , mediates il-1-driven islet failure . other authors have identified oligomers of iapp , as a key trigger for nlrp3 inflammasome activation and the following processing of il-1 . obesity - induced pancreatic -cell death is regulated , at least in part , by the nlrp3 inflammasome , as demonstrated in nlrp3-deficient mice in late - stage obesity , where the ablation of nlrp3 is associated with reduced cell death and increase in pancreatic islet size and local insulin levels . , the nlrp3 inflammasome is activated in adipose tissue in mouse models of obesity and attenuated by calorie restriction . nlrp3 inflammasome levels also correlate with glycaemia in type 2 diabetes patients after weight loss interventions . in contrast , the inflammasome in subcutaneous adipose tissue adipocytes did not seem to be grossly influenced by the presence of the metabolic syndrome . partial depletion of macrophages from adipose tissue of obese animals decreased the expression of the macrophage marker cd68 , with no significant alteration in the expression of caspase-1 , thus suggesting that the effects of the nlrp3-asc - caspase-1 protein complex on adipose tissue are not only exerted though infiltrating macrophages . in addition , adipocyte upregulation of il-1 expression and secretion in response to inflammatory stimuli has been shown to induce hepatic insulin resistance , thus suggesting a further intriguing role for nlrp3 inflammasome activation in the dysfunctional communication between adipocytes and hepatocytes [ 35 , 43 ] . overall these data reveal a novel metabolic function of the nlrp3 inflammasome in adipose tissue , suggesting that its pharmacological modulation in obese and/or patients with type 2 diabetes may restore the metabolic function of adipose tissue and subsequently improve insulin sensitivity . the presence of nlrp3 inflammasome and/or inflammasome activation has been shown in sinusoidal endothelial cells , stellate cells , and hepatocytes . recently , inflammasome activation has been associated with nash , and long - term high - fat diet administration resulted in reduced hepatic steatosis in nlrp3 knockout mice . in contrast , to date , there are no data suggesting a potential role of nlrp3 inflammasome activation on impaired glycogen synthesis and/or augmented glycogenolysis . there is evidence that the inflammasome components are important in the maintenance of the integrity of the intestinal epithelium and the defense against pathogenic organisms that can invade the gastrointestinal tract . the increased host susceptibility to c. rodentium is due to the failure to produce normal levels of il-1 and il-18 in the presence of nlrp3 and caspase-1 deficiency . however , other authors did not show a negative regulatory effect of nlrp3 on colitis , showing that nlrp3-null mice or mice pretreated with the caspase-1 inhibitor pralnacasan had less severe colitis when treated with dss , which was related to decreased il-1 secretion of dss - exposed nlrp3-deficient macrophages in vitro [ 52 , 53 ] . the crucial role of inflammasome components in the impairments of the gut microbiota composition is also suggested by recent studies demonstrating that nlrp3 inflammasome regulates the gastrointestinal microbiome and can thereby affect host susceptibility to diseases beyond the gastrointestinal tract , including obesity and diabetes . in particular , modulation of the intestinal microbiota through multiple inflammasome components has been recently demonstrated to be a critical determinant of nafld / nash progression as well as multiple other aspects of metabolic syndrome such as weight gain and glucose homeostasis . inflammasome - deficiency - associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of tlr4 and tlr9 agonists into the portal circulation , leading to enhanced hepatic tumour - necrosis factor- ( tnf- ) expression that drives nash progression . little is known of the role of the nlrp3 inflammasome complex in the development of renal metabolic damage . this is supported by experimental data showing that inflammasome - regulated cytokines such as il-1 and il-18 are implicated in animal models of chronic kidney disease , including glomerulonephritis and renal ischemic injury . in an animal study aimed to evaluate the renal consequences of the chronic administration of high - fructose corn syrup ( hfcs-55 ) , the major sweetener in foods and soft - drinks , we have recently demonstrated that hfcs-55 feeding caused a significant increase in body weight and more importantly dyslipidemia , hyperinsulinemia , and an increase in insulin resistance due to impaired insulin signaling . most notably , the hfcs-55 diet evoked upregulation of renal nlrp3 expression , resulting in activation of caspase-1 and the subsequent cleavage of pro - il1 to the biologically active secreted form il-1. these effects were due , at least in part , to the marked hyperuricemia afforded by the dietary manipulation , as also confirmed by a previous study demonstrating that increased levels of uric acid directly activate the nlrp3 inflammasome . similarly , rats fed with fructose , which is known to raise uric acid levels , showed a significant increase in renal protein levels of nlrp3 . as several studies have shown that monocyte / macrophage recruitment to the kidney significantly contributes to the renal injury , we can not rule out that the increased nlrp3 activation in the kidney is due to an increase in the infiltrating macrophages or , more likely , to a crosstalk between macrophages and tubular / glomerular cells . although it has been recently proposed that sarcopenia ( loss of muscle mass ) and myosteatosis ( fat infiltration in skeletal muscle ) exert a key role in triggering insulin resistance in obese patients , so far the potential role of nlrp3 complex activation in muscle activity and muscle production of inflammatory mediators has not yet been investigated . however , there is evidence that components of the inflammasome complex are upregulated in dysferlin - deficient human muscle , thus suggesting that skeletal muscle cells can actively participate in inflammasome activation . this is a crucial point as recent studies have demonstrated that skeletal muscle cells produce and release cytokines ( myokines ) that act in an autocrine , paracrine , and/or endocrine manner to modulate metabolic and inflammatory process . however , the interactions between local nlrp3 expression / activity and myokines production as well as the effects of these interactions on muscle structure , function , and insulin - sensitivity in animals and humans have never been investigated . interestingly , both il-1 and il-18 seem to exert a crucial role also in the initiation and progression of the idiopathic inflammatory myopathies , a heterogeneous group of chronic disorders with predominant inflammation in muscle tissue , including dermatomyositis , polymyositis , and myositis [ 6567 ] . individuals with obesity and insulin resistance have an increased burden of cardiovascular disease ( cvd ) . similarly , in the botnia study the risk for coronary heart disease ( chd ) and stroke was shown to be increased threefold and the risk for cardiovascular mortality was increased six fold . studies published during the past decade have convincingly demonstrated a pathophysiological role for the inflammatory response in the development of both insulin resistance and related cvd . the finding a little over a decade ago that the secretion of il-1 and il-18 was increased in an ischemia / reperfusion ( i / r ) model of suprafused human atrial myocardium provided the first clear link between inflammasome activation and cvd development . a recent report described formation of the inflammasome in a mouse model of myocardial i / r , mainly in cardiac fibroblasts and infiltrating cells , and reported that asc knockout mice were protected , with a significant decline in cardiac infiltration of phagocytes , inflammatory cytokine levels , infarct size , and myocardial fibrosis and dysfunction . the inflammasome was also detected in cardiomyocytes bordering the infarct zone during the infarct process , and prevention of inflammasome activation limited infarct size and cardiac enlargement after acute myocardial i / r injury in the mouse . overall , the ability of members of the nlrp3 inflammasome protein complex to target molecular and cellular pathways involved in both metabolic and cardiovascular diseases suggest that selective pharmacological modulation of nlrp3 inflammasome has the potential to exert synergistic effects in the control of metabolic disorders and its cardiovascular complications . thus , this unique therapeutic strategy could decrease the burden of cardiovascular morbidity and mortality in the presence of obesity and insulin resistance , although , to date , there are no clinical data to support this concept . in conclusion , nlrp3 inflammasome is a novel protein complex that integrates multiple exogenous and endogenous danger signals into the immediate secretion of il-1 and il-18 . most recent data suggest that activation of the nlrp3 inflammasome complex contributes to the pathophysiological mechanisms that explain the development of visceral obesity and insulin resistance . thanks to its wide distribution in different tissues and organs , the nlrp3 inflammasome protein complex may represent a crucial signaling pathway that facilitates organ crosstalk and local injury in tissues target of metabolic damage . a better understanding of this novel pathway could help to clarify the crucial role of the molecular mechanisms of interorgan crosstalk during obesity and insulin resistance development . finally , the identification of selective pharmacological tools able to affect expression and/or activity of this novel pathway could represent the ultimate proof of significance of the inflammasome - caspase-1-il-1/18 axis in the development of metabolic inflammation . the effects evoked by these novel pharmacological tools should be compared with effects obtained by targeting selective cytokine receptor activities in order to better elucidate the potential crucial role of nlrp3 inflammasome protein complex in mediating inflammatory diseases . this approach may not only offer a potentially fruitful area of research , but it will also hopefully lead to novel and specific therapies for obesity - related conditions such as insulin resistance and its associated cardiovascular complications .

unintended pregnancy ( including both unwanted and mistimed pregnancy ) is one among the most important issues of reproductive health . from 1995 to 2008 , the unintended pregnancy rate reduced by 17% worldwide ; nonetheless , about 41% of all pregnancies 4 out of 10 pregnancies are unintended . in iran , the rate of unintended pregnancy decreased from 37% in 1992 to 21% in 2009 and has remained constant since then . about 73.3% of iranian families use different types of contraceptive methods ; however , none of the contraceptive methods is reliable . consequently , families still experience unintended pregnancies . lifetime abortion rate in iran is estimated to be one per four women . porter highlighted the importance of religious beliefs in dealing with unintended pregnancy and recommended further studies on this subject area in different ethnic and religious groups . induced abortion has been prohibited ; accordingly , induced abortion is clearly illegal in iran . however , since 2003 , severely high - risk pregnancies have been allowed to be terminated before the ensoulment of the fetus , i.e. before the 19 week of last menstrual period , with the permission of iranian forensic medicine organization . accordingly , the illegality of induced abortion in iran can affect iranian women 's perceptions and experiences of unintended pregnancy . most of the studies on women 's experiences of unintended pregnancy have been conducted on unmarried teens . on the other hand , studies on married women the center for disease control and prevention ( 2009 ) reported that as women 's perceptions and experiences of unintended pregnancy and induced abortion are poorly known , examining the effectiveness of healthcare services provided to women experiencing unintended pregnancy as well as developing new care plans for them are difficult . consequently , exploring women 's experiences of unintended pregnancy and induced abortion in different socio - cultural contexts is a matter of great importance . in iran , many studies have been conducted on the causes and the outcomes of unintended pregnancy and induced abortion ; however , iranian women 's beliefs , attitudes , and experiences surrounding unintended pregnancy and induced abortion are still poorly known . to prevent illegal induced abortions , the aim of the conventional content analysis approach is to study and explain poorly known phenomena . the study setting was all the public and private health clinics as well as private gynecologists and midwives offices located in tabriz . tabriz is one of the five biggest cities of iran that is located in northwest of the country . the study population consisted of all the women having a recent unintended pregnancy who either had decided to continue their pregnancy ( and were in their last 4 months of it or had delivered in the past 40 days ) or had electively aborted it recently ( in the previous 6 months ) . the women who were candidates for obtaining therapeutic abortion and women with established diagnosis of psychological disorders were excluded from the study . the participants were selected through purposeful sampling method from 10 different districts of tabriz city , according to their socioeconomic status . totally , 31 face - to - face unstructured interviews with 23 women were conducted from march to august 2013 . the main interview question was , what were your feelings and concerns about having an unintended pregnancy ? then we employed probing questions to delve into the participants perceptions . sampling and data collection were continued until reaching data saturation , i.e. until the generated concepts and categories were fully developed and refined and no new data was obtained from the interviews . the main and the follow - up interviews lasted for 45 - 80 and 15 - 20 min , respectively . interviews were conducted in a counseling room located in the study setting and recorded using a digital sound recorder . four participants withheld consent to record their voices . consequently , we made note of their narratives . the interviews were transcribed verbatim and reviewed several times to get a basic sense of participants perceptions . then , the generated codes were compared with each other and were categorized according to their differences and similarities . the generated categories were also compared with each other and were categorized into higher - level categories and themes . table 1 shows how meaning units , codes , and sub - categories formed one of the main categories of the study . meaning units , codes , and sub - categories of fear of life difficulty and instability category using several strategies such as maximum variation sampling , member checking , prolonged engagement with the study subject matter , and establishment of an effective communication with the study participants helped improve the credibility of the study findings . in the member - checking process , three participants were asked to check the congruency between their own perceptions and the generated concepts and categories . on the other hand , the peer - checking technique was employed to enhance the dependability of the study findings . the audit team comprised two of the study authors who were experts in qualitative data analysis . they recoded all the interviews and supervised the study . to guarantee the confirmability of the study findings , bracketing was done to prevent preconceptions from affecting the processes of data collection and analysis . finally , transferability of the present study was ensured by rich description in order that other researchers can conduct similar projects and compare the findings . the ethics committee of shahid beheshti university of medical sciences , iran , approved the study . the aim of the study was clearly explained to the study participants and accordingly , their questions and concerns were addressed . both participation in and withdrawal from the study were completely voluntary . moreover , the confidentiality of the participants personal information was guaranteed . the participants were also informed how to access the study findings . finally , immediately before each interview , a written informed consent was obtained . the study participants consisted of 13 women with unwanted pregnancy and 10 women with mistimed pregnancy , between the ages of 18 and 48 , of which 10 women had decided to abort and 13 women had continued to term . five participants had education up to high school , 6 participants had obtained diploma , and 12 participants had some level of college education . eleven participants made reference to employment , and only 3 of the 23 participants were university students while unintended pregnancy was detected . the study data analysis process yielded 214 primary codes , 17 sub - categories , 8 categories , 3 main themes , and the central overarching theme of threat supposition [ table 2 ] . a summary of findings the first theme of the study was the negative effects of unintended pregnancy on daily life . the participants equated unintended pregnancy with deviation from their normal life routines and also with a potential threat to them . they referred to their coming baby as an uninvited guest and a major challenge that could result in disturbed tranquility , life instability , and social deprivations . the four sub - themes of this theme included fear of life difficulty and instability , social deprivations , negative maternal and child outcomes , and inability to fulfill parental commitments and responsibilities . threatening family stability and growing instability and inability to fulfill multiple roles : role conflict constituted a great part of the experiences of the participants , so that one of the women 's concerns of unintended pregnancy was the possibility for encountering financial pressure and difficulty . i felt i would experience financial problems ; i was afraid of potential financial problems . ( obtained elective abortion ) factors such as tenancy , unemployment , home office long distance , marital conflicts , and uncertainty over the prospect of marital life in young newly married couples heightened the participants fear of growing instability after encountering an unintended pregnancy . on the other hand , the perceived difficulty of rearing subsequent little babies , as well as the perceived difficulty of fulfilling multiple conflicting parental and social roles had brought the study participants into role conflict . when i got pregnant , i ( with her face turned sour ) remembered the difficulties of rearing and taking my children to nursery . ( obtained elective abortion ) the second perceived negative effect of unintended pregnancy on women 's daily lives was the probability of suffering from social deprivations . the participants thought that their unintended pregnancy would interfere with their academic and occupational promotion and limit their participation in social activities . i felt that a new baby would cripple me and consequently would hamper the pursuit of my education at phd level . ( obtained elective abortion ) other aspects of social deprivations induced by unintended pregnancy were the restriction imposed on social interactions , recreational activities , and the freedom to travel as well as the inability to attend parties . one of the women said : with two babies , you should do without many things including recreations and parties . you may know that turk men pass on the childrearing responsibilities to their women ; two babies could largely confine me . ( p. 7 ; continued her pregnancy ) another negative effect of unintended pregnancy on women 's daily lives was inability to fulfill parental commitments and responsibilities ( for the previous children ) . women who had either several babies or two subsequent little babies were more worried about being unable to successfully fulfill their parental commitments and responsibilities . ( obtained elective abortion ) being unable to fulfill the coming baby 's emotional and financial needs , as well as being unable to build a high - quality life for him / her were among the other concerns of women over their unintended pregnancy . i know that i can only fulfill the needs of my current two babies and not of a third one . therefore , giving birth to the third baby would be some sort of mercilessness . ( p. 9 ; obtained elective abortion ) negative maternal and child outcomes formed another aspect of the participants perceived negative effects of unintended pregnancy on daily life . an unintended pregnancy may happen at older ages , while a woman is using contraceptive methods or certain types of medications , or is undergoing x - ray studies . all of these conditions have teratogenic potential and may put every fetus at great risk for developing health problems . consequently , the participants noted that they were worried about their fetus 's health status . i am too old to pregnancy ; my baby may be born with mental retardation at this age , which can potentially ruin my life . ( p. 2 ; obtained elective abortion ) besides being worried about their fetus 's health , the participating women were also worried about their own health . pregnancy at ages above 35 years is usually considered as high - risk and can be associated with pregnancy - induced hypertension . ( obtained elective abortion ) the second theme of the study was the fear of being stigmatized with violating social norms . two sub - themes of this theme included violating childbearing norms and the social taboo of abortion . an important dimension of violating childbearing norms was related to the social restrictions of childbearing . in other words , becoming pregnant while having two to three children , very soon after marriage , or at older ages had given the study participants a sense of violating childbearing norms . as the childbearing pattern reflects the couple 's socio - cultural class , violating childbearing norms could lower their social prestige . both of us ( my husband and i ) were worried about other 's outlook ( toward us ) . it seemed as if a repulsive force was saying to us , you are not anymore eligible for childbearing. i thought of others opinion about such a big 17-year gap between our second and the coming children . well , people would think that we are probably of a lower social class that did not take into account such important considerations . ; obtained elective abortion ) in some instances , the fear of others blame and derision had been so great that the participants , at the early stages of pregnancy , had preferred to hide their pregnancy and isolate themselves socially . i avoided going to my parents home and attending ceremonies because i thought people would humiliate me . i refrained going out to avoid people 's talks . ( continued her pregnancy ) the other dimension of violating childbearing norms was related to the prevalence of the only - child practice in some iranian families , which in turn made the continuation of the second pregnancy difficult . in such an unsupportive environment , giving birth to a second baby was not so much admirable . ( obtained elective abortion ) another dimension of social norms violation stigma was related to the social taboo of abortion . an important strategy to get rid of the fears and difficulties of an unintended pregnancy was to obtain an induced abortion . however , relatives and friends disagreement with abortion was in turn the additional source of fear and anxiety for the study participants . fear of others opposition to abortion had compelled the participants to hide their pregnancy and abortion from relatives and friends . i feared my mother , sister , and mother- and father - in - law would notice my pregnancy and my intention to abort it . one of my problems was that i needed to hide it from others because i feared they would prevent me from obtaining abortion . ( p. 1 ; obtained elective abortion ) the other dimension of the social taboo concerning abortion was related to its illegality . due to lack of social support for abortion and its illegality , the participants were worried about having access to abortion services . i had to obtain abortion an illegal , hard - to - access abortion . i did not know what i had to do and where i had to go to obtain abortion . ( p. 5 ; obtained elective abortion ) the third theme of the study was the panic over abortion . women 's abortion panic differed according to their moral and religious beliefs as well as past direct and indirect experiences . this theme consisted of two sub - themes including infringing belief and value systems and fear over difficulty and adverse effects of abortion . moral and religious dilemmas and fear of punishment were some of the major experiences of unintended pregnant women . consequently , they had experienced pangs of guilt and conscience . the severity of their feelings depended on the firmness of their religious beliefs and commitments . i had a twinge of guilt because i did not want the baby and hence decided to terminate my pregnancy . for example , i thought i was ignoring the god 's favor ; it was maybe some sort of ingratitude . ( obtained elective abortion ) some of the participants believed that abortion was equal to deliberate murder and could result in afterlife punishment . i remembered this verse of the holy quran , when the buried - alive female infant is asked , for what sin she was killed? then , i was scared . i thought that my baby would ask me in doomsday , for what sin did you kill me , mum ? ( continued her pregnancy ) women were also reluctant to obtain abortion because they believed that it was against god 's will and could result in worldly punishment . i thought god would curse and chastise my husband , my children , and me in this world . ( continued her pregnancy ) when thinking about abortion , most of the participants had experienced fear and anxiety over its difficulty and pain . ( continued her pregnancy ) on the other hand , women were greatly concerned with the adverse effects and complications of unsafe illegal abortion . however , i dreaded being permanently sterile ; i dreaded the adverse effects of abortion . ( a recently married young woman who had obtained elective abortion ) the three main themes of the study negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic generally reflected participants perceived threat and fear of unintended pregnancy outcomes and complications . it was a common theme emerging from the experiences of both those women who had continued with their pregnancy and those who had obtained elective induced abortion . in other words , participants in both groups had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds . but i also had an inner feeling of fear fear of killing a living being . the first theme of the study was the negative effects of unintended pregnancy on daily life . the participants equated unintended pregnancy with deviation from their normal life routines and also with a potential threat to them . they referred to their coming baby as an uninvited guest and a major challenge that could result in disturbed tranquility , life instability , and social deprivations . the four sub - themes of this theme included fear of life difficulty and instability , social deprivations , negative maternal and child outcomes , and inability to fulfill parental commitments and responsibilities . threatening family stability and growing instability and inability to fulfill multiple roles : role conflict constituted a great part of the experiences of the participants , so that one of the women 's concerns of unintended pregnancy was the possibility for encountering financial pressure and difficulty . i felt i would experience financial problems ; i was afraid of potential financial problems . ( obtained elective abortion ) factors such as tenancy , unemployment , home office long distance , marital conflicts , and uncertainty over the prospect of marital life in young newly married couples heightened the participants fear of growing instability after encountering an unintended pregnancy . on the other hand , the perceived difficulty of rearing subsequent little babies , as well as the perceived difficulty of fulfilling multiple conflicting parental and social roles had brought the study participants into role conflict . when i got pregnant , i ( with her face turned sour ) remembered the difficulties of rearing and taking my children to nursery . ( obtained elective abortion ) the second perceived negative effect of unintended pregnancy on women 's daily lives was the probability of suffering from social deprivations . the participants thought that their unintended pregnancy would interfere with their academic and occupational promotion and limit their participation in social activities . i felt that a new baby would cripple me and consequently would hamper the pursuit of my education at phd level . ( obtained elective abortion ) other aspects of social deprivations induced by unintended pregnancy were the restriction imposed on social interactions , recreational activities , and the freedom to travel as well as the inability to attend parties . one of the women said : with two babies , you should do without many things including recreations and parties . you may know that turk men pass on the childrearing responsibilities to their women ; two babies could largely confine me . ( p. 7 ; continued her pregnancy ) another negative effect of unintended pregnancy on women 's daily lives was inability to fulfill parental commitments and responsibilities ( for the previous children ) . women who had either several babies or two subsequent little babies were more worried about being unable to successfully fulfill their parental commitments and responsibilities . ( obtained elective abortion ) being unable to fulfill the coming baby 's emotional and financial needs , as well as being unable to build a high - quality life for him / her were among the other concerns of women over their unintended pregnancy . i know that i can only fulfill the needs of my current two babies and not of a third one . ( p. 9 ; obtained elective abortion ) negative maternal and child outcomes formed another aspect of the participants perceived negative effects of unintended pregnancy on daily life . an unintended pregnancy may happen at older ages , while a woman is using contraceptive methods or certain types of medications , or is undergoing x - ray studies . all of these conditions have teratogenic potential and may put every fetus at great risk for developing health problems . consequently , the participants noted that they were worried about their fetus 's health status . i am too old to pregnancy ; my baby may be born with mental retardation at this age , which can potentially ruin my life . ( p. 2 ; obtained elective abortion ) besides being worried about their fetus 's health , the participating women were also worried about their own health . pregnancy at ages above 35 years is usually considered as high - risk and can be associated with pregnancy - induced hypertension . threatening family stability and growing instability and inability to fulfill multiple roles : role conflict constituted a great part of the experiences of the participants , so that one of the women 's concerns of unintended pregnancy was the possibility for encountering financial pressure and difficulty . i felt i would experience financial problems ; i was afraid of potential financial problems . ( obtained elective abortion ) factors such as tenancy , unemployment , home office long distance , marital conflicts , and uncertainty over the prospect of marital life in young newly married couples heightened the participants fear of growing instability after encountering an unintended pregnancy . on the other hand , the perceived difficulty of rearing subsequent little babies , as well as the perceived difficulty of fulfilling multiple conflicting parental and social roles had brought the study participants into role conflict . when i got pregnant , i ( with her face turned sour ) remembered the difficulties of rearing and taking my children to nursery . the second perceived negative effect of unintended pregnancy on women 's daily lives was the probability of suffering from social deprivations . the participants thought that their unintended pregnancy would interfere with their academic and occupational promotion and limit their participation in social activities . i felt that a new baby would cripple me and consequently would hamper the pursuit of my education at phd level . ( obtained elective abortion ) other aspects of social deprivations induced by unintended pregnancy were the restriction imposed on social interactions , recreational activities , and the freedom to travel as well as the inability to attend parties . one of the women said : with two babies , you should do without many things including recreations and parties . you may know that turk men pass on the childrearing responsibilities to their women ; two babies could largely confine me . another negative effect of unintended pregnancy on women 's daily lives was inability to fulfill parental commitments and responsibilities ( for the previous children ) . women who had either several babies or two subsequent little babies were more worried about being unable to successfully fulfill their parental commitments and responsibilities . a participant ( no . ( obtained elective abortion ) being unable to fulfill the coming baby 's emotional and financial needs , as well as being unable to build a high - quality life for him / her were among the other concerns of women over their unintended pregnancy . i know that i can only fulfill the needs of my current two babies and not of a third one . therefore , giving birth to the third baby would be some sort of mercilessness . negative maternal and child outcomes formed another aspect of the participants perceived negative effects of unintended pregnancy on daily life . an unintended pregnancy may happen at older ages , while a woman is using contraceptive methods or certain types of medications , or is undergoing x - ray studies . all of these conditions have teratogenic potential and may put every fetus at great risk for developing health problems . consequently , the participants noted that they were worried about their fetus 's health status . i am too old to pregnancy ; my baby may be born with mental retardation at this age , which can potentially ruin my life . ( p. 2 ; obtained elective abortion ) besides being worried about their fetus 's health , the participating women were also worried about their own health . pregnancy at ages above 35 years is usually considered as high - risk and can be associated with pregnancy - induced hypertension . the second theme of the study was the fear of being stigmatized with violating social norms . two sub - themes of this theme included violating childbearing norms and the social taboo of abortion . an important dimension of violating childbearing norms was related to the social restrictions of childbearing . in other words , becoming pregnant while having two to three children , very soon after marriage , or at older ages had given the study participants a sense of violating childbearing norms . as the childbearing pattern reflects the couple 's socio - cultural class , violating childbearing norms could lower their social prestige . both of us ( my husband and i ) were worried about other 's outlook ( toward us ) . it seemed as if a repulsive force was saying to us , you are not anymore eligible for childbearing. i thought of others opinion about such a big 17-year gap between our second and the coming children . well , people would think that we are probably of a lower social class that did not take into account such important considerations . ( p. 3 ; obtained elective abortion ) in some instances , the fear of others blame and derision had been so great that the participants , at the early stages of pregnancy , had preferred to hide their pregnancy and isolate themselves socially . i avoided going to my parents home and attending ceremonies because i thought people would humiliate me . ( continued her pregnancy ) the other dimension of violating childbearing norms was related to the prevalence of the only - child practice in some iranian families , which in turn made the continuation of the second pregnancy difficult . in such an unsupportive environment , giving birth to a second baby was not so much admirable . ( obtained elective abortion ) another dimension of social norms violation stigma was related to the social taboo of abortion . an important strategy to get rid of the fears and difficulties of an unintended pregnancy was to obtain an induced abortion . however , relatives and friends disagreement with abortion was in turn the additional source of fear and anxiety for the study participants . fear of others opposition to abortion had compelled the participants to hide their pregnancy and abortion from relatives and friends . i feared my mother , sister , and mother- and father - in - law would notice my pregnancy and my intention to abort it . one of my problems was that i needed to hide it from others because i feared they would prevent me from obtaining abortion . ( p. 1 ; obtained elective abortion ) the other dimension of the social taboo concerning abortion was related to its illegality . due to lack of social support for abortion and its illegality , i had to obtain abortion an illegal , hard - to - access abortion . i did not know what i had to do and where i had to go to obtain abortion . an important dimension of violating childbearing norms was related to the social restrictions of childbearing . in other words , becoming pregnant while having two to three children , very soon after marriage , or at older ages had given the study participants a sense of violating childbearing norms . as the childbearing pattern reflects the couple 's socio - cultural class , violating childbearing norms could lower their social prestige . both of us ( my husband and i ) were worried about other 's outlook ( toward us ) . it seemed as if a repulsive force was saying to us , you are not anymore eligible for childbearing. i thought of others opinion about such a big 17-year gap between our second and the coming children . well , people would think that we are probably of a lower social class that did not take into account such important considerations . ( p. 3 ; obtained elective abortion ) in some instances , the fear of others blame and derision had been so great that the participants , at the early stages of pregnancy , had preferred to hide their pregnancy and isolate themselves socially . i avoided going to my parents home and attending ceremonies because i thought people would humiliate me . ( continued her pregnancy ) the other dimension of violating childbearing norms was related to the prevalence of the only - child practice in some iranian families , which in turn made the continuation of the second pregnancy difficult . in such an unsupportive environment , giving birth to a second baby was not so much admirable . another dimension of social norms violation stigma was related to the social taboo of abortion . an important strategy to get rid of the fears and difficulties of an unintended pregnancy was to obtain an induced abortion . however , relatives and friends disagreement with abortion was in turn the additional source of fear and anxiety for the study participants . fear of others opposition to abortion had compelled the participants to hide their pregnancy and abortion from relatives and friends . i feared my mother , sister , and mother- and father - in - law would notice my pregnancy and my intention to abort it . one of my problems was that i needed to hide it from others because i feared they would prevent me from obtaining abortion . ( p. 1 ; obtained elective abortion ) the other dimension of the social taboo concerning abortion was related to its illegality . due to lack of social support for abortion and its illegality , i had to obtain abortion an illegal , hard - to - access abortion . i did not know what i had to do and where i had to go to obtain abortion . women 's abortion panic differed according to their moral and religious beliefs as well as past direct and indirect experiences . this theme consisted of two sub - themes including infringing belief and value systems and fear over difficulty and adverse effects of abortion . moral and religious dilemmas and fear of punishment were some of the major experiences of unintended pregnant women . most of the participants had encountered moral and religious dilemmas . consequently , they had experienced pangs of guilt and conscience . the severity of their feelings depended on the firmness of their religious beliefs and commitments . i had a twinge of guilt because i did not want the baby and hence decided to terminate my pregnancy . for example , i thought i was ignoring the god 's favor ; it was maybe some sort of ingratitude . ( obtained elective abortion ) some of the participants believed that abortion was equal to deliberate murder and could result in afterlife punishment . i remembered this verse of the holy quran , when the buried - alive female infant is asked , for what sin she was killed? then , i was scared . i thought that my baby would ask me in doomsday , for what sin did you kill me , mum ? ( continued her pregnancy ) women were also reluctant to obtain abortion because they believed that it was against god 's will and could result in worldly punishment . i thought god would curse and chastise my husband , my children , and me in this world . ( continued her pregnancy ) when thinking about abortion , most of the participants had experienced fear and anxiety over its difficulty and pain . ( continued her pregnancy ) on the other hand , women were greatly concerned with the adverse effects and complications of unsafe illegal abortion . however , i dreaded being permanently sterile ; i dreaded the adverse effects of abortion . ( a recently married young woman who had obtained elective abortion ) the three main themes of the study negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic generally reflected participants perceived threat and fear of unintended pregnancy outcomes and complications . these themes constituted the broader overarching theme of threat supposition . it was a common theme emerging from the experiences of both those women who had continued with their pregnancy and those who had obtained elective induced abortion . in other words , participants in both groups had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds . but i also had an inner feeling of fear fear of killing a living being . moral and religious dilemmas and fear of punishment were some of the major experiences of unintended pregnant women . consequently , they had experienced pangs of guilt and conscience . the severity of their feelings depended on the firmness of their religious beliefs and commitments . i had a twinge of guilt because i did not want the baby and hence decided to terminate my pregnancy . for example , i thought i was ignoring the god 's favor ; it was maybe some sort of ingratitude . ( obtained elective abortion ) some of the participants believed that abortion was equal to deliberate murder and could result in afterlife punishment . i remembered this verse of the holy quran , when the buried - alive female infant is asked , for what sin she was killed? then , i was scared . i thought that my baby would ask me in doomsday , for what sin did you kill me , mum ? ( continued her pregnancy ) women were also reluctant to obtain abortion because they believed that it was against god 's will and could result in worldly punishment . i thought god would curse and chastise my husband , my children , and me in this world . when thinking about abortion , most of the participants had experienced fear and anxiety over its difficulty and pain . therefore , i dreaded abortion . ( continued her pregnancy ) on the other hand , women were greatly concerned with the adverse effects and complications of unsafe illegal abortion . however , i dreaded being permanently sterile ; i dreaded the adverse effects of abortion . ( a recently married young woman who had obtained elective abortion ) the three main themes of the study negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic generally reflected participants perceived threat and fear of unintended pregnancy outcomes and complications . it was a common theme emerging from the experiences of both those women who had continued with their pregnancy and those who had obtained elective induced abortion . in other words , participants in both groups had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds . but i also had an inner feeling of fear fear of killing a living being . the aim of this study was to explore iranian azeri women 's perceptions of unintended pregnancy . the participating women were mainly worried about continuing with an unintended pregnancy because of life difficulty and instability , inability to fulfill parental commitments and responsibilities , negative outcomes for mother and baby , social deprivations , and violating childbearing norms . fear of life difficulty and instability was one of the most remarkable perceptions of women about unintended pregnancy . in other studies , different dimensions of the above - mentioned threat , such as the perceived financial burden of unintended pregnancy , uncertainty over the prospect of marital life , and perceived role conflicts , were among the most common factors contributing to the obtainment of elective abortion . another important finding of the study was the women 's fear over the inability to fulfill parental commitments and responsibilities . hoogen reported that unintended pregnancy challenges women 's perception of being a good mother . in that study , women had a holistic family - oriented outlook rather than a one - dimensional fetus - oriented one toward motherhood and believed that a good mother is responsible for the welfare of the whole family . in a study conducted on 11 mexican immigrant women , wilson also reported that women 's main reason for pregnancy planning was to create a better life and future for their children . in this study , the participants were also concerned with social deprivations induced by unintended pregnancy and subsequent childrearing . consequently , strengthening women 's support system as well as providing them with better educational and employment opportunities throughout and after their pregnancy are recommended . another finding of the study was the women 's fear of negative outcomes of unintended pregnancy for themselves and their baby . fear of maternal health problems has also been reported in african sub - saharan area and southwest asia ; however , the fear of birth defects has been reported only in some studies conducted in asia . the main reason for such fears is probably women 's lack of health information as well as their limited access to modern screening tools for identifying birth defects in developing countries . these findings confirm the importance of healthcare providers role in providing education and counseling to women experiencing an unintended pregnancy . efficient counseling and strong social support alongside comprehensive birth defect risk assessment can help decrease the rate of unsafe illegal abortions . another aspect of the participants perceptions of unintended pregnancy was the fear of violating childbearing norms . in 1980s , population control policies under the slogan daughter or son , two children are enough was adopted in iran . accordingly , having only one or two children gradually became an accepted norm . the adverse long - term effect of the aforementioned policies was the increment of elderly population in the country . however , the only- and two - child traditions are still dominant in the iranian society . consequently , the participants considered having two or more children as a clear violation against this tradition and a potential damage to their social prestige . this important finding implies that abortion is not performed in a social vacuum ; rather , the society 's cultural climate can sometimes prevent women from continuing an unintended pregnancy . other studies conducted in iran also support the effect of public attitude toward the number of children on the acceptance of and adaptation with an unintended pregnancy . another type of threat perceived by the participants was related to the barriers to abortion . legal obstacles to abortion and subsequent inaccessibility to safe abortion services , as well as the complications of an unsafe backstreet abortion were the other sources of fear and anxiety for women . moreover , the participants were extremely concerned with the important others probable opposition to abortion . the findings of another study conducted in iran also revealed that legal obstacles and negative social attitude to elective abortion increased the severity of the threat perceived by women having an unintended pregnancy . in that study , women weighted their ability to obtain abortion against political obstacles and others reactions . lie et al . also reported that inadequate social and emotional support could result in women 's fear and anxiety over illegal abortion - induced sterility and death . the study findings also demonstrated that when thinking about aborting an unintended pregnancy , the participants had experienced pangs of guilt and conscience and fear over worldly and afterlife punishment . fear of worldly punishment and probable god - inflicting damage to other family members were among the most important inner feelings of women . an important limitation of this study was the reluctance of women having unwed , unintended pregnancy to participate in the study . pregnancy and childbearing are inherently interesting experiences that guarantee the continuation of human race ; however , when happening unintentionally , women perceive it as a real threat to their personal , familial , and social life . an unintended pregnancy can threaten women 's lives through depriving them of educational and employment opportunities , interfering with their ability to fulfill parental responsibilities , imposing financial burden on them and growing instability , and even by putting both mother and baby at great risk for serious physical and psychosocial problems . on the other hand , an unsafe illegal abortion would have potentially life - threatening complications . healthcare providers and policy - makers can fulfill such women 's need for support by developing pre - abortion counseling services and providing them with professional counseling .

background : many women , throughout their life cycle , experience unintended pregnancy and its subsequent induced abortion . nonetheless , women 's perceptions of this phenomenon particularly in countries prohibiting elective abortion are poorly known . the aim of this study was to explore iranian azeri women 's perceptions of unintended pregnancy.materials and methods : this was a conventional content analysis study conducted in tabriz , iran . the data were collected through 31 semi - structured interviews with 23 women who had recently experienced an unintended pregnancy . the study participants were recruited using the purposive sampling method . sampling started in march 2013 and continued until reaching data saturation , i.e. till august 2013 . data analysis was carried out concurrently with data collection . maxqda 10.0 software was employed for managing the study data.results:the study data analysis process yielded the formation of three main themes including negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic , which in turn constituted the broader overarching theme of threat supposition . in other words , following an unintended pregnancy , the study participants had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds.conclusions:women perceive unintended pregnancy as a challenging and threatening situation . an unintended pregnancy can threaten women 's lives through social deprivations , growing instability , and putting both mother and baby at risk for physical and psychosocial problems . on the other hand , an unsafe illegal abortion could have potentially life - threatening complications . to cope with such a situation , women need strong social support . healthcare providers can fulfill such women 's need for support by developing pre - abortion counseling services and providing them with professional counseling . also , strengthening women 's support system by policy - makers is recommended .

I M R Negative effects of unintended pregnancy on daily life Fear of life difficulty and instability Social deprivations Inability to fulfill parental commitments and responsibilities Negative maternal and child outcomes Fear of being stigmatized with violating social norms Violating childbearing norms The social taboo of abortion Abortion panic Infringing belief and value systems Fear over difficulty and adverse effects of abortion D C

from 1995 to 2008 , the unintended pregnancy rate reduced by 17% worldwide ; nonetheless , about 41% of all pregnancies 4 out of 10 pregnancies are unintended . in iran , the rate of unintended pregnancy decreased from 37% in 1992 to 21% in 2009 and has remained constant since then . porter highlighted the importance of religious beliefs in dealing with unintended pregnancy and recommended further studies on this subject area in different ethnic and religious groups . accordingly , the illegality of induced abortion in iran can affect iranian women 's perceptions and experiences of unintended pregnancy . most of the studies on women 's experiences of unintended pregnancy have been conducted on unmarried teens . on the other hand , studies on married women the center for disease control and prevention ( 2009 ) reported that as women 's perceptions and experiences of unintended pregnancy and induced abortion are poorly known , examining the effectiveness of healthcare services provided to women experiencing unintended pregnancy as well as developing new care plans for them are difficult . consequently , exploring women 's experiences of unintended pregnancy and induced abortion in different socio - cultural contexts is a matter of great importance . in iran , many studies have been conducted on the causes and the outcomes of unintended pregnancy and induced abortion ; however , iranian women 's beliefs , attitudes , and experiences surrounding unintended pregnancy and induced abortion are still poorly known . to prevent illegal induced abortions , the aim of the conventional content analysis approach is to study and explain poorly known phenomena . the study population consisted of all the women having a recent unintended pregnancy who either had decided to continue their pregnancy ( and were in their last 4 months of it or had delivered in the past 40 days ) or had electively aborted it recently ( in the previous 6 months ) . the women who were candidates for obtaining therapeutic abortion and women with established diagnosis of psychological disorders were excluded from the study . the participants were selected through purposeful sampling method from 10 different districts of tabriz city , according to their socioeconomic status . totally , 31 face - to - face unstructured interviews with 23 women were conducted from march to august 2013 . the main interview question was , what were your feelings and concerns about having an unintended pregnancy ? sampling and data collection were continued until reaching data saturation , i.e. meaning units , codes , and sub - categories of fear of life difficulty and instability category using several strategies such as maximum variation sampling , member checking , prolonged engagement with the study subject matter , and establishment of an effective communication with the study participants helped improve the credibility of the study findings . on the other hand , the peer - checking technique was employed to enhance the dependability of the study findings . the audit team comprised two of the study authors who were experts in qualitative data analysis . to guarantee the confirmability of the study findings , bracketing was done to prevent preconceptions from affecting the processes of data collection and analysis . the ethics committee of shahid beheshti university of medical sciences , iran , approved the study . the aim of the study was clearly explained to the study participants and accordingly , their questions and concerns were addressed . the study participants consisted of 13 women with unwanted pregnancy and 10 women with mistimed pregnancy , between the ages of 18 and 48 , of which 10 women had decided to abort and 13 women had continued to term . eleven participants made reference to employment , and only 3 of the 23 participants were university students while unintended pregnancy was detected . the study data analysis process yielded 214 primary codes , 17 sub - categories , 8 categories , 3 main themes , and the central overarching theme of threat supposition [ table 2 ] . a summary of findings the first theme of the study was the negative effects of unintended pregnancy on daily life . they referred to their coming baby as an uninvited guest and a major challenge that could result in disturbed tranquility , life instability , and social deprivations . the four sub - themes of this theme included fear of life difficulty and instability , social deprivations , negative maternal and child outcomes , and inability to fulfill parental commitments and responsibilities . threatening family stability and growing instability and inability to fulfill multiple roles : role conflict constituted a great part of the experiences of the participants , so that one of the women 's concerns of unintended pregnancy was the possibility for encountering financial pressure and difficulty . ( obtained elective abortion ) factors such as tenancy , unemployment , home office long distance , marital conflicts , and uncertainty over the prospect of marital life in young newly married couples heightened the participants fear of growing instability after encountering an unintended pregnancy . on the other hand , the perceived difficulty of rearing subsequent little babies , as well as the perceived difficulty of fulfilling multiple conflicting parental and social roles had brought the study participants into role conflict . ( obtained elective abortion ) the second perceived negative effect of unintended pregnancy on women 's daily lives was the probability of suffering from social deprivations . ( obtained elective abortion ) other aspects of social deprivations induced by unintended pregnancy were the restriction imposed on social interactions , recreational activities , and the freedom to travel as well as the inability to attend parties . ( p. 7 ; continued her pregnancy ) another negative effect of unintended pregnancy on women 's daily lives was inability to fulfill parental commitments and responsibilities ( for the previous children ) . women who had either several babies or two subsequent little babies were more worried about being unable to successfully fulfill their parental commitments and responsibilities . ( obtained elective abortion ) being unable to fulfill the coming baby 's emotional and financial needs , as well as being unable to build a high - quality life for him / her were among the other concerns of women over their unintended pregnancy . ( p. 9 ; obtained elective abortion ) negative maternal and child outcomes formed another aspect of the participants perceived negative effects of unintended pregnancy on daily life . an unintended pregnancy may happen at older ages , while a woman is using contraceptive methods or certain types of medications , or is undergoing x - ray studies . ( p. 2 ; obtained elective abortion ) besides being worried about their fetus 's health , the participating women were also worried about their own health . ( obtained elective abortion ) the second theme of the study was the fear of being stigmatized with violating social norms . in other words , becoming pregnant while having two to three children , very soon after marriage , or at older ages had given the study participants a sense of violating childbearing norms . as the childbearing pattern reflects the couple 's socio - cultural class , violating childbearing norms could lower their social prestige . ; obtained elective abortion ) in some instances , the fear of others blame and derision had been so great that the participants , at the early stages of pregnancy , had preferred to hide their pregnancy and isolate themselves socially . ( continued her pregnancy ) the other dimension of violating childbearing norms was related to the prevalence of the only - child practice in some iranian families , which in turn made the continuation of the second pregnancy difficult . an important strategy to get rid of the fears and difficulties of an unintended pregnancy was to obtain an induced abortion . however , relatives and friends disagreement with abortion was in turn the additional source of fear and anxiety for the study participants . fear of others opposition to abortion had compelled the participants to hide their pregnancy and abortion from relatives and friends . i feared my mother , sister , and mother- and father - in - law would notice my pregnancy and my intention to abort it . due to lack of social support for abortion and its illegality , the participants were worried about having access to abortion services . ( p. 5 ; obtained elective abortion ) the third theme of the study was the panic over abortion . ( continued her pregnancy ) when thinking about abortion , most of the participants had experienced fear and anxiety over its difficulty and pain . ( continued her pregnancy ) on the other hand , women were greatly concerned with the adverse effects and complications of unsafe illegal abortion . ( a recently married young woman who had obtained elective abortion ) the three main themes of the study negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic generally reflected participants perceived threat and fear of unintended pregnancy outcomes and complications . it was a common theme emerging from the experiences of both those women who had continued with their pregnancy and those who had obtained elective induced abortion . in other words , participants in both groups had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds . the first theme of the study was the negative effects of unintended pregnancy on daily life . they referred to their coming baby as an uninvited guest and a major challenge that could result in disturbed tranquility , life instability , and social deprivations . the four sub - themes of this theme included fear of life difficulty and instability , social deprivations , negative maternal and child outcomes , and inability to fulfill parental commitments and responsibilities . threatening family stability and growing instability and inability to fulfill multiple roles : role conflict constituted a great part of the experiences of the participants , so that one of the women 's concerns of unintended pregnancy was the possibility for encountering financial pressure and difficulty . ( obtained elective abortion ) factors such as tenancy , unemployment , home office long distance , marital conflicts , and uncertainty over the prospect of marital life in young newly married couples heightened the participants fear of growing instability after encountering an unintended pregnancy . on the other hand , the perceived difficulty of rearing subsequent little babies , as well as the perceived difficulty of fulfilling multiple conflicting parental and social roles had brought the study participants into role conflict . ( obtained elective abortion ) the second perceived negative effect of unintended pregnancy on women 's daily lives was the probability of suffering from social deprivations . ( obtained elective abortion ) other aspects of social deprivations induced by unintended pregnancy were the restriction imposed on social interactions , recreational activities , and the freedom to travel as well as the inability to attend parties . ( p. 7 ; continued her pregnancy ) another negative effect of unintended pregnancy on women 's daily lives was inability to fulfill parental commitments and responsibilities ( for the previous children ) . women who had either several babies or two subsequent little babies were more worried about being unable to successfully fulfill their parental commitments and responsibilities . ( obtained elective abortion ) being unable to fulfill the coming baby 's emotional and financial needs , as well as being unable to build a high - quality life for him / her were among the other concerns of women over their unintended pregnancy . ( p. 9 ; obtained elective abortion ) negative maternal and child outcomes formed another aspect of the participants perceived negative effects of unintended pregnancy on daily life . an unintended pregnancy may happen at older ages , while a woman is using contraceptive methods or certain types of medications , or is undergoing x - ray studies . threatening family stability and growing instability and inability to fulfill multiple roles : role conflict constituted a great part of the experiences of the participants , so that one of the women 's concerns of unintended pregnancy was the possibility for encountering financial pressure and difficulty . ( obtained elective abortion ) factors such as tenancy , unemployment , home office long distance , marital conflicts , and uncertainty over the prospect of marital life in young newly married couples heightened the participants fear of growing instability after encountering an unintended pregnancy . on the other hand , the perceived difficulty of rearing subsequent little babies , as well as the perceived difficulty of fulfilling multiple conflicting parental and social roles had brought the study participants into role conflict . the second perceived negative effect of unintended pregnancy on women 's daily lives was the probability of suffering from social deprivations . ( obtained elective abortion ) other aspects of social deprivations induced by unintended pregnancy were the restriction imposed on social interactions , recreational activities , and the freedom to travel as well as the inability to attend parties . another negative effect of unintended pregnancy on women 's daily lives was inability to fulfill parental commitments and responsibilities ( for the previous children ) . women who had either several babies or two subsequent little babies were more worried about being unable to successfully fulfill their parental commitments and responsibilities . ( obtained elective abortion ) being unable to fulfill the coming baby 's emotional and financial needs , as well as being unable to build a high - quality life for him / her were among the other concerns of women over their unintended pregnancy . negative maternal and child outcomes formed another aspect of the participants perceived negative effects of unintended pregnancy on daily life . the second theme of the study was the fear of being stigmatized with violating social norms . in other words , becoming pregnant while having two to three children , very soon after marriage , or at older ages had given the study participants a sense of violating childbearing norms . ( p. 3 ; obtained elective abortion ) in some instances , the fear of others blame and derision had been so great that the participants , at the early stages of pregnancy , had preferred to hide their pregnancy and isolate themselves socially . ( continued her pregnancy ) the other dimension of violating childbearing norms was related to the prevalence of the only - child practice in some iranian families , which in turn made the continuation of the second pregnancy difficult . ( obtained elective abortion ) another dimension of social norms violation stigma was related to the social taboo of abortion . an important strategy to get rid of the fears and difficulties of an unintended pregnancy was to obtain an induced abortion . however , relatives and friends disagreement with abortion was in turn the additional source of fear and anxiety for the study participants . fear of others opposition to abortion had compelled the participants to hide their pregnancy and abortion from relatives and friends . due to lack of social support for abortion and its illegality , i had to obtain abortion an illegal , hard - to - access abortion . in other words , becoming pregnant while having two to three children , very soon after marriage , or at older ages had given the study participants a sense of violating childbearing norms . ( p. 3 ; obtained elective abortion ) in some instances , the fear of others blame and derision had been so great that the participants , at the early stages of pregnancy , had preferred to hide their pregnancy and isolate themselves socially . ( continued her pregnancy ) the other dimension of violating childbearing norms was related to the prevalence of the only - child practice in some iranian families , which in turn made the continuation of the second pregnancy difficult . an important strategy to get rid of the fears and difficulties of an unintended pregnancy was to obtain an induced abortion . however , relatives and friends disagreement with abortion was in turn the additional source of fear and anxiety for the study participants . fear of others opposition to abortion had compelled the participants to hide their pregnancy and abortion from relatives and friends . due to lack of social support for abortion and its illegality , i had to obtain abortion an illegal , hard - to - access abortion . ( continued her pregnancy ) when thinking about abortion , most of the participants had experienced fear and anxiety over its difficulty and pain . ( continued her pregnancy ) on the other hand , women were greatly concerned with the adverse effects and complications of unsafe illegal abortion . however , i dreaded being permanently sterile ; i dreaded the adverse effects of abortion . ( a recently married young woman who had obtained elective abortion ) the three main themes of the study negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic generally reflected participants perceived threat and fear of unintended pregnancy outcomes and complications . these themes constituted the broader overarching theme of threat supposition . it was a common theme emerging from the experiences of both those women who had continued with their pregnancy and those who had obtained elective induced abortion . in other words , participants in both groups had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds . when thinking about abortion , most of the participants had experienced fear and anxiety over its difficulty and pain . ( continued her pregnancy ) on the other hand , women were greatly concerned with the adverse effects and complications of unsafe illegal abortion . ( a recently married young woman who had obtained elective abortion ) the three main themes of the study negative effects of unintended pregnancy on daily life , fear of being stigmatized with violating social norms , and abortion panic generally reflected participants perceived threat and fear of unintended pregnancy outcomes and complications . it was a common theme emerging from the experiences of both those women who had continued with their pregnancy and those who had obtained elective induced abortion . in other words , participants in both groups had experienced different levels of fear and threat depending on their personal , family , and socio - cultural backgrounds . the aim of this study was to explore iranian azeri women 's perceptions of unintended pregnancy . the participating women were mainly worried about continuing with an unintended pregnancy because of life difficulty and instability , inability to fulfill parental commitments and responsibilities , negative outcomes for mother and baby , social deprivations , and violating childbearing norms . fear of life difficulty and instability was one of the most remarkable perceptions of women about unintended pregnancy . in other studies , different dimensions of the above - mentioned threat , such as the perceived financial burden of unintended pregnancy , uncertainty over the prospect of marital life , and perceived role conflicts , were among the most common factors contributing to the obtainment of elective abortion . another important finding of the study was the women 's fear over the inability to fulfill parental commitments and responsibilities . hoogen reported that unintended pregnancy challenges women 's perception of being a good mother . in a study conducted on 11 mexican immigrant women , wilson also reported that women 's main reason for pregnancy planning was to create a better life and future for their children . in this study , the participants were also concerned with social deprivations induced by unintended pregnancy and subsequent childrearing . consequently , strengthening women 's support system as well as providing them with better educational and employment opportunities throughout and after their pregnancy are recommended . another finding of the study was the women 's fear of negative outcomes of unintended pregnancy for themselves and their baby . fear of maternal health problems has also been reported in african sub - saharan area and southwest asia ; however , the fear of birth defects has been reported only in some studies conducted in asia . these findings confirm the importance of healthcare providers role in providing education and counseling to women experiencing an unintended pregnancy . efficient counseling and strong social support alongside comprehensive birth defect risk assessment can help decrease the rate of unsafe illegal abortions . another aspect of the participants perceptions of unintended pregnancy was the fear of violating childbearing norms . this important finding implies that abortion is not performed in a social vacuum ; rather , the society 's cultural climate can sometimes prevent women from continuing an unintended pregnancy . other studies conducted in iran also support the effect of public attitude toward the number of children on the acceptance of and adaptation with an unintended pregnancy . legal obstacles to abortion and subsequent inaccessibility to safe abortion services , as well as the complications of an unsafe backstreet abortion were the other sources of fear and anxiety for women . the findings of another study conducted in iran also revealed that legal obstacles and negative social attitude to elective abortion increased the severity of the threat perceived by women having an unintended pregnancy . also reported that inadequate social and emotional support could result in women 's fear and anxiety over illegal abortion - induced sterility and death . the study findings also demonstrated that when thinking about aborting an unintended pregnancy , the participants had experienced pangs of guilt and conscience and fear over worldly and afterlife punishment . an important limitation of this study was the reluctance of women having unwed , unintended pregnancy to participate in the study . pregnancy and childbearing are inherently interesting experiences that guarantee the continuation of human race ; however , when happening unintentionally , women perceive it as a real threat to their personal , familial , and social life . an unintended pregnancy can threaten women 's lives through depriving them of educational and employment opportunities , interfering with their ability to fulfill parental responsibilities , imposing financial burden on them and growing instability , and even by putting both mother and baby at great risk for serious physical and psychosocial problems . on the other hand , an unsafe illegal abortion would have potentially life - threatening complications . healthcare providers and policy - makers can fulfill such women 's need for support by developing pre - abortion counseling services and providing them with professional counseling .

ulcerative colitis ( uc ) and crohn 's disease ( cd ) are usually referred to with the common label of inflammatory bowel diseases ( ibd ) due to their similar inflammatory nature and unknown cause . however , many differences in the clinical and pathologic features of these two chronic intestinal diseases have been found . ulcerative colitis involves the rectum and may affect part or all of colon , and the inflammation is typically restricted to the mucosa . crohn 's disease , on the other hand , is generally limited to the ileum and colon , not rarely in a patchy manner , and the inflammation is mostly transmural , with consequent stenosis and fistulae . ibd result from an impaired barrier function of the intestinal mucosa characterized by increased permeability and defective regulation of tight junctions . the failure of this barrier , determining an exposition to fecal antigens , may induce an inappropriate activation of the acquired mucosal immune system . as a matter of fact , antibodies against intestinal bacteria are frequently detected in serum of patients with ibd but , although many pathogens have been incriminated , none has been demonstrated to play a causative role . the initial fast , generic response to intestinal microbes is supplied by the innate immune system , while the adaptive immune system recognizes individual bacteria through antigen - specific receptors . a number of inflammatory cells and mediators and genetic alterations involved in the pathogenesis of ibd have been identified in recent years . the th1 cytokines interleukin ( il)-12 , interferon- , and tumor necrosis factor ( tnf)- are the main mediators of inflammation found in cd , whereas in uc an increased expression of th2 cytokine il-5 is often seen . elevated levels of il-23 and th17 cytokines have been found in both cd and uc . as to the innate immune system , genetic factors play an important role in the development of ibd , particularly in cd . in this respect , the first identified locus is located at the chromosome position 16q12 and the different genes included within this locus are named nod2 . the nod2 system encodes an intracellular sensor of peptidoglycan ( a component of bacterial cell wall ) and activates components of the innate immune system . the stimulation of the nod2 leads to the production of cytokines and antimicrobial peptides , and an impaired response at this level is now recognized as the likely cause of ibd . in fact , in healthy subjects nod2 , primed by microbial peptidoglycans , activates nuclear factor(nf)-b , which in turn enhances the production of antibacterial cytokines . however , the acute stimulation of nod2 by bacterial peptidoglycans is also modulated by inhibitory cytokines , like transforming growth factor ( tgf)- and il-10 , which downregulate the proinflammatory cytokines , and this downregulation is impaired in subjects with ibd , who are thus exposed to a chronic intestinal inflammation . three nod2 polymorphisms have been seen in about 30% of european patients , each impairing responses to peptidoglycans . carriers of a nod2 polymorphism are more likely than non carriers to have an ileal involvement . in addition , autophagy , a mechanism aimed to clear microbes , and its gene atg16l1 , are involved in cd , where abnormal paneth - cell morphology has also been found . the autophagy is implicated in t - cell tolerance ; thus in ibd an increased susceptibility to intestinal inflammation occurs , due to defects in t - cell tolerance . respiratory diseases are a possible complication of ibd , even if pulmonary alterations are often overlooked , especially when respiratory symptoms are already present before the diagnosis of ibd . the first recognition of a correlation between the diseases of the two districts is attributable to kraft , who in 1976 reported a series of patients with unexplained bronchial suppuration . since then , numerous reports have outlined the association between ibd and respiratory pathologies . the rate of extra - intestinal manifestations in patients with ibd ranges from 21 to 41% , increasing with the duration of the intestinal disease , and being greater in cd than in uc . however , the true prevalence of lung involvement in ibd remains unknown and it seems rather variable , because in some series only few cases of respiratory complications have been found . in fact , in 624 patients no respiratory complication was reported , and only 3/1400 cases of ibd were found by rodgers et al . , it can be difficult to establish a relationship between ibd and rd in patients who are already affected with pulmonary disease at the diagnosis of ibd , or are current smokers . respiratory diseases occurring in ibd may consist merely in a subclinical abnormal lung function or , in contrast , they may manifest as clear interstitial lung disease . an ongoing bowel inflammation is not a prerequisite for the onset of respiratory alterations , since bronchopulmonary diseases that developed after colectomy have been reported . the respiratory changes can occur at any time in the history of ibd , but they generally follow it by a time interval varying from days to decades . concerning the " respiratory dysfunction in asymptomatic patients " , mohamed - hussein et al . reported that 57.6% of their patients with uc had at least one abnormal lung function test ( pft ) result : restrictive dysfunction in 30.7% and obstructive in 11.5% . the impairment of pft was more pronounced and significant in patients with active uc than in controls or in patients with the inactive form . in a review including over 600 patients with uc , more than 50% of patients showed abnormal pft results when compared to healthy controls , and the decrease in diffusion capacity of the lung for carbon monoxide ( dlco ) was the most common defect . first reported a series of 26 children with cd in acute or quiescent phase , all non - smokers and asymptomatic . ten of them were studied before the start of therapy so as to exclude possible influence of therapy itself on the respiratory abnormalities . during the remission the value of dlco resulted higher than during the acute phase ( p < 0.0001 ) even if 50% of patients still presented an abnormal dlco . with the aim of investigating possible differences in respiratory function abnormalities between the two ibd identities , tzanakis et al . an increased , though not significant , percentage of abnormal pft results among both patient groups was observed compared to controls . a statistically significant difference was found only for the dlco decrease in cd patients when compared to controls ( p < 0.05 ) , and a slightly greater , but significant , reduction of dlco in the active compared to the inactive cd and uc . in a study of 66 ibd patients ( 35 cd and 31 uc ) , herrlinger et al . found 42% with at least one abnormal pulmonary function test compared to 3% of controls , and with no significant difference between both diseases . the forced expiratory volume in one second ( fev1 ) and the inspiratory vital capacity ( ivc ) resulted significantly dependent on the disease activity , whereas the dlco was not . dierkes - globisch and mohr investigated 44 ibd patients without pulmonary symptoms , 20% of whom showed an obstructive or restrictive ventilatory defect compared to 5% of controls ( p < 0.05% ) , but with no correlation to activity and duration of bowel disease or to smoking habit . from all these reports it seems clear that the pulmonary involvement in ibd is more frequent than believed , has no clear relationship with the activity of the intestinal disease and often is asymptomatic and detectable only at the lung function investigation . this is further supported by the finding by wallaert et al . of a high proportion of latent lymphocytic pulmonary alveolitis in the bronchoalveolar lavage ( bal ) of 18 consecutive patients with cd , all free from respiratory symptoms and showing a normal chest x - ray . in fact , an abnormal percentage of alveolar lymphocytes was observed in 11 of 18 patients ( 61% ) with a t - lymphocyte prevalence . the same was found by sarioglou et al . at the bal fluid examination , where a lymphocytic alveolitis was evident in 46.6% of ibd cases . a number of clinical lung diseases can be found in the course of ibd , but the true frequency of these conditions is unknown , although it has been shown that the incidence is higher in patients with uc than in those with cd ( 35 ) . respiratory diseases in patients affected by ibd can be classified as " airways disease " : tracheobronchial stenosis , bronchiectasis , chronic bronchial suppuration , chronic bronchitis , copd , asthma and bronchiolitis ; " parenchymal disease " : cryptogenic organizing pneumonia ( cop ) , interstitial lung disease , localized interstitial fibrosis , necrotic nodule and eosinophilic pneumonitis ; " pleural effusions " are uncommon as a manifestation of ibd ( table 1 ) . respiratory alterations in ibd definition of abbreviations : bhr , bronchial hyperresponsiveness ; cop , cryptogenic organizing pneumonia ; copd , chronic obstructive pulmonary disease ; dlco , diffusion capacity of the lung for carbon monoxide ; ibd , inflammatory bowel diseases . according to black et al . , who report 171 respiratory pathologies in 155 patients , the " large airways " are the most common site of involvement in ibd , accounting for 39% of all cases , and bronchiectasis was present in 66% of these cases . the second most frequently reported respiratory disease in ibd patients is chronic bronchitis , detected in a surprisingly high proportion ( 81% ) of nonsmoker patients . this led the authors to affirm that in these patients the bronchial inflammation is mainly caused by a diffusion of the bowel inflammatory condition to the bronchial tree . where 15/33 patients ( 45.4% ) with ibd had large airways involvement , six of them ( 40% ) in the form of bronchiectasis ; 9 of the 15 cases were postcolectomy . bronchiectasis was also the predominant finding in the report of spira et al . regarding 7 patients with ibd and large airways involvement , where three of them developed respiratory symptoms in 1 - 4 months after colectomy . finally , kelly et al . , in a retrospective analysis of patients with ibd and respiratory manifestations , identified 10 patients with bronchiectasis , 8 of whom post colectomy . these authors also reported that bronchiectasis in ibd patients is more severe , more rapidly progressive and more responsive to steroids than in non ibd patients . moreover , since colectomy has no effect on the respiratory involvement , they formulated the hypothesis that the discontinuation of anti - inflammatory drugs after the surgical resection of an intestinal tract is probably able to uncover or worsen the pulmonary manifestations . the involvement of the " small airways " in ibd , considered rare in both entities , has proved more frequent than was supposed in the days before expiratory high resolution computed tomography ( hrct ) of the chest was introduced . this imaging tool has allowed the detection of the small airways involvement in these patients , some of whom also with normal pft results . in like manner , examined with pft and inspiratory and expiratory hrct 36 patients with ibd ( 23 uc and 13 cd , only one with cd a current smoker ) , and 14 healthy non - smokers , free from respiratory symptoms . nineteen ( 53% ) patients had hrct abnormalities , with features suggestive of air trapping in expiratory ct scan in 12 ( 33% ) patients , 6 of whom were symptomatic . no significant correlation was found between hrct abnormalities and pft results , whereas there was a difference between the two types of ibd as to the incidence of ct abnormalities , that was 92% in cd and 39% in uc . tzakanis et al . investigated the function of small airways using the density dependence of airflow technique in 30 patients with ibd ( 18 uc and 12 cd ) and 16 healthy subjects . they found that both groups of patients with ibd had an increased volume of isoflow ( viso v ) despite their normal baseline spirometric values . the average viso v was lower in active vs. inactive uc ( p < 0.05 ) , while there was no difference between the two forms of ibd . the mean value of viso v in untreated patients was significantly greater than in control subjects ( p < 0.05 ) " bronchiolitis " is the most commonly reported disease involving the small airways in patients with ibd , and it is frequently associated with peribronchiolar granuloma [ 36,42 - 46 ] . camus et al . reported two cases diagnosed with this pathological manifestation among 33 patients . they were young men affected by uc , in an inactive phase in one case , who complained of productive cough , and showed diffuse small opacities at chest x - ray and an obstructive pattern at pft . in this patient the open lung biopsy revealed fibrosing and stenosing bronchiolitis and inflammatory lesions . both vandenplas et al . and trow et al . reported one case each of granulomatous bronchiolitis in women with cd who had undergone bowel resection . the respiratory alteration presented with nonproductive cough and progressive dyspnea , and chest pain the ct of the chest demonstrated irregularly dispersed micronodular densities , and the lung biopsy revealed an infiltrate of mononuclear cells associated with non - necrotizing epithelioid granulomas and giant cells in the bronchiolar walls . a cd4:cd8cell ratio of 3:1 in bal was found in the first patient , whereas in the second patient there was a peribronchial lymphocytic infiltrate and poorly organized granuloma with a normal bal cd4:cd8ratio . sometimes patients complaining of symptoms due to bronchiolitis may erroneously be thought to be affected with asthma , as reported by bentur et al . in a 13-year - old girl with cd since 8 years of age . the repeated episodes of shortness of breath , which started at age 9 years in this patient , had been interpreted as asthma . ct scan of the chest revealed irregularly diffused ground glass , and lung biopsy showed bronchiolitis obliterans and mononuclear inflammation with noncaseating granulomas , not related to sulfasalazine , because the pulmonary status did not improve when sulfasalazine was stopped . also the incidence of " asthma " is far more frequent than previously considered and the disease has a more severe course in ibd patients . a prevalence of 7.1 - 7.8% is reported by bernstein et al . , with a higher percentage in women . the airway inflammation sometimes may not be detectable by routine pft and only an asymptomatic increase in bronchial hyperresponsiveness ( bhr ) together with high ige levels may be found during the chronic bowel inflammation despite the absence of atopic symptoms . studied 14 cd children without extraintestinal manifestations , 5 of whom in a phase of clinical remission , with the aim to evaluate the bronchial responsiveness to methacholine in comparison with 10 healthy nonatopic subjects and 10 with mild bronchial asthma . in patients with cd , bhr was present in a very high proportion ( 71% of cases ) even in the absence of clinical and functional evidence of airways disease . the dose of bronchoconstrictor agent causing a 20% reduction of fev1 compared to baseline ( pd20 ) was not related either to the baseline fev1 or to total blood eosinophil count or serum ige levels ; however the pd20 values found in these patients were higher than those seen in asthmatic individuals . bhr was evident both in atopic patients with cd and in 7 of the 10 nonatopic cd subjects . the higher prevalence of bhr reported in this study than in others may likely depend on the different mean age of the study group and this accords with the observation that bhr progressively decreases with age . bhr was found in several studies with an incidence ranging from 17 to 45% . in a study comparing 30 ibd patients with 16 controls without gastrointestinal disease the respiratory and allergic symptoms were more prevalent in ibd patients ( p < 0.04 and p < 0.007 respectively ) than in the controls , and particularly in uc patients ( p < 0.004 ) . abnormal pfts were present in 27% of patients ( p < 0.04 vs. controls ) and bhr in 17% . skin prick tests were positive in 50% ibd but no difference was found between the two ibd subgroups . in 4 of the patients the pd20 value of methacholine ( mch ) test was < 16 mg / ml , while negative in all control subjects , and this hyperreactivity index was unrelated to the activity of the intestinal disease . . found a greater ( p = 0.026 ) positivity with methacholine challenge in 15 patients with cd than in 20 healthy controls , and a higher ( p = 0.011 ) level of lymphocytes in induced sputum . these results strengthen the possibility that a subclinical inflammation in absence of pulmonary symptoms can occur . . demonstrated a higher prevalence of positive skin - prick tests ( p < 0.02 ) and increased allergic symptoms ( p < 0.007 ) among 30 patients with ibd ( 19 uc and 11 cd ) compared to 16 controls without gastrointestinal disease . alterations affecting the " lung parenchyma " are relatively rare in ibd patients , and cryptogenic organizing pneumonia ( cop ) is the most common reported manifestation . the current pathogenetic hypothesis of ibd is an impairment of the mucosal immune regulation of the gastrointestinal system concerning intraluminal bacterial antigens in genetically predisposed persons . as a matter of fact , antibodies against intestinal bacteria are frequently detected in serum of patients with ibd ( 2 ) , and increased titers of anti - saccharomyces cerevisiae antibodies to baker 's yeast have been seen in cd patients . one hypothesis to explain the respiratory involvement in ibd is that these products cross - react with common antigens outside the bowel in the human body . furthermore , a common embryologic origin of both gastrointestinal and bronchial tree may support the onset of inflammation at two different sites with common embryological origin . respiratory symptoms may also be due to an aberrant homing of inflammatory cells to the lungs from the primary site of chronic inflammation , and this might also explain why the large airways disease is frequently not cured by colectomy . thus , according to this hypothesis , the inflammatory process would shift from the gastrointestinal tract to the airways . a recent study has shown that patients with cd have increased pulmonary permeability which appears unrelated to the disease activity . the same conclusions were reached by gursoy et al . in their study on uc patients without respiratory symptoms , who underwent the increase in permeability might even provide a satisfactory explanation for the occurrence of bronchial hyperresponsiveness ( bhr ) . it should also be taken into account that not rarely the side effects of treatments may be a cause of pulmonary disease in ibd patients , in the form of hypersensitivity reactions and pulmonary edema [ 64 - 66 ] . usually , most reactions related to sulfasalazine and 5-asa are seen between 2 and 6 months after commencement of drug administration . also a relapse or onset of pulmonary tuberculosis may be caused by the immunosuppressant monoclonal antibodies and other anti - inflammatory drugs used to control the chronic intestinal inflammatory process [ 67 - 69 ] . the involvement of the airways in the course of ibd may manifest itself with persistent productive cough , exertional dyspnea , obstructive and/or restrictive ventilatory deficit with or without abnormal chest radiograph . interestingly , a severe tracheobronchial stenosis has been reported in a young patient with cd . his respiratory symptoms developed two years after colectomy , with a productive cough initially controlled by beclomethasone . however , ten years later there was an increase in the volume of sputum with purulent features and fever , and a ct scan of the chest showed a marked distortion of the large bronchi with mucoid impaction in the small bronchi and alveolar micronodules . fiberoptic bronchoscopy ( fob ) revealed mucosal inflammation , purulent secretions and tracheal and bronchial deformities , with a severe stenosis of both mainstem bronchi , and marked airflow obstruction at the pft . the patient was initially treated with antibiotics and oral steroids , tapered over a period of 13 months . after 12 months ct and fob appeared unchanged , but no mucosal inflammation was present , and symptoms had disappeared at six weeks following the start of therapy ; an improvement on pft was measured at 28 months of switch therapy with inhaled budesonide . this case probably represents an advanced stage of tracheo - bronchial involvement which had developed over a long period of time . symptoms due to bronchiectasis and chronic bronchitis are the most frequent clinical presentation of airways pathology in ibd , generally following the onset of bowel disease and sometimes after the colectomy ( 71 ) . also in the series reported by kelly et al . , 80% of ibd patients with bronchiectasis developed their symptoms after surgery for ibd , supporting kinnear 's and higenbottam 's hypothesis that inflammatory process may shift from the bowel to the airway and manifest or increase after the bowel inflammation has been eliminated . in a series collected by higenbottam et al . six out of these had normal chest radiograph and three a minor obstructive ventilatory defect on pft . in one of these patients the remaining 4 patients developed a marked exertional dyspnea and bilateral pulmonary shadows on the chest x - ray with a mixed ventilatory defect . there was no correlation with sulfasalazine therapy and in 3 patients the symptoms began after colectomy . in 7 patients however the presence of respiratory symptoms may be highly variable in ibd patients and completely absent in those with milder disease . in fact , hrct performed in 17 ibd patients ( 3 out of 17 were cd patients ) revealed bronchiectasis in 13 ( 76% ) , in the absence of sputum production in those with a lesser extent of bronchial alteration , while 9 patients presented signs of air trapping , and 5 a " tree in bud " pattern at ct . as regards asthma symptoms , bernstein et al . report an increased risk for this disease in either form of ibd in a population - based study screening for a number of other chronic inflammations . asthma was the second most common extraintestinal comorbidity , after arthritis , without significant differences in relation to the type of ibd and gender , although a slightly greater prevalence in women was present . sometimes patients complain of dyspnea on exertion or even at rest , nonproductive cough , fever and general malaise due to the involvement of lung parenchyma in the form of interstitial and alveolar diffuse changes . in these cases it is necessary to distinguish the alterations directly consequent to the bowel disease from those caused by adverse reactions to the drugs used for ibd . alterations of the lung parenchyma are more frequent in uc and in females , cryptogenic organizing pneumonia ( cop ) being the most reported single case . in most patients [ 21,35,75 - 78 ] cop generally follows the onset of ibd , and it presents with fever , dyspnea , dry cough , pleuritic chest pain and weight loss . areas of peribronchiolar inflammation and fibrosis and/or fibrosis of the peribronchial region or subpleural opacities have been seen on ct scan , with a tendency to migrate and consolidate . at physical examination of the chest , inspiratory crackles are found on both sides of the chest , and pulmonary function tests show a restrictive pattern . the culture of bronchoalveolar lavage fluid generally does not lead to the growth of bacteria , acid - fast bacilli or fungi , and the cytologic study shows an increased percentage of lymphocytes with a variable , although usually normal , cd4/cd8 ratio . a normal cd4/cd8 ratio is reported by simon , whereas carrat found it increased . in this case an open - lung biopsy revealed isolated foci of non - specific chronic inflammation , myofibroblastic proliferation and peripheral endoalveolar foamy macrophages . rarely , cases of pulmonary infiltrates and eosinophilia have been reported both before and during or after the diagnosis of ibd . dry cough was the only symptom referred by a 38-year - old woman affected by cd and treated with mesalazine . chest x - ray showed some alveolar opacities in the right middle lobe , that were interpreted as pneumonitis and successfully treated with antimicrobial drugs . bal was performed and the cellular examination revealed a marked increase ( 30% ) in eosinophilis . at surgical biopsy a pattern of interstitial fibrosis with few non - caseating epithelioid granulomas was demonstrated , together with sparse lesions of bronchiolitis obliterans and desquamative interstitial pneumonia . this was the first description of a necrobiotic pulmonary nodule associated with interstitial pneumonia and eosinophilia . however , the hypothesis - albeit unlikely - that mesalazine treatment was responsible was difficult to confirm or exclude because , while the first pneumonia resolved without the withdrawl of the drug , the relapse improved after corticosteroid therapy and discontinuation of treatment with 5-asa . as to the pulmonary complications derived from the use of drugs for ibd , sulfasalazine was first prescribed in the management of these chronic bowel diseases in the 1940s . it is known that the drug may have clinically important side - effects , including pulmonary toxicity . in this respect , a retrospective study on published reports between 1972 and 1999 revealed that lung toxicity caused by sulfasalazine is more common in ulcerative colitis , where it mainly manifests with dyspnea ( 80% of cases ) , fever ( 70% ) and cough ( 64% ) . interestingly , the respiratory function assessment showed abnormalities in 28 out of 29 tests performed , consisting in a restrictive defect in 66% of cases . chest radiography was abnormal in all cases , showing mainly pulmonary infiltrates , that were confirmed at ct scan , where it revealed typical infiltrates , ground glass opacities , and pulmonary nodules with central cavitation . bal was performed in 11 patients , with 5 ( 45.4% ) demonstrating an increase in eosinophils ranging from 23 to 69% . based on the histological findings , the final diagnosis was eosinophilic pneumonia in 11 cases , and in 4 fibrosing alveolitis . other less common histological diagnoses included cop , desquamative interstitial pneumonia ( dip ) and less specified pneumopathies , like sulfasalazine lung disease or pulmonary hypersensitivity reaction . averbuch et al . reported a case of rash urticaria and generalized angioedema in a 58-year - old man suffering from uc and treated with sulfasalazine therapy , where the adverse symptoms resolved with the discontinuation of the drug . the patient under - went a protocol of desensitization ( gradually increasing doses of sulfasalazine with progressive tapering of prednisone ) , and after two months he developed a dry cough , fever and progressive dyspnea . a chest x - ray showed diffuse bilateral interstitial infiltrates which dramatically improved with sulfasalazine discontinuation and administration of high dose prednisone . in this case the first manifestations may be interpreted as a typical allergic reaction , whereas the parenchymal lung alterations likely reflect a direct toxicity of the drug . alternatively , both features could be due to the same steroid - responsive mechanism , but with higher doses of prednisone necessary for the prevention of the interstitial pneumonia . in a case of eosinophilic pneumonia in a 35-year - old woman treated with oral mesalazine , who improved after discontinuation of therapy , the lymphocyte stimulation test with the drug was positive for mesalazine and negative for sulfasalazine and sulphapyridine . only few cases of pneumonitis have been reported in patients treated with methotrexate for ibd , whereas one series reported 70 cases of tuberculosis in ibd patients given therapy with infliximab ( anti - tumor necrosis factor monoclonal anti - body ) . inhaled steroids are very effective , also for the long term control of the respiratory disease , in patients with chronic bronchitis , in symptomatic asthmatic patients , and in upper airways disease too . oral corticosteroids can improve interstitial disease , cop , granulomatous bronchiolitis , and drug - induced pneumonia , while intravenous administration is the initial management of life - threatening subglottis stenosis or extensive interstitial lung disease . a surgical approach is sometimes necessary to repair the sequels of destructive inflammatory processes , like tracheal and bronchial stenosis . despite the marked improvement in medical and surgical therapy , it remains a debated issue whether the death risk is greater in ibd patients compared to the general population , also because dissimilar results have been reported in many studies , where the most severe patients , bearing the worse prognosis , are prevalently included . other factors like malnutrition , infection , and side effects of medical and surgical therapy may increase the fragility of these patients . cucino et al . , with the aim to analyze the comorbidities possibly contributing to increased mortality of patients with ibd , pooled the data from 6 consecutive years ( 1991 - 1996 ) and analyzed them together . in patients affected with uc the comorbidities were most frequently represented by shock and protein / calorie malnutrition , volume depletion and anemia , septicemia , and peritonitis , whereas cd patients often showed nutritional , volume and electrolyte disturbances , besides surgical complication and pulmonary insufficiency . a meta - analysis was carried out to ascertain the overall and cause - specific mortality in a cohort of uc patients published in the literature from 1965 to 2006 . five scandinavian studies included in this meta - analysis reported higher mortality rates ( smr 1.2 , p = 0.001 ) than did non - scandinavian countries , similarly to an ec - ibd study ( european collaborative group of inflammatory bowel disease ) that reported a slightly higher mortality in north europe than in southern europe . the studies which presented smrs stratified for age at diagnosis did not show significant differences , whereas those which considered the extent of uc at diagnosis showed a higher smr when the inflammation was close to the rectum ( 1.2 ) . similarly , the smr was higher during the first 5 years after the diagnosis ( 1.4 ) and especially during the first year ( 2.2 ) ( 88 ) . lower smr ( 0.8 ) was encountered in uc patients who had not been given immunosuppressive drugs ( 6-mercaptopurine , azathioprine or infliximab ) and in non - colectomized patients ( 0.9 ) . the mean percentage of deaths ascribed to uc was 17% ( ranging from 11% to 30% ) , the most common causes being colorectal cancer and surgical or post - operative complications . . a significantly lower risk of dying from lung cancer was observed ( smr 0.3 , p = 0.04 ) , whereas a greater , but not significant , risk of dying from copd ( smr 1.6 , p = 0.26 ) and a significant risk for pneumonia ( smr 3.1 , p > only one study estimated the risk of dying from pulmonary embolism , reporting a significantly higher smr of 4.0 . the lower smr reported for lung cancer may be due to the fact that uc patients often are non smokers , so that the difference in smoking habits between the uc patients and the healthy population may act as a confounding factor . the other reported causes of death include cardiovascular , renal and liver diseases , leukemia and non - hodgkin 's lymphoma . the ec - ibd study on mortality in uc in europe did not find a higher mortality risk in uc patients compared with the normal population . the mortality was slightly higher in females than in males ( smr 1.39 and 0.92 respectively ) and slightly higher in older patients . a comparison of disease specific mortality between north european region and south european region also revealed a higher smr only for pulmonary disease ( smr 1.19 and 0,82 respectively ) . similarly , investigating the overall and cause - specific mortality in cd , duricova et al . examined the results from nine studies , six of which also reported cause - specific mortality . a slightly but significantly increased pooled smr ( 1.39 ) for mortality in general was observed , with no significant gender difference although it was higher in women , whereas a higher significant smr for mortality due to pulmonary diseases was found in cd compared to the general population and uc patients . in this metaanalysis a significantly increased risk of death from lung cancer was detected ( smr 2.72 ) , as well as from copd ( smr 2.55 ) , that led to a tendency of risk of overall death from respiratory disease to be increased ( smr 1.44 ) . this can likely be explained by a higher prevalence of smokers among cd patients compared to the general population and to uc patients , and it is known that smoking has detrimental effects on the clinical course of cd . , performed in the same type of patients , revealed higher smr in hospital and referral centers with respect to community - based studies . in conclusion , the inflammatory process of ibd is not necessarily restricted to the gastrointestinal tract , since many studies have demonstrated that latent or patent pulmonary involvement can occur in these patients even in the complete absence of symptoms . the available diagnostic tools may make us able to detect early ibd - related respiratory syndromes . a bronchial hyperresponsiveness in patients affected with uc or cd and no respiratory symptoms can easily be detected by methacholine challenge , so indicating that in the airways an inflammation not detectable by routine pfts may exist . use of the density dependence of air - flow technique may help to clarify if small airways are affected , and a defect in the transfer of respiratory gases , as expressed by the decrease in the dlco , suggests the presence of a process involving the interstitial pulmonary district possibly not identifiable by standard chest radiographs . in this context , hrct scan obtained at full expiration is a valid tool to show air trapping mainly in patients with emphysema , asthma and hypersensitivity pneumonia even in the absence of recognizable morphologic abnormalities on inspiratory scans . this method can be useful to detect small airways involvement also in asymptomatic ibd patients . as previously reported , pulmonary involvement , encountered in about 60% of ibd patients , can range from a simple defect of pulmonary function without symptoms to fibrosing alveolitis with a greater risk of mortality . for this reason the early detection of latent abnormalities of pulmonary function is important to prevent future and more severe respiratory impairment . early detection is all the more warranted in view of the fact that ibd - related respiratory syndromes generally respond well to inhaled or systemic corticosteroid treatment . none of the authors has any conflict of interest to declare in relation to the subject of this manuscript .

inflammatory bowel diseases ( ibd ) include ulcerative colitis ( uc ) and crohn 's disease ( cd ) and are due to a dysregulation of the antimicrobial defense normally provided by the intestinal mucosa . this inflammatory process may extend outside the bowel to many organs and also to the respiratory tract . the respiratory involvement in ibd may be completely asymptomatic and detected only at lung function assessment , or it may present as bronchial disease or lung parenchymal alterations . corticosteroids , both systemic and aerosolized , are the mainstay of the therapeutical approach , while antibiotics must be also administered in the case of infectious and suppurative processes , whose sequels sometimes require surgical intervention . the relatively high incidence of bronchopulmonary complications in ibd suggests the need for a careful investigation of these patients in order to detect a possible respiratory involvement , even when they are asymptomatic .

Definition and pathogenesis of IBD Incidence of respiratory diseases (RD) in IBD and of IBD in RD Pathogenesis of respiratory complications in IBD Clinical manifestations and treatment of RD in IBD Causes of mortality in IBD Conclusions Conflict of Interest Statement

ulcerative colitis ( uc ) and crohn 's disease ( cd ) are usually referred to with the common label of inflammatory bowel diseases ( ibd ) due to their similar inflammatory nature and unknown cause . however , many differences in the clinical and pathologic features of these two chronic intestinal diseases have been found . ulcerative colitis involves the rectum and may affect part or all of colon , and the inflammation is typically restricted to the mucosa . crohn 's disease , on the other hand , is generally limited to the ileum and colon , not rarely in a patchy manner , and the inflammation is mostly transmural , with consequent stenosis and fistulae . ibd result from an impaired barrier function of the intestinal mucosa characterized by increased permeability and defective regulation of tight junctions . the failure of this barrier , determining an exposition to fecal antigens , may induce an inappropriate activation of the acquired mucosal immune system . the initial fast , generic response to intestinal microbes is supplied by the innate immune system , while the adaptive immune system recognizes individual bacteria through antigen - specific receptors . a number of inflammatory cells and mediators and genetic alterations involved in the pathogenesis of ibd have been identified in recent years . the th1 cytokines interleukin ( il)-12 , interferon- , and tumor necrosis factor ( tnf)- are the main mediators of inflammation found in cd , whereas in uc an increased expression of th2 cytokine il-5 is often seen . as to the innate immune system , genetic factors play an important role in the development of ibd , particularly in cd . the nod2 system encodes an intracellular sensor of peptidoglycan ( a component of bacterial cell wall ) and activates components of the innate immune system . the stimulation of the nod2 leads to the production of cytokines and antimicrobial peptides , and an impaired response at this level is now recognized as the likely cause of ibd . the autophagy is implicated in t - cell tolerance ; thus in ibd an increased susceptibility to intestinal inflammation occurs , due to defects in t - cell tolerance . respiratory diseases are a possible complication of ibd , even if pulmonary alterations are often overlooked , especially when respiratory symptoms are already present before the diagnosis of ibd . the first recognition of a correlation between the diseases of the two districts is attributable to kraft , who in 1976 reported a series of patients with unexplained bronchial suppuration . the rate of extra - intestinal manifestations in patients with ibd ranges from 21 to 41% , increasing with the duration of the intestinal disease , and being greater in cd than in uc . however , the true prevalence of lung involvement in ibd remains unknown and it seems rather variable , because in some series only few cases of respiratory complications have been found . respiratory diseases occurring in ibd may consist merely in a subclinical abnormal lung function or , in contrast , they may manifest as clear interstitial lung disease . the respiratory changes can occur at any time in the history of ibd , but they generally follow it by a time interval varying from days to decades . reported that 57.6% of their patients with uc had at least one abnormal lung function test ( pft ) result : restrictive dysfunction in 30.7% and obstructive in 11.5% . in a review including over 600 patients with uc , more than 50% of patients showed abnormal pft results when compared to healthy controls , and the decrease in diffusion capacity of the lung for carbon monoxide ( dlco ) was the most common defect . ten of them were studied before the start of therapy so as to exclude possible influence of therapy itself on the respiratory abnormalities . a statistically significant difference was found only for the dlco decrease in cd patients when compared to controls ( p < 0.05 ) , and a slightly greater , but significant , reduction of dlco in the active compared to the inactive cd and uc . in a study of 66 ibd patients ( 35 cd and 31 uc ) , herrlinger et al . the forced expiratory volume in one second ( fev1 ) and the inspiratory vital capacity ( ivc ) resulted significantly dependent on the disease activity , whereas the dlco was not . dierkes - globisch and mohr investigated 44 ibd patients without pulmonary symptoms , 20% of whom showed an obstructive or restrictive ventilatory defect compared to 5% of controls ( p < 0.05% ) , but with no correlation to activity and duration of bowel disease or to smoking habit . from all these reports it seems clear that the pulmonary involvement in ibd is more frequent than believed , has no clear relationship with the activity of the intestinal disease and often is asymptomatic and detectable only at the lung function investigation . this is further supported by the finding by wallaert et al . a number of clinical lung diseases can be found in the course of ibd , but the true frequency of these conditions is unknown , although it has been shown that the incidence is higher in patients with uc than in those with cd ( 35 ) . respiratory alterations in ibd definition of abbreviations : bhr , bronchial hyperresponsiveness ; cop , cryptogenic organizing pneumonia ; copd , chronic obstructive pulmonary disease ; dlco , diffusion capacity of the lung for carbon monoxide ; ibd , inflammatory bowel diseases . , who report 171 respiratory pathologies in 155 patients , the " large airways " are the most common site of involvement in ibd , accounting for 39% of all cases , and bronchiectasis was present in 66% of these cases . the second most frequently reported respiratory disease in ibd patients is chronic bronchitis , detected in a surprisingly high proportion ( 81% ) of nonsmoker patients . this led the authors to affirm that in these patients the bronchial inflammation is mainly caused by a diffusion of the bowel inflammatory condition to the bronchial tree . where 15/33 patients ( 45.4% ) with ibd had large airways involvement , six of them ( 40% ) in the form of bronchiectasis ; 9 of the 15 cases were postcolectomy . regarding 7 patients with ibd and large airways involvement , where three of them developed respiratory symptoms in 1 - 4 months after colectomy . moreover , since colectomy has no effect on the respiratory involvement , they formulated the hypothesis that the discontinuation of anti - inflammatory drugs after the surgical resection of an intestinal tract is probably able to uncover or worsen the pulmonary manifestations . the involvement of the " small airways " in ibd , considered rare in both entities , has proved more frequent than was supposed in the days before expiratory high resolution computed tomography ( hrct ) of the chest was introduced . this imaging tool has allowed the detection of the small airways involvement in these patients , some of whom also with normal pft results . no significant correlation was found between hrct abnormalities and pft results , whereas there was a difference between the two types of ibd as to the incidence of ct abnormalities , that was 92% in cd and 39% in uc . investigated the function of small airways using the density dependence of airflow technique in 30 patients with ibd ( 18 uc and 12 cd ) and 16 healthy subjects . the respiratory alteration presented with nonproductive cough and progressive dyspnea , and chest pain the ct of the chest demonstrated irregularly dispersed micronodular densities , and the lung biopsy revealed an infiltrate of mononuclear cells associated with non - necrotizing epithelioid granulomas and giant cells in the bronchiolar walls . a cd4:cd8cell ratio of 3:1 in bal was found in the first patient , whereas in the second patient there was a peribronchial lymphocytic infiltrate and poorly organized granuloma with a normal bal cd4:cd8ratio . sometimes patients complaining of symptoms due to bronchiolitis may erroneously be thought to be affected with asthma , as reported by bentur et al . ct scan of the chest revealed irregularly diffused ground glass , and lung biopsy showed bronchiolitis obliterans and mononuclear inflammation with noncaseating granulomas , not related to sulfasalazine , because the pulmonary status did not improve when sulfasalazine was stopped . also the incidence of " asthma " is far more frequent than previously considered and the disease has a more severe course in ibd patients . the airway inflammation sometimes may not be detectable by routine pft and only an asymptomatic increase in bronchial hyperresponsiveness ( bhr ) together with high ige levels may be found during the chronic bowel inflammation despite the absence of atopic symptoms . in patients with cd , bhr was present in a very high proportion ( 71% of cases ) even in the absence of clinical and functional evidence of airways disease . the dose of bronchoconstrictor agent causing a 20% reduction of fev1 compared to baseline ( pd20 ) was not related either to the baseline fev1 or to total blood eosinophil count or serum ige levels ; however the pd20 values found in these patients were higher than those seen in asthmatic individuals . the higher prevalence of bhr reported in this study than in others may likely depend on the different mean age of the study group and this accords with the observation that bhr progressively decreases with age . in a study comparing 30 ibd patients with 16 controls without gastrointestinal disease the respiratory and allergic symptoms were more prevalent in ibd patients ( p < 0.04 and p < 0.007 respectively ) than in the controls , and particularly in uc patients ( p < 0.004 ) . abnormal pfts were present in 27% of patients ( p < 0.04 vs. controls ) and bhr in 17% . in 4 of the patients the pd20 value of methacholine ( mch ) test was < 16 mg / ml , while negative in all control subjects , and this hyperreactivity index was unrelated to the activity of the intestinal disease . demonstrated a higher prevalence of positive skin - prick tests ( p < 0.02 ) and increased allergic symptoms ( p < 0.007 ) among 30 patients with ibd ( 19 uc and 11 cd ) compared to 16 controls without gastrointestinal disease . the current pathogenetic hypothesis of ibd is an impairment of the mucosal immune regulation of the gastrointestinal system concerning intraluminal bacterial antigens in genetically predisposed persons . one hypothesis to explain the respiratory involvement in ibd is that these products cross - react with common antigens outside the bowel in the human body . respiratory symptoms may also be due to an aberrant homing of inflammatory cells to the lungs from the primary site of chronic inflammation , and this might also explain why the large airways disease is frequently not cured by colectomy . thus , according to this hypothesis , the inflammatory process would shift from the gastrointestinal tract to the airways . it should also be taken into account that not rarely the side effects of treatments may be a cause of pulmonary disease in ibd patients , in the form of hypersensitivity reactions and pulmonary edema [ 64 - 66 ] . also a relapse or onset of pulmonary tuberculosis may be caused by the immunosuppressant monoclonal antibodies and other anti - inflammatory drugs used to control the chronic intestinal inflammatory process [ 67 - 69 ] . the involvement of the airways in the course of ibd may manifest itself with persistent productive cough , exertional dyspnea , obstructive and/or restrictive ventilatory deficit with or without abnormal chest radiograph . however , ten years later there was an increase in the volume of sputum with purulent features and fever , and a ct scan of the chest showed a marked distortion of the large bronchi with mucoid impaction in the small bronchi and alveolar micronodules . symptoms due to bronchiectasis and chronic bronchitis are the most frequent clinical presentation of airways pathology in ibd , generally following the onset of bowel disease and sometimes after the colectomy ( 71 ) . also in the series reported by kelly et al . , 80% of ibd patients with bronchiectasis developed their symptoms after surgery for ibd , supporting kinnear 's and higenbottam 's hypothesis that inflammatory process may shift from the bowel to the airway and manifest or increase after the bowel inflammation has been eliminated . in one of these patients the remaining 4 patients developed a marked exertional dyspnea and bilateral pulmonary shadows on the chest x - ray with a mixed ventilatory defect . in 7 patients however the presence of respiratory symptoms may be highly variable in ibd patients and completely absent in those with milder disease . in fact , hrct performed in 17 ibd patients ( 3 out of 17 were cd patients ) revealed bronchiectasis in 13 ( 76% ) , in the absence of sputum production in those with a lesser extent of bronchial alteration , while 9 patients presented signs of air trapping , and 5 a " tree in bud " pattern at ct . sometimes patients complain of dyspnea on exertion or even at rest , nonproductive cough , fever and general malaise due to the involvement of lung parenchyma in the form of interstitial and alveolar diffuse changes . in these cases it is necessary to distinguish the alterations directly consequent to the bowel disease from those caused by adverse reactions to the drugs used for ibd . areas of peribronchiolar inflammation and fibrosis and/or fibrosis of the peribronchial region or subpleural opacities have been seen on ct scan , with a tendency to migrate and consolidate . the culture of bronchoalveolar lavage fluid generally does not lead to the growth of bacteria , acid - fast bacilli or fungi , and the cytologic study shows an increased percentage of lymphocytes with a variable , although usually normal , cd4/cd8 ratio . chest x - ray showed some alveolar opacities in the right middle lobe , that were interpreted as pneumonitis and successfully treated with antimicrobial drugs . however , the hypothesis - albeit unlikely - that mesalazine treatment was responsible was difficult to confirm or exclude because , while the first pneumonia resolved without the withdrawl of the drug , the relapse improved after corticosteroid therapy and discontinuation of treatment with 5-asa . as to the pulmonary complications derived from the use of drugs for ibd , sulfasalazine was first prescribed in the management of these chronic bowel diseases in the 1940s . in this respect , a retrospective study on published reports between 1972 and 1999 revealed that lung toxicity caused by sulfasalazine is more common in ulcerative colitis , where it mainly manifests with dyspnea ( 80% of cases ) , fever ( 70% ) and cough ( 64% ) . interestingly , the respiratory function assessment showed abnormalities in 28 out of 29 tests performed , consisting in a restrictive defect in 66% of cases . other less common histological diagnoses included cop , desquamative interstitial pneumonia ( dip ) and less specified pneumopathies , like sulfasalazine lung disease or pulmonary hypersensitivity reaction . reported a case of rash urticaria and generalized angioedema in a 58-year - old man suffering from uc and treated with sulfasalazine therapy , where the adverse symptoms resolved with the discontinuation of the drug . in this case the first manifestations may be interpreted as a typical allergic reaction , whereas the parenchymal lung alterations likely reflect a direct toxicity of the drug . alternatively , both features could be due to the same steroid - responsive mechanism , but with higher doses of prednisone necessary for the prevention of the interstitial pneumonia . in a case of eosinophilic pneumonia in a 35-year - old woman treated with oral mesalazine , who improved after discontinuation of therapy , the lymphocyte stimulation test with the drug was positive for mesalazine and negative for sulfasalazine and sulphapyridine . inhaled steroids are very effective , also for the long term control of the respiratory disease , in patients with chronic bronchitis , in symptomatic asthmatic patients , and in upper airways disease too . a surgical approach is sometimes necessary to repair the sequels of destructive inflammatory processes , like tracheal and bronchial stenosis . despite the marked improvement in medical and surgical therapy , it remains a debated issue whether the death risk is greater in ibd patients compared to the general population , also because dissimilar results have been reported in many studies , where the most severe patients , bearing the worse prognosis , are prevalently included . other factors like malnutrition , infection , and side effects of medical and surgical therapy may increase the fragility of these patients . a meta - analysis was carried out to ascertain the overall and cause - specific mortality in a cohort of uc patients published in the literature from 1965 to 2006 . similarly , the smr was higher during the first 5 years after the diagnosis ( 1.4 ) and especially during the first year ( 2.2 ) ( 88 ) . lower smr ( 0.8 ) was encountered in uc patients who had not been given immunosuppressive drugs ( 6-mercaptopurine , azathioprine or infliximab ) and in non - colectomized patients ( 0.9 ) . the lower smr reported for lung cancer may be due to the fact that uc patients often are non smokers , so that the difference in smoking habits between the uc patients and the healthy population may act as a confounding factor . a slightly but significantly increased pooled smr ( 1.39 ) for mortality in general was observed , with no significant gender difference although it was higher in women , whereas a higher significant smr for mortality due to pulmonary diseases was found in cd compared to the general population and uc patients . in this metaanalysis a significantly increased risk of death from lung cancer was detected ( smr 2.72 ) , as well as from copd ( smr 2.55 ) , that led to a tendency of risk of overall death from respiratory disease to be increased ( smr 1.44 ) . in conclusion , the inflammatory process of ibd is not necessarily restricted to the gastrointestinal tract , since many studies have demonstrated that latent or patent pulmonary involvement can occur in these patients even in the complete absence of symptoms . the available diagnostic tools may make us able to detect early ibd - related respiratory syndromes . a bronchial hyperresponsiveness in patients affected with uc or cd and no respiratory symptoms can easily be detected by methacholine challenge , so indicating that in the airways an inflammation not detectable by routine pfts may exist . use of the density dependence of air - flow technique may help to clarify if small airways are affected , and a defect in the transfer of respiratory gases , as expressed by the decrease in the dlco , suggests the presence of a process involving the interstitial pulmonary district possibly not identifiable by standard chest radiographs . in this context , hrct scan obtained at full expiration is a valid tool to show air trapping mainly in patients with emphysema , asthma and hypersensitivity pneumonia even in the absence of recognizable morphologic abnormalities on inspiratory scans . as previously reported , pulmonary involvement , encountered in about 60% of ibd patients , can range from a simple defect of pulmonary function without symptoms to fibrosing alveolitis with a greater risk of mortality . early detection is all the more warranted in view of the fact that ibd - related respiratory syndromes generally respond well to inhaled or systemic corticosteroid treatment . none of the authors has any conflict of interest to declare in relation to the subject of this manuscript .

serious complications including damage to the neural elements , stroke and death have been reported with epidural corticosteroid injections ( esis ) but are mostly anecdotal . their true incidence is unknown , but such outcomes seem to be rare.vascular penetration is possible , relatively more frequent in the cervical segments and potentially hazardous . intravascular injection can be reduced by use of injected contrast media.the use of a blunt needle , live fluoroscopy , digital subtraction angiography and the administration of a small test dose initially could help reduce the adverse effects.many complications can be avoided by a thorough understanding of the anatomy , accurate placement of the needle and familiarisation of the contrast patterns on fluoroscopy.more research must be performed regarding the benefits versus risk , techniques and outcome of esis . epidural corticosteroid injections ( esis ) have been used for decades as a therapeutic modality in the management of spinal pain syndromes attributed to disc pathology and spinal stenosis . although the exact pathophysiology of these conditions remains obscure , it has been suggested they occur through an ectopic firing action potentials mechanism in nerve roots derived from the mechanical compression . this mechanical compression could stimulate a local inflammatory process , which forms the rationale behind the administration of the corticosteroids . this theory is further strengthened by findings suggesting that the lavage of inflammatory mediators may reduce pain and inflammation . the interlaminar route is considered to be non - specific and the injectate is free to spread within the posterior epidural space with possible flow anteriorly , cephalad and caudally . this could be influenced by tissue fibrosis , scarring or hypertrophy , which may occur in spinal pathology . esi administered through this route could in theory deposit a larger mass of corticosteroid close to the pain generators at the ventral epidural space allowing a greater degree of drug diffusion , so transforaminal esi may be more efficacious in alleviating patients pain . however , several prospective randomised studies have failed to demonstrate a statistically significant difference in terms of pain reduction and functional score improvement between the transforaminal and interlaminar approaches [ 5 , 6 ] . in a recent systematic literature review of comparative studies involving patients with lumbosacral radicular pain , chang - chien et al . , suggested that both approaches are equally effective and demonstrated only minor non - significant differences between them . in contrast to the interlaminar and transforaminal routes , caudal epidural injections require relatively higher volumes of corticosteroids but are considered to be easier and safer and are preferred in patients after spinal surgery . the modality of imaging may influence the efficacy of esi . currently , fluoroscopy , ultrasound and computed tomography ( ct ) imaging have been used and their utilisation continues to increase . the choice amongst them partly lies in personal preference but also on the availability and prior training on the device . limited evidence currently exists in terms of the effectiveness and safety differences between these techniques . for instance , a recent literature review by bui and bogduk concluded that ct - guided lumbar transforaminal injection of corticosteroids is neither more effective nor safer than the fluoroscopy - guided injections but that ct is associated with significantly higher radiation doses than conventional fluoroscopy . ultrasound has gained popularity and maybe a safe alternative to the other radiological imaging modalities . several authors have questioned the overall efficacy of esis for the management of radicular pain [ 912 ] . in a systematic review of the available literature in 2009 by chou et al . , esis were moderately effective for short - term symptom relief in patients with low back pain but conferred no long - term benefit . in a similar manuscript , pinto et al . concluded that epidural corticosteroid injections offer only short - term relief of leg pain and disability for patients with sciatica . the authors questioned the clinical justification of this procedure when comparing the benefits with the risks . furthermore , in a systematic review including data from the cochrane central register of controlled trials , staal et al . concluded that there is insufficient evidence to support the use of injection therapy in subacute and chronic low back pain . these conclusions have been challenged by several other trials and systematic reviews [ 1318 ] . in patients with lumbar radicular pain caused by contained disc herniations , macvicar et al . suggested that lumbar transforaminal injection of corticosteroids is effective in reducing pain , restoring function , reducing the need for other healthcare modalities and avoiding surgery . in line with these deductions , quraishi concluded that in patients with lumbar radiculopathy , esis result in an improvement in pain but not disability . friedly et al . suggested that epidural injection of glucocorticoids plus lidocaine offered minimal or no short - term benefit as compared with epidural injection of lidocaine in the treatment of lumbar spinal stenosis alone . esis were found to have significant effect in relieving chronic intractable pain of cervical origin , providing long - term relief . some meta - analyses suggested that there is good evidence for the effectiveness of cervical interlaminar epidural injections in managing radiculitis secondary to disc herniation and fair evidence in managing axial or discogenic pain , pain of central spinal stenosis and pain of post - surgery syndrome [ 15 , 17 ] . the same authors concluded that the evidence is poor for cervical transforaminal epidural injections . it should be mentioned , however , that several of these studies have been criticised for flaws and deficiencies , adding further overall confusion . in addition to the controversy surrounding the efficacy of esis , some authors have raised concerns regarding potential adverse events . on 23 april 2014 the us food and drug administration ( fda ) issued a warning to the medical community covering the potential risks of these injections . the warning states that injection of corticosteroids into the epidural space of the spine may result in rare but serious adverse events , including loss of vision , stroke , paralysis , and death . this systematic review aims to scrutinise the available literature , present the available data and documentation from several authors , and analyse the risks involved with the esis in the cervical and lumbar spine . data were documented according to a standardised protocol , where objectives and inclusion criteria were specified in detail . publications from the last 20 years ( september 1994 to september 2014 ) were considered for further analysis . studies selected were original articles , in the english language , publishing results on complications related to the technique used for the esis . only cervical and lumbar esis were included and the studies had to specify the approach used for injection . studies were identified by searching the following resources / databases to retrieve all available relevant articles : pubmed medline , ovid medline , embase , scopus , google scholar and the cochrane library . transforaminal , interlaminar , adverse events , complication and side effect. the identified articles and their bibliographies including any relevant reviews were manually searched for additional potential eligible studies . two of the authors ( ippokratis pountos and gavin walters ) of this systematic review performed 208 the assessment , in an independent , unblinded and standardised manner . most citations were excluded on the basis of information provided by their respective title or abstract . in any other case , of 3255 papers initially identified , 162 met the inclusion criteria ( fig . 1 ) . this included 58 studies , of which 38 recorded complications while the remaining 20 state that 217 no complications were encountered [ 2178 ] . 101 1flowchart of study selection flowchart of study selection the review of the available literature identified 11 manuscripts presenting complications following interlaminar cervical esis ( table 1 ) [ 21 , 22 , 24 , 26 , 2834 ] . one manuscript that reports no complications has also been identified but only includes 14 interlaminar cervical esis .table 2003 retrospective cohort study157 pts , 345 injections , c67 or c7t1triamcinolone acetonide 80 mgfloverall 16.8 % : 23 increased neck pain ( 6.7 % ) 16 non - positional headaches ( 4.6 % ) 6 insomnia ( 1.7 % ) 6 vasovagal reactions ( 1.7 % ) 5 facial flushing ( 1.5 % ) 1 pyrexia ( 0.3 % ) 1 dural puncture ( 0.3 % ) derby et al . , 2003 retrospective survey4389 injectionsnana3 dural punctures ( 0.07 % ) 17 vagal symptoms ( 0.4 % ) 3 paraesthesia and numbness ( 0.07 % ) goel and pollan , 2006 prospective cohort29 pts , 65 injectionsnafl16.9 % headaches21.5 % insomnia , flushing of the face , temperature6.2 % of increased painkwon et al . , 2007 retrospective cohort study76 pts , 76 injectionstriamcinolone acetonide 40 mgfl2 dural punctures ( 2.6 % ) kranz et al . , 2011 retrospective cohort study50 pts , 53 injectionsbetamethasonect1 intra - thecal injectionmanchikanti et al . , 2012 prospective cohort study2376 injectionsnafl100 intravascular placement of needle ( 4.2 % ) 24 dural puncture ( 1 % ) 6 transient nerve root irritation ( 0.25 % ) 5 transient spinal cord irritation ( 0.21 % ) 16 profuse bleeding ( 0.7 % ) 1 vasovagal ( 0.04 % ) 2 facial flushing ( 0.08 % ) lee et al . , 2012 prospective cohort study~127 injectionsdexamethasone sodium phosphate 10 mgfl1 vasovagal and syncope ( 0.8 % ) 1 dural puncture ( 0.8 % ) beyaz and eman , 2013 retrospective cohort study65 ptsnafl1 vasovagal ( 1.54 % ) 1 transient increase of pain ( 1.54 % ) manchikanti et al . , 2013 rct120 pts , 654 injectionsbetamethasone 6 mg ( n = 60)fl2 subarachnoid punctures ( 0.3 % ) 4 intravascular penetrations ( 0.6 % ) 5 nerve root irritations ( 0.76 % ) 1 pain lasting 1 week ( 0.15 % ) lee et al . , 2014 retrospective cohort study143 ptstriamcinolone acetonide 40 mg1 itching sensations1 facial flushing1 dry mouth1 erectile dysfunctionmanchikanti et al . , 2014 rct120 pts , 688 injectionsbetamethasone 6 mg ( n = 60)fl6 subarachnoid punctures ( 0.3 % ) 10 intravascular penetrations ( 0.6 % ) 3 nerve root irritations ( 0.76 % ) 1 pain lasting 1 week ( 0.15 % ) ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with interlaminar cervical epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial based on the available studies , the incidence of dural puncture ranged between 0.07 and 2.6 % . vasovagal reactions ranged between 0.04 and 1.7 % . in a prospective study including 2376 injections , manchikanti et al . reported 100 cases where intravascular placement of the needle occurred . however , complications that could potentially be correlated with inadvertent intravascular injection of corticosteroids were low and included 11 cases of transient nerve root or spinal cord irritation , one vasovagal event and two cases of facial flushing . in a retrospective analysis of the results of 345 c67 or c7t1 injections , botwin et al a large proportion of these adverse events were related to an increase of neck pain , headache , insomnia and vasovagal reactions . furman et al . presented 504 cervical ( c3c8 ) transforaminal esis performed on 337 patients . they reported identification of 98 intravascular injections that did not result in any adverse effects . similarly , other authors have reported no complications [ 27 , 31 , 3739 ] . in a retrospective review of 1579 injections , derby et al . reported two cases of aggravated radicular pain , two cases of prolonged paraesthesias and the development of skin rash in one patient . in another study including 43 esis with prednisolone , 19 % of patients experienced minor neurovegetative manifestations . conducted an anonymous survey asking the us physician members of the american pain society about their experience with regards to serious complications following cervical transforaminal epidural corticosteroid injections ( tesis ) . from the 287 replies , 78 complications were reported , among which there were 30 brain or spinal cord infarcts and 24 neurologic complications including death of unsuspected aetiology ( n = 5 ) , high spinal anaesthesia ( n = 3 ) , transient ischaemic attacks ( n = 3 ) , and spinal cord or brainstem oedema ( n = 3 ) . the literature search found 11 studies that present adverse effects following interlaminar lumbar esis ( table 2 ) [ 3436 , 40 , 42 , 44 , 45 , 4752 ] . in addition , four studies that involve more than 250 patients have reported no adverse events following interlaminar lumbar epidural esis [ 46 , 54 , 55 , 60 ] . in a prospective cohort study including 1450 injections , manchikanti et al . reported an incidence of 0.8 % for dural puncture and profuse bleeding following the injection . a prospective , randomised blinded study including 106 patients has reported a rather high number of minor adverse effects . in particular , 26 % of the patients experienced discomfort and pain at the injection site , 18 % had non - positional headache and 10 % suffered from nausea after the injection . in an analysis of 6631 interlaminar lumbar esis , huang et al . the physician recognised the lumbar facet joint injection . a similar study design reported by candido et al . reported the incidence of intradiscal injection to be one in 4723 .table 2complications reported with interlaminar lumbar epidural corticosteroid injectionsstudy , yeardesignptsmedicationsimagingcomplicationscarette et al . , 1997 prospective randomised blinded study78 pts , 162 injectionsmethylprednisolone 80 mgbl1 dural puncture ( 0.6 % ) 27 transient headachekraemer et al . , 1997 rct87 pts , 87 injectionstriamcinolone 10 mgct1.93.6 % headachevalat et al . , 2003 rct39 pts , 117 injectionsprednisolone acetate 50 mgbl2 headachearden et al . , 2005 rct115 pts , 3 injections eachtriamcinolone acetonide 80 mgbl4 non - specific headache2 postdural puncture headache , nausea5 otherkim et al . , 2010 retrospective cohort study150 pts , 150 injectionsdexamethasone 16 mgfl42 facial flushing ( 28 % ) kim et al . , 2011 prospective randomised study60 pts , 120 injectionsdexamethasone phosphate 15 mg or methylprednisolone acetate 80 mgfl1 intrathecal injectionmanchikanti et al . , 2012 [ 34 , 40]prospective randomised blinded study120 pts , 213 injectionsbetamethasone 1 ml ( n = 60)fl3 subarachnoid punctures ( 1.4 % ) manchikanti et al . , 2012 prospective cohort study1450 injectionsnafl7 intravascular placement of needle ( 0.5 % ) 4 transient nerve root irritation ( 0.28 % ) 11 dural punctures ( 0.8 % ) 11 profuse bleeding ( 0.8 % ) 4 local haematoma ( 0.28 % ) 1 headache ( 0.07 % ) 2 facial flushing ( 0.13 % ) bartynski et al . , 2013 retrospective cohort study276 pts , 392 injectionmethylprednisolone acetate 80 mgfl1 dural puncture1 transient paraparesiscandido el al . 2013 prospective randomised blinded study106 pts , l3s1methylprednisolone acetate 120 mgfl26 % discomfort and pain at the injection site18 % non - positional headache10 % nauseaevansa et al . , 2015 prospective randomised study120 pts , 120 injectionsmethylprednisolone acetate 80 mgfl ( n = 56 ) , us ( n = 56)15 dizziness or pain at injection site or facial flushing1 intrathecal injection bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound complications reported with interlaminar lumbar epidural corticosteroid injections bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound fourteen studies were identified that presented adverse effects following transforaminal esis ( table 3 ) [ 34 , 50 , 53 , 5659 , 61 , 63 , 6569 ] . in contrast , no adverse events were presented by a number of other authors [ 25 , 62 , 7078 ] . the most common complication reported was increased pain , which was encountered in half of the patients . two prospective studies analysing a large number of transforaminal lumbar esis reported an incidence of intravascular penetration of between 7.4 and 7.9 % [ 34 , 57 ] . the main difference was an 8.7 % rate of vasovagal episodes reported by karaman et al . . a similar rate was reported by ploumis et al . , although only 20 patients were included in that study . identified six intradiscal injections among 249 transforaminal esis , which represents an incidence of 2.4 % . a lower incidence of one in 402 injections was reported by candido et al . , although this was tenfold higher than after interlaminar esi .table , 2000 retrospective cohort study207 pts , 322 injectionseither betamethasone 912 mg or methylprednisolone 80 mgfl10 non - positional headaches ( 3.1 % ) 8 increased back pain ( 2.4 % ) 2 increased leg pain ( 0.6 % ) 4 facial flushing ( 1.2 % ) 1 vasovagal reaction ( 0.3 % ) 1 increased blood sugar ( 258 mg / dl ) in an insulin - dependent patient with diabetes mellitus ( 0.3 % ) 1 intraoperative hypertension ( 0.3 % ) ahadian et al . , 2011 prospective randomised study98 pts , 98 injectionsdexamethasone 4 , 8 or 12 mgfl1 pain at injection site5 vascular uptake6 paraesthesia during procedurekaraman et al . , 2011 prospective cohort study562 pts , 1305 injectionstriamcinolone acetonidefl97 vascular penetration ( 7.4 % ) 8.7 % vasovagal episodes5 transient erectile dysfunction ( 0.9 % ) 5 facial flushing ( 0.9 % ) mcgrath et al . , 2011 retrospective cohort study1667 pts , 3964 injectionsnafl42 increased pain6 numbness9 pain at injection sitemanchikanti et al . , 2012 prospective cohort study1310 injectionsnafl104 intravascular placement of needle ( 7.9 % ) 16 transient nerve root irritation ( 4.6 % ) 8 profuse bleeding ( 1 % ) 1 vasovagal ( 0.08 % ) 2 facial flushing ( 0.15 % ) cansever et al . , 2012 cohort study37 pts , 65 injectionstriamcinolone 40 mgct3 transient weakness14 increased low back pain2 low blood pressure post - injectionkoh et al . , 2013 rct53 ptstriamcinolone 20 mgfl1 burning at injection site ( 1.9 % ) manson et al . , 2013 retrospective cohort study91 pts , 106 injectionstriamcinalone 40 mgfl2 vasovagal episodeshong et al . , 2014 prospective cohort study239 pts , 249 injectionsdexamethasone 5 mg and mepivacaine 3 mlfl6 intradiscal injectionmanchikanti et al . , 2014 rct120 pts , 601 injectionsbetamethasone 3 mgfl28 intravascular infiltrations ( 4.6 % ) 9 nerve root irritations ( 1.5 % ) kraiwattanapong et al . , 2014 prospective cohort study38 pts , 72 injectionsmethylprednisolone 80 mgfl3 worsening of leg painploumis et al . , 2014 prospective cohort study20 pts , l4s1betamethasone 9 mgfl2 vasovagal episodes ( 10 % ) tauheed et al . , 2014 rct60 ptsmethylprednisolone 60 mgfl3 transient paraesthesia of nerve distribution ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with transforaminal lumbar epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial a large number of case reports presenting rare adverse events following esis exist ( fig . the available literature has described deaths following esis [ 89 , 93 , 97 ] . reviewing the available case reports , the most common and devastating complication was infarction of the spinal cord , cerebellum , brain and brainstem [ 97 , 141 ] . infarctions could occur due to damage to the blood vessels , or either to vasospasm or an emboli from particulate matter associated with the corticosteroid injection . damage to the blood vessels could result in haematomas , and subdural and epidural haematomas have been reported [ 124 , 135 , 144 , 146 , 155 , 162 , 164 ] . direct damage to the spinal cord by trauma or direct injection of esi medications into the cervical spinal cord has been also documented [ 140 , 148 , 160 , 173 ] . such a complication can occur with an absence of pain being reported by the patient when the spinal cord structures were punctured . subdural and intrathecal spread or diffusion of the injected mixture of corticosteroids , anaesthetic and contrast dye could result in cauda equina and conus medularis syndromes , arachnoiditis , meningitis and temporary respiratory depression [ 139 , 148 , 153 , 171 ] . furthermore , cases of pneumocephalus , pneumorrhachis and cerebrospinal fluid ( csf ) leak can occur [ 90 , 94 , 95 , 147 , 151 , 153 , 170].fig . 2reported major complications and approach used reported major complications and approach used infections and abscesses have been also reported following esi [ 108114 , 117127 , 130 , 132 , 133 , 164 ] . with the exception of a fungal infection outbreak in the usa in 2012 , infection rates vary following an epidural injection , but , on average , are reported to be one in 60,000100,000 epidural injections . the documented outbreak in 2012 was possibly caused by a contaminated glucocorticoid product used for epidural and paraspinal injection [ 108 , 109 ] . in single case reports , cases of meningitis , vertebral osteomyelitis , and spinal and paraspinal abscesses have been reported that are caused by microorganisms including aspergillus spp . , staphylococcus aureus and methicillin - resistant s. aureus [ 111 , 113 , 126 ] . blindness after esi has been reported multiple times [ 8284 , 86 , 87 , 154 , 167 ] . it has been hypothesised that this complication is caused by an abrupt rise in the csf pressure caused by the volume of the injected pharmaceutical agents . in the cervical spine , this complication can be the result of the administration of radio contrast agents administered in the intracranial vasculature . the patient s vision returned to normal within 1 year of follow - up in some studies [ 82 , 83 ] , but permanent visual impairment in patients vision was reported by some authors [ 84 , 86 , 87 , 167 ] . vaginal bleeding has been reported as a potential complication of esi [ 99 , 101 ] . have retrospectively reviewed 8166 esi procedures and reported an incidence of 2.5 % ( n = 201 ; 197 patients ) for abnormal vaginal bleeding . of these women , 70 % were premenopausal and 30 % were postmenopausal . ovarian axis causing anovulatory cycles has been hypothesised to be the mechanism for this adverse effect . case reports have also presented other complications including iatrogenic cushing s syndrome [ 131 , 166 ] , persistent hiccups [ 128 , 152 ] , convulsions , reversible posterior leukoencephalopathy syndrome , epidural granuloma formation , subdural block , brown squard syndrome , herpes zoster outbreak , steroid myopathy and corticosteroid - induced psychosis following esi . spinal epidural lipomatosis is a rare condition of adipose tissue hypertrophy in the epidural space and has been reported to occur after esi [ 138 , 143 , 161 ] . in addition , cardiopulmonary arrest following esi and anaphylaxis and other adverse events due to the epidural corticosteroid compounds can occur [ 134 , 136 , 149 , 169 , 175 ] . complex regional pain syndrome and development of neuropathic pain following esi have also occurred [ 158 , 163 ] . the review of the available literature identified 11 manuscripts presenting complications following interlaminar cervical esis ( table 1 ) [ 21 , 22 , 24 , 26 , 2834 ] . one manuscript that reports no complications has also been identified but only includes 14 interlaminar cervical esis .table 2003 retrospective cohort study157 pts , 345 injections , c67 or c7t1triamcinolone acetonide 80 mgfloverall 16.8 % : 23 increased neck pain ( 6.7 % ) 16 non - positional headaches ( 4.6 % ) 6 insomnia ( 1.7 % ) 6 vasovagal reactions ( 1.7 % ) 5 facial flushing ( 1.5 % ) 1 pyrexia ( 0.3 % ) 1 dural puncture ( 0.3 % ) derby et al . , 2003 retrospective survey4389 injectionsnana3 dural punctures ( 0.07 % ) 17 vagal symptoms ( 0.4 % ) 3 paraesthesia and numbness ( 0.07 % ) goel and pollan , 2006 prospective cohort29 pts , 65 injectionsnafl16.9 % headaches21.5 % insomnia , flushing of the face , temperature6.2 % of increased painkwon et al . , 2007 retrospective cohort study76 pts , 76 injectionstriamcinolone acetonide 40 mgfl2 dural punctures ( 2.6 % ) kranz et al . , , 2012 prospective cohort study2376 injectionsnafl100 intravascular placement of needle ( 4.2 % ) 24 dural puncture ( 1 % ) 6 transient nerve root irritation ( 0.25 % ) 5 transient spinal cord irritation ( 0.21 % ) 16 profuse bleeding ( 0.7 % ) 1 vasovagal ( 0.04 % ) 2 facial flushing ( 0.08 % ) lee et al . , 2012 prospective cohort study~127 injectionsdexamethasone sodium phosphate 10 mgfl1 vasovagal and syncope ( 0.8 % ) 1 dural puncture ( 0.8 % ) beyaz and eman , 2013 retrospective cohort study65 ptsnafl1 vasovagal ( 1.54 % ) 1 transient increase of pain ( 1.54 % ) manchikanti et al . , 2013 rct120 pts , 654 injectionsbetamethasone 6 mg ( n = 60)fl2 subarachnoid punctures ( 0.3 % ) 4 intravascular penetrations ( 0.6 % ) 5 nerve root irritations ( 0.76 % ) 1 pain lasting 1 week ( 0.15 % ) lee et al . , 2014 retrospective cohort study143 ptstriamcinolone acetonide 40 mg1 itching sensations1 facial flushing1 dry mouth1 erectile dysfunctionmanchikanti et al . , 2014 rct120 pts , 688 injectionsbetamethasone 6 mg ( n = 60)fl6 subarachnoid punctures ( 0.3 % ) 10 intravascular penetrations ( 0.6 % ) 3 nerve root irritations ( 0.76 % ) 1 pain lasting 1 week ( 0.15 % ) ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with interlaminar cervical epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial based on the available studies , the incidence of dural puncture ranged between 0.07 and 2.6 % . vasovagal reactions ranged between 0.04 and 1.7 % . in a prospective study including 2376 injections , manchikanti et al . reported 100 cases where intravascular placement of the needle occurred . however , complications that could potentially be correlated with inadvertent intravascular injection of corticosteroids were low and included 11 cases of transient nerve root or spinal cord irritation , one vasovagal event and two cases of facial flushing . in a retrospective analysis of the results of 345 c67 or c7t1 injections , botwin et al . reported an overall incidence of complications of 16.8 % . a large proportion of these adverse events were related to an increase of neck pain , headache , insomnia and vasovagal reactions . furman et al . presented 504 cervical ( c3c8 ) transforaminal esis performed on 337 patients . they reported identification of 98 intravascular injections that did not result in any adverse effects . similarly , other authors have reported no complications [ 27 , 31 , 3739 ] . in a retrospective review of 1579 injections , derby et al . reported two cases of aggravated radicular pain , two cases of prolonged paraesthesias and the development of skin rash in one patient . in another study including 43 esis with prednisolone , 19 % of patients experienced minor neurovegetative manifestations . conducted an anonymous survey asking the us physician members of the american pain society about their experience with regards to serious complications following cervical transforaminal epidural corticosteroid injections ( tesis ) . from the 287 replies , 78 complications were reported , among which there were 30 brain or spinal cord infarcts and 24 neurologic complications including death of unsuspected aetiology ( n = 5 ) , high spinal anaesthesia ( n = 3 ) , transient ischaemic attacks ( n = 3 ) , and spinal cord or brainstem oedema ( n = 3 ) . the literature search found 11 studies that present adverse effects following interlaminar lumbar esis ( table 2 ) [ 3436 , 40 , 42 , 44 , 45 , 4752 ] . in addition , four studies that involve more than 250 patients have reported no adverse events following interlaminar lumbar epidural esis [ 46 , 54 , 55 , 60 ] . in a prospective cohort study including 1450 injections , manchikanti et al . reported an incidence of 0.8 % for dural puncture and profuse bleeding following the injection . a prospective , randomised blinded study including 106 patients has reported a rather high number of minor adverse effects . in particular , 26 % of the patients experienced discomfort and pain at the injection site , 18 % had non - positional headache and 10 % suffered from nausea after the injection . in an analysis of 6631 interlaminar lumbar esis , huang et al . the physician recognised the lumbar facet joint injection . a similar study design reported by candido et al . reported the incidence of intradiscal injection to be one in 4723 .table 2complications reported with interlaminar lumbar epidural corticosteroid injectionsstudy , yeardesignptsmedicationsimagingcomplicationscarette et al . , 1997 prospective randomised blinded study78 pts , 162 injectionsmethylprednisolone 80 mgbl1 dural puncture ( 0.6 % ) 27 transient headachekraemer et al . , 1997 rct87 pts , 87 injectionstriamcinolone 10 mgct1.93.6 % headachevalat et al . , 2003 rct39 pts , 117 injectionsprednisolone acetate 50 mgbl2 headachearden et al . , 2005 rct115 pts , 3 injections eachtriamcinolone acetonide 80 mgbl4 non - specific headache2 postdural puncture headache , nausea5 otherkim et al . , 2010 retrospective cohort study150 pts , 150 injectionsdexamethasone 16 mgfl42 facial flushing ( 28 % ) kim et al . , 2011 prospective randomised study60 pts , 120 injectionsdexamethasone phosphate 15 mg or methylprednisolone acetate 80 mgfl1 intrathecal injectionmanchikanti et al . , 2012 [ 34 , 40]prospective randomised blinded study120 pts , 213 injectionsbetamethasone 1 ml ( n = 60)fl3 subarachnoid punctures ( 1.4 % ) manchikanti et al . , 2012 prospective cohort study1450 injectionsnafl7 intravascular placement of needle ( 0.5 % ) 4 transient nerve root irritation ( 0.28 % ) 11 dural punctures ( 0.8 % ) 11 profuse bleeding ( 0.8 % ) 4 local haematoma ( 0.28 % ) 1 headache ( 0.07 % ) 2 facial flushing ( 0.13 % ) bartynski et al . , 2013 retrospective cohort study276 pts , 392 injectionmethylprednisolone acetate 80 mgfl1 dural puncture1 transient paraparesiscandido el al . 2013 prospective randomised blinded study106 pts , l3s1methylprednisolone acetate 120 mgfl26 % discomfort and pain at the injection site18 % non - positional headache10 % nauseaevansa et al . , 2015 prospective randomised study120 pts , 120 injectionsmethylprednisolone acetate 80 mgfl ( n = 56 ) , us ( n = 56)15 dizziness or pain at injection site or facial flushing1 intrathecal injection bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound complications reported with interlaminar lumbar epidural corticosteroid injections bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound fourteen studies were identified that presented adverse effects following transforaminal esis ( table 3 ) [ 34 , 50 , 53 , 5659 , 61 , 63 , 6569 ] . in contrast , no adverse events were presented by a number of other authors [ 25 , 62 , 7078 ] . the most common complication reported was increased pain , which was encountered in half of the patients . two prospective studies analysing a large number of transforaminal lumbar esis reported an incidence of intravascular penetration of between 7.4 and 7.9 % [ 34 , 57 ] . the main difference was an 8.7 % rate of vasovagal episodes reported by karaman et al . . a similar rate was reported by ploumis et al . , although only 20 patients were included in that study . identified six intradiscal injections among 249 transforaminal esis , which represents an incidence of 2.4 % . a lower incidence of one in 402 injections was reported by candido et al . , although this was tenfold higher than after interlaminar esi .table 2000 retrospective cohort study207 pts , 322 injectionseither betamethasone 912 mg or methylprednisolone 80 mgfl10 non - positional headaches ( 3.1 % ) 8 increased back pain ( 2.4 % ) 2 increased leg pain ( 0.6 % ) 4 facial flushing ( 1.2 % ) 1 vasovagal reaction ( 0.3 % ) 1 increased blood sugar ( 258 mg / dl ) in an insulin - dependent patient with diabetes mellitus ( 0.3 % ) 1 intraoperative hypertension ( 0.3 % ) ahadian et al . , 2011 prospective randomised study98 pts , 98 injectionsdexamethasone 4 , 8 or 12 mgfl1 pain at injection site5 vascular uptake6 paraesthesia during procedurekaraman et al . , 2011 prospective cohort study562 pts , 1305 injectionstriamcinolone acetonidefl97 vascular penetration ( 7.4 % ) 8.7 % vasovagal episodes5 transient erectile dysfunction ( 0.9 % ) 5 facial flushing ( 0.9 % ) mcgrath et al . , 2011 retrospective cohort study1667 pts , 3964 injectionsnafl42 increased pain6 numbness9 pain at injection sitemanchikanti et al . , 2012 prospective cohort study1310 injectionsnafl104 intravascular placement of needle ( 7.9 % ) 16 transient nerve root irritation ( 4.6 % ) 8 profuse bleeding ( 1 % ) 1 vasovagal ( 0.08 % ) 2 facial flushing ( 0.15 % ) cansever et al . 2012 cohort study37 pts , 65 injectionstriamcinolone 40 mgct3 transient weakness14 increased low back pain2 low blood pressure post - injectionkoh et al . , 2013 rct53 ptstriamcinolone 20 mgfl1 burning at injection site ( 1.9 % ) manson et al . , 2013 retrospective cohort study91 pts , 106 injectionstriamcinalone 40 mgfl2 vasovagal episodeshong et al . , 2014 prospective cohort study239 pts , 249 injectionsdexamethasone 5 mg and mepivacaine 3 mlfl6 intradiscal injectionmanchikanti et al . , 2014 rct120 pts , 601 injectionsbetamethasone 3 mgfl28 intravascular infiltrations ( 4.6 % ) 9 nerve root irritations ( 1.5 % ) kraiwattanapong et al . , 2014 prospective cohort study38 pts , 72 injectionsmethylprednisolone 80 mgfl3 worsening of leg painploumis et al . , 2014 prospective cohort study20 pts , l4s1betamethasone 9 mgfl2 vasovagal episodes ( 10 % ) tauheed et al . , 2014 rct60 ptsmethylprednisolone 60 mgfl3 transient paraesthesia of nerve distribution ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with transforaminal lumbar epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial a large number of case reports presenting rare adverse events following esis exist ( fig . the available literature has described deaths following esis [ 89 , 93 , 97 ] . reviewing the available case reports , the most common and devastating complication was infarction of the spinal cord , cerebellum , brain and brainstem [ 97 , 141 ] . infarctions could occur due to damage to the blood vessels , or either to vasospasm or an emboli from particulate matter associated with the corticosteroid injection . damage to the blood vessels could result in haematomas , and subdural and epidural haematomas have been reported [ 124 , 135 , 144 , 146 , 155 , 162 , 164 ] . direct damage to the spinal cord by trauma or direct injection of esi medications into the cervical spinal cord has been also documented [ 140 , 148 , 160 , 173 ] . such a complication can occur with an absence of pain being reported by the patient when the spinal cord structures were punctured . subdural and intrathecal spread or diffusion of the injected mixture of corticosteroids , anaesthetic and contrast dye could result in cauda equina and conus medularis syndromes , arachnoiditis , meningitis and temporary respiratory depression [ 139 , 148 , 153 , 171 ] . furthermore , cases of pneumocephalus , pneumorrhachis and cerebrospinal fluid ( csf ) leak can occur [ 90 , 94 , 95 , 147 , 151 , 153 , 170].fig . 2reported major complications and approach used reported major complications and approach used infections and abscesses have been also reported following esi [ 108114 , 117127 , 130 , 132 , 133 , 164 ] . with the exception of a fungal infection outbreak in the usa in 2012 , infection rates vary following an epidural injection , but , on average , are reported to be one in 60,000100,000 epidural injections . the documented outbreak in 2012 was possibly caused by a contaminated glucocorticoid product used for epidural and paraspinal injection [ 108 , 109 ] . in single case reports , cases of meningitis , vertebral osteomyelitis , and spinal and paraspinal abscesses , staphylococcus aureus and methicillin - resistant s. aureus [ 111 , 113 , 126 ] . blindness after esi has been reported multiple times [ 8284 , 86 , 87 , 154 , 167 ] . it has been hypothesised that this complication is caused by an abrupt rise in the csf pressure caused by the volume of the injected pharmaceutical agents . in the cervical spine , this complication can be the result of the administration of radio contrast agents administered in the intracranial vasculature . the patient s vision returned to normal within 1 year of follow - up in some studies [ 82 , 83 ] , but permanent visual impairment in patients vision was reported by some authors [ 84 , 86 , 87 , 167 ] . vaginal bleeding has been reported as a potential complication of esi [ 99 , 101 ] . have retrospectively reviewed 8166 esi procedures and reported an incidence of 2.5 % ( n = 201 ; 197 patients ) for abnormal vaginal bleeding . of these women , 70 % were premenopausal and 30 % were postmenopausal . ovarian axis causing anovulatory cycles has been hypothesised to be the mechanism for this adverse effect . case reports have also presented other complications including iatrogenic cushing s syndrome [ 131 , 166 ] , persistent hiccups [ 128 , 152 ] , convulsions , reversible posterior leukoencephalopathy syndrome , epidural granuloma formation , subdural block , brown squard syndrome , herpes zoster outbreak , steroid myopathy and corticosteroid - induced psychosis following esi . spinal epidural lipomatosis is a rare condition of adipose tissue hypertrophy in the epidural space and has been reported to occur after esi [ 138 , 143 , 161 ] . in addition , cardiopulmonary arrest following esi and anaphylaxis and other adverse events due to the epidural corticosteroid compounds can occur [ 134 , 136 , 149 , 169 , 175 ] . complex regional pain syndrome and development of neuropathic pain following esi have also occurred [ 158 , 163 ] . esis have been used for more than 60 years since lievre et al . reported the use of epidural hydrocortisone in a series of 20 patients . over the years the number of epidural injections has increased by 106.3 % in the decade between 1997 and 2006 . currently , esis are the most common intervention performed for the management of chronic low back pain in the usa . indications for esi are not robust and the outcome could not be correlated with the extent of the underlying pathology , e.g. the degree of lumbar spinal stenosis , but could be determined by factors such as age , sex and the preceding opioid use [ 183185 ] . collectively , this systematic review contains data from more than 100,000 esis reported in prospective or retrospective studies . major events have been reported anecdotally and it is impossible to comment on their true incidence based on the available results in the literature . overall , the potential causes of adverse events could be categorised into three distinct categories : ( 1 ) direct damage to the blood vessels or adjacent anatomical structures during the procedure ; ( 2 ) intravascular administration of the injectate ; and ( 3 ) a local or systemic reaction including bacterial contamination . direct damage to the blood vessels or adjacent structure is an inherent risk for any injection , including esis . direct damage to the spinal cord by the needle and the injection of corticosteroids into the cervical spinal cord has been also documented in a very limited number of case reports . clinically significant haematomas derived from piercing or damage to the blood vessels can occur and the reported incidence for all epidurals is less than one in 150,000 . this complication is increased in patients with coagulopathy and patients on anticoagulant medications [ 186188 ] . inadvertent dural punctures can occur after esis and csf flashback is pathognomonic of this complication . other complications , including intracranial subdural haematoma after accidental dural puncture and cases of pneumocephalus , pneumorrhachis and csf leak , have been presented in case reports [ 90 , 94 , 95 , 98 ] . intravascular injection of the corticosteroids , carrier and/or the local anaesthetic could account for the large majority of the serious adverse effects . the reported incidence of inadvertent intravascular injection with fluoroscopically guided tesi is reported to range from 9 % to as high as 32.8 % [ 25 , 189192 ] . furman et al . [ 25 , 189 ] reported an incidence of fluoroscopically confirmed intravascular penetration of 19.4 % for cervical tesis , 8.1 % for lumbar tesis and 21.3 % for tesis at the s1 level . in addition , sullivan et al . suggested that intravascular uptake is twice as likely to occur in patients over rather than under 50 years of age . vascular embolic events from intra - arterial injection of particulate corticosteroids have been found to account for serious complications including spinal cord infarction , paraplegia and death [ 93 , 100 , 102 , 103 , 105 ] . houten et al . presented three cases of paraplegia which ensued suddenly after instillation of the corticosteroid solution in the artery of adamkiewicz . it should be mentioned that intra - articular injections pose a higher degree of danger , while venous uptake has been considered benign [ 193 , 194 ] . in terms of the injectate , medium - sized particles between 51 and 1000 m have the potential to enter and occlude a blood vessel . irrespective of the size , it has been suggested that when corticosteroid particles enter a blood vessel they could coalesce and precipitate , forming larger particles . the injection of the particulate corticosteroid methylprednisolone into the vertebral artery of four pigs resulted in permanent loss of consciousness , while the animals receiving dexamethasone and prednisolone recovered fully . . demonstrated that methylprednisolone and its non - particulate carrier can produce significant injury to the blood the authors also suggested that in addition to the cerebral microvasculature occlusion by the particulate corticosteroids , damage can occur via toxicity of the carrier or the corticosteroid . based on several observations that failed to highlight any difference in the efficacy of particulate and non - particulate corticosteroids , we would recommend the use of soluble non - particulant agents [ 196198 ] . a transient blockage of the neural conduction is expected ; however , the reported central canal , conus medularis and cauda equina syndromes must have an underlying cause , i.e. hematoma , infarct , etc . transient systemic reactions including headaches , vasovagal reactions and facial flushing have been reported ; these reactions occurred shortly after the esi and could represent a reaction to the injected anaesthetic agents and/or corticosteroids . it is rather unclear whether esis pose a long - term risk of certain conditions and whether a cumulative effect of prolonged exposure exists . if that is true , epidural corticosteroids could have similar systemic effects to that of long - term corticosteroids administered through other routes . for instance , a significant number of the patients with chronic back pain conditions are treated with repeated injections over prolonged period of time . corticosteroids are known to interfere with calcium homeostasis , reducing bone formation and increasing bone breakdown . osteoporosis and an increased fracture risk could theoretically occur ; however , the available literature does not support this theory . prospectively evaluated 100 patients receiving epidural injections and reported no change in bone mineral density . insulin resistance is another adverse effect associated with corticosteroid administration ; however , studies looking specifically at patients receiving esis did not find any changes in the fasting glucose levels [ 200 , 201 ] . adrenal axis suppression has been demonstrated to occur after esis [ 200 , 202 ] . . showed that following esi with dexamethasone a profound decrease in the serum levels of adrenocorticotropic hormone ( acth ) occurs . hypertension can also occur following esis ; a mean systolic blood pressure increase of 5 mmhg has been previously reported following esis . finally , corticosteroid administration represents a risk factor for wound complications postoperatively and poses an increased risk for infections . esis are frequently performed prior to spinal surgery , either as a disgnostic tool or for pain management , but their contribution to complications of such procedures is currently unknown . severe infections are rare after spinal injections and have an incidence of 0.10.01 % . the only exception is a fungal infection outbreak in the usa in 2012 . according to the centers for disease control and prevention , 25 deaths due to epidural corticosteroid - related meningitis ( many due to aspergillosis ) were identified : 337 patients were affected in 18 us states and 14,000 patients were probably exposed to contaminated corticosteroids . in the authors opinion , several recommendations can be made with the aim of minimise the incidence of major complications after esis . first , esis should be performed under fluoroscopic guidance and the needle position should be confirmed in at least two planes , typically anteroposterior and either an oblique or lateral plane . aspiration prior to the injection is specific but not sensitive at detecting intravascular needle placement , being unable to produce a flashback of blood in 74 % of cases in which the needle was ultimately determined to be intravascular . injection of contrast media is recommended and operators must be able to distinguish between intravascular , epidural and subdural contrast flow patterns . the use of a blunt needle and the suggestion that a small test dose of the medication should be inject initially has been proposed [ 204207 ] . dynamic live fluoroscopy was found to perform better than static intermittent fluoroscopy , which was found to miss 57 % of the intravascular injections . digital subtraction angiography can be used as a radiologic adjunct to identify vascular compromise during the injection . however , in a case report by chang et al . , an anaesthetic test dose and digital subtraction angiography performed twice did not prevent a catastrophic spinal cord infarction and the resultant paraplegia . it is under debate whether the transforaminal route poses a higher risk of serious complications when performed by an experience physician . given the lack of evidence , one could argue that it is reasonable to consider the transforaminal approach only when the interlaminar route has failed . finally , informed consent should be taken and the patient should be aware of the potential risk and benefits of this procedure . the survey of cervical injections conducted by scanlon et al . is the only manuscript that presents a high number of serious and fatal cases . possible mechanisms explaining these events include the intra - arterial injection of particulate corticosteroid or trauma causing embolisation to the distal basilar or vertebral arteries . despite the fact that the study presents the extreme end of potential complications , it is unclear what the true incidence of these events is . as previously mentioned , it would be of enormous educational interest to have further details regarding these events , especially details of the technique and imaging used as well as the training and experience of the physician . the warning issued by the fda regarding esis merits further discussion and analysis , and the use of corticosteroids for injections in the epidural space for spinal pain syndromes the fda mentions that , despite their use , the effectiveness of esis has been challenged and could potentially result in serious adverse events including death , stroke and paralysis . in support of these arguments , the fda has published several case reports . major adverse events can occur with esis , but such events are rare , their true incidence is unknown and they have only been presented in case reports . for instance , none of the studies included in tables 1 , 2 and 3 presents such major devastating complications . conclusion stating that the fda s warning is an additional burden on patient access to pain - relieving treatments . should the fda s warning letter be replaced by an evidence - based educational guidance to safeguard the best clinical practice ? a large proportion of the available case reports and studies provide insufficient documentation , i.e. the approach used for esi , symptom duration , volume injected or even the number of injections . in addition , the majority of the studies report adverse effects incidentally as their main aim is to report the efficacy of the injections . furthermore , the available studies were heterogeneous with regards to the outcome measures , and in several manuscripts the surgical technique , corticosteroid dosage and the addition of other medications are not reported . it is of note that many studies did not look at or record the complications or adverse effects of corticosteroid exposure but present results from several pain and functional scores . therefore , it is possible that both the short- and long - term adverse effects of corticosteroid exposure remain unreported . esis are relatively safe ; however , although major complications of esis have been reported , their true incidence remains obscure . vascular penetration and administration of pharmaceutical agents intra - arterially could account for a large proportion of the adverse events reported . with accurate placement of the needle , use of non - particulate corticosteroids , live fluoroscopy , digital subtraction angiography and familiarisation of the contrast patterns on fluoroscopy these risks should be minimised . further research is required to shed more light on the best clinical practice for the use of esis and the true incidence of complications relating to them .

background and objectiveepidural corticosteroid injections ( esis ) have been used for several decades and now represent the most common intervention performed for the management of back pain with a radicular component . however , several reports have presented devastating complications and adverse effects , which fuelled concerns over the risk versus clinical effectiveness . the authors offer a comprehensive review of the available literature and analyse the data derived from studies and case reports.methodsstudies were identified by searching pubmed medline , ovid medline , embase , scopus , google scholar and the cochrane library to retrieve all available relevant articles . publications from the last 20 years ( september 1994 to september 2014 ) were considered for further analysis . studies selected were english - language original articles publishing results on complications related to the technique used for cervical and lumbar esis . the studies had to specify the approach used for injection . all studies that did not fulfil these eligibility criteria were excluded from further analysis.resultsoverall , the available literature supports the view that serious complications following injections of corticosteroid suspensions into the cervical and lumbar epidural space are uncommon , but if they occur they can be devastating.conclusionsthe true incidence of such complications remains unclear . direct vascular injury and/or administration of injectates intra - arterially represent a major concern and could account for the vast majority of the adverse events reported . accurate placement of the needle , use of a non - particulate corticosteroid , live fluoroscopy , digital subtraction angiography , and familiarisation of the operator with contrast patterns on fluoroscopy should minimise these risks . the available literature has several limitations including incomplete documentation , unreported data and inherent bias . large registries and well - structured observational studies are needed to determine the true incidence of adverse events and address the safety concerns .

Key Points Introduction Risk Versus Efficacy of Epidural Corticosteroid Injections (ESIs) Methods Results Interlaminar Cervical ESIs Transforaminal Cervical ESIs Interlaminar Lumbar ESIs Transforaminal Lumbar ESIs Adverse Event Case Reports According to the Approach Used Discussion Conclusions

serious complications including damage to the neural elements , stroke and death have been reported with epidural corticosteroid injections ( esis ) but are mostly anecdotal . their true incidence is unknown , but such outcomes seem to be rare.vascular penetration is possible , relatively more frequent in the cervical segments and potentially hazardous . intravascular injection can be reduced by use of injected contrast media.the use of a blunt needle , live fluoroscopy , digital subtraction angiography and the administration of a small test dose initially could help reduce the adverse effects.many complications can be avoided by a thorough understanding of the anatomy , accurate placement of the needle and familiarisation of the contrast patterns on fluoroscopy.more research must be performed regarding the benefits versus risk , techniques and outcome of esis . epidural corticosteroid injections ( esis ) have been used for decades as a therapeutic modality in the management of spinal pain syndromes attributed to disc pathology and spinal stenosis . although the exact pathophysiology of these conditions remains obscure , it has been suggested they occur through an ectopic firing action potentials mechanism in nerve roots derived from the mechanical compression . this mechanical compression could stimulate a local inflammatory process , which forms the rationale behind the administration of the corticosteroids . the interlaminar route is considered to be non - specific and the injectate is free to spread within the posterior epidural space with possible flow anteriorly , cephalad and caudally . esi administered through this route could in theory deposit a larger mass of corticosteroid close to the pain generators at the ventral epidural space allowing a greater degree of drug diffusion , so transforaminal esi may be more efficacious in alleviating patients pain . however , several prospective randomised studies have failed to demonstrate a statistically significant difference in terms of pain reduction and functional score improvement between the transforaminal and interlaminar approaches [ 5 , 6 ] . currently , fluoroscopy , ultrasound and computed tomography ( ct ) imaging have been used and their utilisation continues to increase . several authors have questioned the overall efficacy of esis for the management of radicular pain [ 912 ] . in a systematic review of the available literature in 2009 by chou et al . concluded that there is insufficient evidence to support the use of injection therapy in subacute and chronic low back pain . the warning states that injection of corticosteroids into the epidural space of the spine may result in rare but serious adverse events , including loss of vision , stroke , paralysis , and death . this systematic review aims to scrutinise the available literature , present the available data and documentation from several authors , and analyse the risks involved with the esis in the cervical and lumbar spine . publications from the last 20 years ( september 1994 to september 2014 ) were considered for further analysis . studies selected were original articles , in the english language , publishing results on complications related to the technique used for the esis . only cervical and lumbar esis were included and the studies had to specify the approach used for injection . studies were identified by searching the following resources / databases to retrieve all available relevant articles : pubmed medline , ovid medline , embase , scopus , google scholar and the cochrane library . 101 1flowchart of study selection flowchart of study selection the review of the available literature identified 11 manuscripts presenting complications following interlaminar cervical esis ( table 1 ) [ 21 , 22 , 24 , 26 , 2834 ] . , 2014 rct120 pts , 688 injectionsbetamethasone 6 mg ( n = 60)fl6 subarachnoid punctures ( 0.3 % ) 10 intravascular penetrations ( 0.6 % ) 3 nerve root irritations ( 0.76 % ) 1 pain lasting 1 week ( 0.15 % ) ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with interlaminar cervical epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial based on the available studies , the incidence of dural puncture ranged between 0.07 and 2.6 % . reported 100 cases where intravascular placement of the needle occurred . in a retrospective analysis of the results of 345 c67 or c7t1 injections , botwin et al a large proportion of these adverse events were related to an increase of neck pain , headache , insomnia and vasovagal reactions . they reported identification of 98 intravascular injections that did not result in any adverse effects . conducted an anonymous survey asking the us physician members of the american pain society about their experience with regards to serious complications following cervical transforaminal epidural corticosteroid injections ( tesis ) . from the 287 replies , 78 complications were reported , among which there were 30 brain or spinal cord infarcts and 24 neurologic complications including death of unsuspected aetiology ( n = 5 ) , high spinal anaesthesia ( n = 3 ) , transient ischaemic attacks ( n = 3 ) , and spinal cord or brainstem oedema ( n = 3 ) . the literature search found 11 studies that present adverse effects following interlaminar lumbar esis ( table 2 ) [ 3436 , 40 , 42 , 44 , 45 , 4752 ] . in addition , four studies that involve more than 250 patients have reported no adverse events following interlaminar lumbar epidural esis [ 46 , 54 , 55 , 60 ] . reported the incidence of intradiscal injection to be one in 4723 .table 2complications reported with interlaminar lumbar epidural corticosteroid injectionsstudy , yeardesignptsmedicationsimagingcomplicationscarette et al . , 2015 prospective randomised study120 pts , 120 injectionsmethylprednisolone acetate 80 mgfl ( n = 56 ) , us ( n = 56)15 dizziness or pain at injection site or facial flushing1 intrathecal injection bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound complications reported with interlaminar lumbar epidural corticosteroid injections bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound fourteen studies were identified that presented adverse effects following transforaminal esis ( table 3 ) [ 34 , 50 , 53 , 5659 , 61 , 63 , 6569 ] . the most common complication reported was increased pain , which was encountered in half of the patients . , 2014 rct60 ptsmethylprednisolone 60 mgfl3 transient paraesthesia of nerve distribution ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with transforaminal lumbar epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial a large number of case reports presenting rare adverse events following esis exist ( fig . the available literature has described deaths following esis [ 89 , 93 , 97 ] . reviewing the available case reports , the most common and devastating complication was infarction of the spinal cord , cerebellum , brain and brainstem [ 97 , 141 ] . damage to the blood vessels could result in haematomas , and subdural and epidural haematomas have been reported [ 124 , 135 , 144 , 146 , 155 , 162 , 164 ] . direct damage to the spinal cord by trauma or direct injection of esi medications into the cervical spinal cord has been also documented [ 140 , 148 , 160 , 173 ] . 2reported major complications and approach used reported major complications and approach used infections and abscesses have been also reported following esi [ 108114 , 117127 , 130 , 132 , 133 , 164 ] . with the exception of a fungal infection outbreak in the usa in 2012 , infection rates vary following an epidural injection , but , on average , are reported to be one in 60,000100,000 epidural injections . in single case reports , cases of meningitis , vertebral osteomyelitis , and spinal and paraspinal abscesses have been reported that are caused by microorganisms including aspergillus spp . in the cervical spine , this complication can be the result of the administration of radio contrast agents administered in the intracranial vasculature . case reports have also presented other complications including iatrogenic cushing s syndrome [ 131 , 166 ] , persistent hiccups [ 128 , 152 ] , convulsions , reversible posterior leukoencephalopathy syndrome , epidural granuloma formation , subdural block , brown squard syndrome , herpes zoster outbreak , steroid myopathy and corticosteroid - induced psychosis following esi . the review of the available literature identified 11 manuscripts presenting complications following interlaminar cervical esis ( table 1 ) [ 21 , 22 , 24 , 26 , 2834 ] . , 2014 rct120 pts , 688 injectionsbetamethasone 6 mg ( n = 60)fl6 subarachnoid punctures ( 0.3 % ) 10 intravascular penetrations ( 0.6 % ) 3 nerve root irritations ( 0.76 % ) 1 pain lasting 1 week ( 0.15 % ) ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with interlaminar cervical epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial based on the available studies , the incidence of dural puncture ranged between 0.07 and 2.6 % . reported 100 cases where intravascular placement of the needle occurred . they reported identification of 98 intravascular injections that did not result in any adverse effects . conducted an anonymous survey asking the us physician members of the american pain society about their experience with regards to serious complications following cervical transforaminal epidural corticosteroid injections ( tesis ) . the literature search found 11 studies that present adverse effects following interlaminar lumbar esis ( table 2 ) [ 3436 , 40 , 42 , 44 , 45 , 4752 ] . in addition , four studies that involve more than 250 patients have reported no adverse events following interlaminar lumbar epidural esis [ 46 , 54 , 55 , 60 ] . , 2015 prospective randomised study120 pts , 120 injectionsmethylprednisolone acetate 80 mgfl ( n = 56 ) , us ( n = 56)15 dizziness or pain at injection site or facial flushing1 intrathecal injection bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound complications reported with interlaminar lumbar epidural corticosteroid injections bl blind , ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial , us ultrasound fourteen studies were identified that presented adverse effects following transforaminal esis ( table 3 ) [ 34 , 50 , 53 , 5659 , 61 , 63 , 6569 ] . the most common complication reported was increased pain , which was encountered in half of the patients . identified six intradiscal injections among 249 transforaminal esis , which represents an incidence of 2.4 % . , although this was tenfold higher than after interlaminar esi .table 2000 retrospective cohort study207 pts , 322 injectionseither betamethasone 912 mg or methylprednisolone 80 mgfl10 non - positional headaches ( 3.1 % ) 8 increased back pain ( 2.4 % ) 2 increased leg pain ( 0.6 % ) 4 facial flushing ( 1.2 % ) 1 vasovagal reaction ( 0.3 % ) 1 increased blood sugar ( 258 mg / dl ) in an insulin - dependent patient with diabetes mellitus ( 0.3 % ) 1 intraoperative hypertension ( 0.3 % ) ahadian et al . , 2014 rct60 ptsmethylprednisolone 60 mgfl3 transient paraesthesia of nerve distribution ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial complications reported with transforaminal lumbar epidural corticosteroid injections ct computed tomography , fl fluoroscopy , na not available , pts patients , rct randomised controlled trial a large number of case reports presenting rare adverse events following esis exist ( fig . the available literature has described deaths following esis [ 89 , 93 , 97 ] . reviewing the available case reports , the most common and devastating complication was infarction of the spinal cord , cerebellum , brain and brainstem [ 97 , 141 ] . damage to the blood vessels could result in haematomas , and subdural and epidural haematomas have been reported [ 124 , 135 , 144 , 146 , 155 , 162 , 164 ] . direct damage to the spinal cord by trauma or direct injection of esi medications into the cervical spinal cord has been also documented [ 140 , 148 , 160 , 173 ] . 2reported major complications and approach used reported major complications and approach used infections and abscesses have been also reported following esi [ 108114 , 117127 , 130 , 132 , 133 , 164 ] . in the cervical spine , this complication can be the result of the administration of radio contrast agents administered in the intracranial vasculature . esis have been used for more than 60 years since lievre et al . currently , esis are the most common intervention performed for the management of chronic low back pain in the usa . major events have been reported anecdotally and it is impossible to comment on their true incidence based on the available results in the literature . overall , the potential causes of adverse events could be categorised into three distinct categories : ( 1 ) direct damage to the blood vessels or adjacent anatomical structures during the procedure ; ( 2 ) intravascular administration of the injectate ; and ( 3 ) a local or systemic reaction including bacterial contamination . direct damage to the spinal cord by the needle and the injection of corticosteroids into the cervical spinal cord has been also documented in a very limited number of case reports . clinically significant haematomas derived from piercing or damage to the blood vessels can occur and the reported incidence for all epidurals is less than one in 150,000 . intravascular injection of the corticosteroids , carrier and/or the local anaesthetic could account for the large majority of the serious adverse effects . [ 25 , 189 ] reported an incidence of fluoroscopically confirmed intravascular penetration of 19.4 % for cervical tesis , 8.1 % for lumbar tesis and 21.3 % for tesis at the s1 level . vascular embolic events from intra - arterial injection of particulate corticosteroids have been found to account for serious complications including spinal cord infarction , paraplegia and death [ 93 , 100 , 102 , 103 , 105 ] . the injection of the particulate corticosteroid methylprednisolone into the vertebral artery of four pigs resulted in permanent loss of consciousness , while the animals receiving dexamethasone and prednisolone recovered fully . demonstrated that methylprednisolone and its non - particulate carrier can produce significant injury to the blood the authors also suggested that in addition to the cerebral microvasculature occlusion by the particulate corticosteroids , damage can occur via toxicity of the carrier or the corticosteroid . based on several observations that failed to highlight any difference in the efficacy of particulate and non - particulate corticosteroids , we would recommend the use of soluble non - particulant agents [ 196198 ] . a transient blockage of the neural conduction is expected ; however , the reported central canal , conus medularis and cauda equina syndromes must have an underlying cause , i.e. transient systemic reactions including headaches , vasovagal reactions and facial flushing have been reported ; these reactions occurred shortly after the esi and could represent a reaction to the injected anaesthetic agents and/or corticosteroids . for instance , a significant number of the patients with chronic back pain conditions are treated with repeated injections over prolonged period of time . osteoporosis and an increased fracture risk could theoretically occur ; however , the available literature does not support this theory . according to the centers for disease control and prevention , 25 deaths due to epidural corticosteroid - related meningitis ( many due to aspergillosis ) were identified : 337 patients were affected in 18 us states and 14,000 patients were probably exposed to contaminated corticosteroids . in the authors opinion , several recommendations can be made with the aim of minimise the incidence of major complications after esis . the use of a blunt needle and the suggestion that a small test dose of the medication should be inject initially has been proposed [ 204207 ] . dynamic live fluoroscopy was found to perform better than static intermittent fluoroscopy , which was found to miss 57 % of the intravascular injections . digital subtraction angiography can be used as a radiologic adjunct to identify vascular compromise during the injection . , an anaesthetic test dose and digital subtraction angiography performed twice did not prevent a catastrophic spinal cord infarction and the resultant paraplegia . possible mechanisms explaining these events include the intra - arterial injection of particulate corticosteroid or trauma causing embolisation to the distal basilar or vertebral arteries . despite the fact that the study presents the extreme end of potential complications , it is unclear what the true incidence of these events is . the warning issued by the fda regarding esis merits further discussion and analysis , and the use of corticosteroids for injections in the epidural space for spinal pain syndromes the fda mentions that , despite their use , the effectiveness of esis has been challenged and could potentially result in serious adverse events including death , stroke and paralysis . major adverse events can occur with esis , but such events are rare , their true incidence is unknown and they have only been presented in case reports . for instance , none of the studies included in tables 1 , 2 and 3 presents such major devastating complications . a large proportion of the available case reports and studies provide insufficient documentation , i.e. the approach used for esi , symptom duration , volume injected or even the number of injections . in addition , the majority of the studies report adverse effects incidentally as their main aim is to report the efficacy of the injections . furthermore , the available studies were heterogeneous with regards to the outcome measures , and in several manuscripts the surgical technique , corticosteroid dosage and the addition of other medications are not reported . it is of note that many studies did not look at or record the complications or adverse effects of corticosteroid exposure but present results from several pain and functional scores . esis are relatively safe ; however , although major complications of esis have been reported , their true incidence remains obscure . vascular penetration and administration of pharmaceutical agents intra - arterially could account for a large proportion of the adverse events reported . with accurate placement of the needle , use of non - particulate corticosteroids , live fluoroscopy , digital subtraction angiography and familiarisation of the contrast patterns on fluoroscopy these risks should be minimised . further research is required to shed more light on the best clinical practice for the use of esis and the true incidence of complications relating to them .

the survey addressed information on antimicrobial drug resistance testing , phage typing , or both . since most countries do not routinely apply these typing methods , the questionnaire was not simply sent to all world health organization ( who ) member states . instead , an invitation to participate in the survey was distributed through the who global salm - surv ( gss ) network ( 25 ) and directly to all enter - net ( 26 ) network countries , plus a group of large countries known or assumed to have resistance testing or phage typing as an integrated part of their national salmonella surveillance system . a total of 52 questionnaires were sent out in june and july 2002 . of these , 44 were sent directly to countries known or assumed to have resistance testing or phage typing , and 8 were sent out to member states responding to the gss invitation . of the 52 questionnaires , 32 were sent to countries in the european region , 6 to the american region , 7 to the asian region , 5 to the african region , and 2 to the oceania region . country names and names of geographic regions and subregions are used as described in the united nations classification system ( 27 ) . each country was requested to give information on the total annual number of laboratory - verified episodes of nontyphoidal salmonellosis , s. typhimurium , mdr s. typhimurium , and s. typhimurium dt104 from 1992 to 2001 . the participating countries were also asked to give details on whether the reported number of salmonella isolates that formed the basis for further phage typing or antimicrobial drug susceptibility testing included all national isolates or if they were a subset of isolates . if only a subset of isolates were tested , the countries were asked to state the proportion of s. typhimurium strains tested and what the criteria for choosing the isolates were . we also asked participants to describe methods used for antimicrobial drug resistance testing and the drugs included in their tests . multidrug - resistance was defined as isolates being resistant or intermediately susceptible towards 4 separate classes of drugs . this group includes isolates of r - type acssut , the so - called classical penta - resistant phenotype . when information was available , participants were asked to give the numbers of isolates exhibiting r - types extending the acssut - complex and the number of isolates resistant to clinically relevant antimicrobial drugs ( quinolones , trimethoprim , and cephalosporins ) . in countries where only a subset of salmonella isolates had been submitted for phage typing , antimicrobial drug - resistance testing , or both , the total number of dt104 or mdr s. typhimurium isolates was extrapolated from the reported numbers of isolates and the proportion of tested isolates . the survey addressed information on antimicrobial drug resistance testing , phage typing , or both . since most countries do not routinely apply these typing methods , the questionnaire was not simply sent to all world health organization ( who ) member states . instead , an invitation to participate in the survey was distributed through the who global salm - surv ( gss ) network ( 25 ) and directly to all enter - net ( 26 ) network countries , plus a group of large countries known or assumed to have resistance testing or phage typing as an integrated part of their national salmonella surveillance system . a total of 52 questionnaires were sent out in june and july 2002 . of these , 44 were sent directly to countries known or assumed to have resistance testing or phage typing , and 8 were sent out to member states responding to the gss invitation . of the 52 questionnaires , 32 were sent to countries in the european region , 6 to the american region , 7 to the asian region , 5 to the african region , and 2 to the oceania region . country names and names of geographic regions and subregions are used as described in the united nations classification system ( 27 ) . each country was requested to give information on the total annual number of laboratory - verified episodes of nontyphoidal salmonellosis , s. typhimurium , mdr s. typhimurium , and s. typhimurium dt104 from 1992 to 2001 . the participating countries were also asked to give details on whether the reported number of salmonella isolates that formed the basis for further phage typing or antimicrobial drug susceptibility testing included all national isolates or if they were a subset of isolates . if only a subset of isolates were tested , the countries were asked to state the proportion of s. typhimurium strains tested and what the criteria for choosing the isolates were . we also asked participants to describe methods used for antimicrobial drug resistance testing and the drugs included in their tests . multidrug - resistance was defined as isolates being resistant or intermediately susceptible towards 4 separate classes of drugs . this group includes isolates of r - type acssut , the so - called classical penta - resistant phenotype . when information was available , participants were asked to give the numbers of isolates exhibiting r - types extending the acssut - complex and the number of isolates resistant to clinically relevant antimicrobial drugs ( quinolones , trimethoprim , and cephalosporins ) . in countries where only a subset of salmonella isolates had been submitted for phage typing , antimicrobial drug - resistance testing , or both , the total number of dt104 or mdr s. typhimurium isolates was extrapolated from the reported numbers of isolates and the proportion of tested isolates . the questionnaire was sent to 52 countries , and a completed questionnaire was received from 29 , a response rate of 56% ( figure 1 ) . of the 52 invited countries , 23 were members of or affiliated with enter - net ; from these a positive feedback was received from 20 ( 87% ) . these countries were australia , austria , belgium , canada , denmark , england and wales , finland , germany , greece , ireland , japan , luxembourg , the netherlands , new zealand , norway , scotland , south africa , spain , sweden , and switzerland . the 8 countries and 1 regional center ( 31% response rate ) that participated in the survey that were not associated with enter - net when data were collected were brazil , the czech republic , hungary , israel , latvia , malta , republic of south korea , united states , and the caribbean epidemiology centre ( carec ) , a regional center that represents 21 countries in the caribbean ( anguilla , antigua and barbuda , aruba , bahamas , barbados , belize , bermuda , british virgin islands , cayman islands , dominica , grenada , guyana , jamaica , montserrat , netherlands antilles , st . kitts and nevis , st . vincent and the grenadines , suriname , trinidad and tobago , and turks and caicos ) . for the purpose of this study , participating countries in the survey of multidrug - resistant salmonella enterica serotype typhimurium , 19922001 , internationally ( a ) and in europe ( b ) . the proportion of salmonella isolates forwarded from local health laboratories to national or regional institutions varied from 5% to 100% . none of the participating countries restricted the submission of isolates to strains from selected patients , e.g. , in case of septicemia or outbreak situations . all 29 participating countries performed serotyping on 85% to 100% of salmonella isolates received at the national reference laboratory . table 1 shows the number of nontyphoidal salmonella isolates and the proportion hereof that were s. typhimurium in each country from 1992 to 2001 presented as 2-year intervals . the table also shows the estimated incidence of laboratory - confirmed cases of s. typhimurium in 2001 . throughout the study period , the proportion of s. typhimurium among nontyphoidal salmonellae has been relatively stable . in 1992 , 16% of isolates were s. typhimurium , compared to 17% in 2001 . however , large differences between countries and variations from year to year within each country made comparison difficult . nevertheless , some trends emerged when the countries were aggregated into geographic regions . in the caribbean region , south america , eastern asia , and europe , a general decrease in the proportion of s. typhimurium was observed . in north america , on the other hand , the situation has remained relatively stable , whereas both australia and new zealand have seen an increase in the number and proportion of s. typhimurium cases from 1992 to 2001 . * pop . , population 10 ; ist , incidence of s. typhimurium per 10 population . data on s. typhimurium and incidence are based on data from the new federal states of germany and the city of berlin . submission of strains for serotyping at the central laboratory was not compulsory in switzerland at the time of data collection . # according to the united nations classification , israel belongs to the western asian region . in this study phage typing was carried out in 6 of the participating countries ; by 2001 , this number had risen to 22 . five countries ( brazil , japan , norway , switzerland , and the united states ) only performed phage typing when an mdr strain was found or in outbreak situations . although not all countries performed phage typing in accordance with the colindale scheme ( 28 ) , the countries using different standards provided information that allowed for comparison with results obtained using the colindale scheme ( 2931 ) . the proportion of s. typhimurium strains that were dt104 and the proportion thereof that were found to be mdr are shown for each country in table 2 . in general , the incidence and proportion of dt104 increased throughout the period . in 1992 , 8.7% of s. typhimurium isolates were dt104 , but in 2001 this proportion had increased to 33% . again , the incidence peaked in 1996 and then decreased . in most other european countries and north america , the relative numbers of dt104 strains had increased throughout the period . in australia and new zealand , the proportion of s. typhimurium strains that were dt104 is depicted in figure 2 ( the countries are aggregated into 8 regions ) . * mdr , multidrug - resistant ; na , data not available . ; no . of dt104 ; and no . of dt104 that are mdr . according to the united nations classification , israel belongs to the western asian region . in this study , israel has been grouped with southern european countries . salmonella enterica serovar typhimurium dt104 as percentage of all s. typhimurium in 8 world regions , 19922001 . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : england and wales , scotland , ireland ; scandinavia : denmark , finland , norway , sweden ; western europe : austria , germany , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : spain and israel ; north america : canada , united states ; eastern asia : the republic of korea ; oceania : australia and new zealand . antimicrobial susceptibility testing became an increasingly common constituent of the surveillance during the study period . in 1992 , susceptibility testing was performed in 14 of the 29 participating countries and in all but one in 2000 . all countries routinely tested a minimum of 6 antimicrobial drugs , all belonging to different drug classes . the majority of countries ( 73% ) used disc diffusion testing according to nccls standards . listed by frequency , the most common antimicrobial drug classes included in the test panel were ( fluoro)quinolones , broad - spectrum penicillins , phenicols , aminoglycosides , tetracyclines , cephalosporins , sulfonamides , and trimethoprim . in 21 countries , antimicrobial susceptibility testing was performed independently of phage typing results , but in 2 countries ( finland until 1999 and new zealand ) , testing was only performed on strains found to be dt104 . table 2 shows the distribution of mdr s. typhimurium presented as 2-year time - bands . figure 3 depicts the general trend , with countries divided into 9 different regions . in 1992 15% of all s. typhimurium isolates were mdr ; by 2001 , this percentage had increased 3-fold to 42% . once again , large variations occurred between countries and within countries from year to year . in most european countries and north america , mdr s. typhimurium was common and , with the exception of the united kingdom and ireland , multidrug resistance increased from the mid-1990s to the end of the study period . for example , in 2001 multidrug resistance ranged from 22% ( greece ) to 72% ( ireland ) . only limited data were available from brazil , the caribbean region , south africa , and the republic of korea . however , in these regions multidrug resistance was far less common , ranging from zero in the caribbean region to 19% in the republic of korea . in australia , multidrug resistance remained low throughout the period , with 1.0% of strains in 1997 and 3.6% in 2001 . multidrug - resistant salmonella enterica serovar typhimurium as a percentage of all s. typhimurium in 9 world regions , 19922001 . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : scotland and ireland ; scandinavia and the baltics : denmark , finland , norway , and latvia ; western europe : austria , germany , luxembourg , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : greece , malta , and spain ; north america : canada , united states ; south america : brazil ; eastern asia : the republic of korea ; oceania : australia . mdr dt104 was a frequent subtype of mdr s. typhimurium in most of the countries . in 1992 , between 11% ( germany ) and 77% ( scotland ) of the mdr strains were dt104 ; in 2001 this ranged from 22% ( australia ) to 94% ( the netherlands ) . overall , the proportion of mdr dt104 of all dt104 has remained fairly stable ; 84% of dt104 were classified as mdr in 2001 , compared with 72% in 1992 . among mdr dt104 , the classical penta - resistant phenotype , r - type acssut , was by far the most common phenotype throughout the period . it was found in 99% of mdr dt104 strains in 1992 and in 94% of such strains in 2001 ( data not shown ) . finally , we looked at trends in development of additional resistance in the classical penta - resistant phenotype ( r - type acssut ) , with focus on 3 clinically important antimicrobial drug classes : quinolones , cephalosporins , and trimethoprim . both quinolone and trimethoprim resistance increased in mdr dt104 throughout the study period . in 1992 , nalidixic acid susceptibility testing was performed on a total of 194 mdr dt104 isolates in 4 different countries ; no resistant isolates were found . in 2001 , nalidixic acid susceptibility testing of 1,812 mdr dt104 strains in 11 countries identified 109 ( 6.0% ) resistant strains . similarly , trimethoprim resistance was found in 1.2% of the 180 mdr dt104 strains tested in 1992 , but in 6.6% of 1,855 mdr dt104 strains tested in 2001 . cephalosporin - resistant mdr dt104 remained rare , with only 0.5% resistant strains in 2001 , and no clear trend observable . figure 4 shows the overall trend of resistance to quinolone , trimethoprim , and cephalosporins from 1992 to 2001 . the increase in quinolone resistance seen in 1996 and in 1998 was caused by a general increase in quinolone - resistant mdr dt104 in scotland and an outbreak of quinolone - resistant mdr dt104 in denmark ( 14 ) , respectively . the increase in trimethoprim resistance from 1995 to 1996 was also caused by a general increase in trimethoprim - resistant mdr dt104 in scotland . proportion of multidrug - resistant salmonella enterica serovar typhimurium with additional resistance to quinolones , cephalosporin , or trimethoprim , 19922001 . the proportion of salmonella isolates forwarded from local health laboratories to national or regional institutions varied from 5% to 100% . none of the participating countries restricted the submission of isolates to strains from selected patients , e.g. , in case of septicemia or outbreak situations . all 29 participating countries performed serotyping on 85% to 100% of salmonella isolates received at the national reference laboratory . table 1 shows the number of nontyphoidal salmonella isolates and the proportion hereof that were s. typhimurium in each country from 1992 to 2001 presented as 2-year intervals . the table also shows the estimated incidence of laboratory - confirmed cases of s. typhimurium in 2001 . throughout the study period , the proportion of s. typhimurium among nontyphoidal salmonellae has been relatively stable . in 1992 , 16% of isolates were s. typhimurium , compared to 17% in 2001 . however , large differences between countries and variations from year to year within each country made comparison difficult . nevertheless , some trends emerged when the countries were aggregated into geographic regions . in the caribbean region , south america , eastern asia , and europe , a general decrease in the proportion of s. typhimurium was observed . in north america , on the other hand , the situation has remained relatively stable , whereas both australia and new zealand have seen an increase in the number and proportion of s. typhimurium cases from 1992 to 2001 . * pop . , population 10 ; ist , incidence of s. typhimurium per 10 population . data on s. typhimurium and incidence are based on data from the new federal states of germany and the city of berlin . submission of strains for serotyping at the central laboratory was not compulsory in switzerland at the time of data collection . # according to the united nations classification , israel belongs to the western asian region . in this study in 1992 , phage typing was carried out in 6 of the participating countries ; by 2001 , this number had risen to 22 . five countries ( brazil , japan , norway , switzerland , and the united states ) only performed phage typing when an mdr strain was found or in outbreak situations . although not all countries performed phage typing in accordance with the colindale scheme ( 28 ) , the countries using different standards provided information that allowed for comparison with results obtained using the colindale scheme ( 2931 ) . the proportion of s. typhimurium strains that were dt104 and the proportion thereof that were found to be mdr are shown for each country in table 2 . in general , the incidence and proportion of dt104 increased throughout the period . in 1992 , 8.7% of s. typhimurium the incidence peaked in 1996 and then decreased . in most other european countries and north america , the relative numbers of dt104 strains had increased throughout the period . in australia and new zealand , the proportion of s. typhimurium strains that were dt104 is depicted in figure 2 ( the countries are aggregated into 8 regions ) . * ; no . of dt104 ; and no . of dt104 that are mdr . according to the united nations classification , israel belongs to the western asian region . in this study salmonella enterica serovar typhimurium dt104 as percentage of all s. typhimurium in 8 world regions , 19922001 . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : england and wales , scotland , ireland ; scandinavia : denmark , finland , norway , sweden ; western europe : austria , germany , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : spain and israel ; north america : canada , united states ; eastern asia : the republic of korea ; oceania : australia and new zealand . antimicrobial susceptibility testing became an increasingly common constituent of the surveillance during the study period . in 1992 , susceptibility testing was performed in 14 of the 29 participating countries and in all but one in 2000 . all countries routinely tested a minimum of 6 antimicrobial drugs , all belonging to different drug classes . the majority of countries ( 73% ) used disc diffusion testing according to nccls standards . listed by frequency , the most common antimicrobial drug classes included in the test panel were ( fluoro)quinolones , broad - spectrum penicillins , phenicols , aminoglycosides , tetracyclines , cephalosporins , sulfonamides , and trimethoprim . in 21 countries , antimicrobial susceptibility testing was performed independently of phage typing results , but in 2 countries ( finland until 1999 and new zealand ) , testing was only performed on strains found to be dt104 . table 2 shows the distribution of mdr s. typhimurium presented as 2-year time - bands . figure 3 depicts the general trend , with countries divided into 9 different regions . in 1992 15% of all s. typhimurium isolates were mdr ; by 2001 , this percentage had increased 3-fold to 42% . once again , large variations occurred between countries and within countries from year to year . in most european countries and north america , mdr s. typhimurium was common and , with the exception of the united kingdom and ireland , multidrug resistance increased from the mid-1990s to the end of the study period . for example , in 2001 multidrug resistance ranged from 22% ( greece ) to 72% ( ireland ) . only limited data were available from brazil , the caribbean region , south africa , and the republic of korea . however , in these regions multidrug resistance was far less common , ranging from zero in the caribbean region to 19% in the republic of korea . in australia , multidrug resistance remained low throughout the period , with 1.0% of strains in 1997 and 3.6% in 2001 . multidrug - resistant salmonella enterica serovar typhimurium as a percentage of all s. typhimurium in 9 world regions , 19922001 . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : scotland and ireland ; scandinavia and the baltics : denmark , finland , norway , and latvia ; western europe : austria , germany , luxembourg , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : greece , malta , and spain ; north america : canada , united states ; south america : brazil ; eastern asia : the republic of korea ; oceania : australia . mdr dt104 was a frequent subtype of mdr s. typhimurium in most of the countries . in 1992 , between 11% ( germany ) and 77% ( scotland ) of the mdr strains were dt104 ; in 2001 this ranged from 22% ( australia ) to 94% ( the netherlands ) . overall , the proportion of mdr dt104 of all dt104 has remained fairly stable ; 84% of dt104 were classified as mdr in 2001 , compared with 72% in 1992 . among mdr dt104 , the classical penta - resistant phenotype , r - type acssut , was by far the most common phenotype throughout the period . it was found in 99% of mdr dt104 strains in 1992 and in 94% of such strains in 2001 ( data not shown ) . finally , we looked at trends in development of additional resistance in the classical penta - resistant phenotype ( r - type acssut ) , with focus on 3 clinically important antimicrobial drug classes : quinolones , cephalosporins , and trimethoprim . both quinolone and trimethoprim resistance increased in mdr dt104 throughout the study period . in 1992 , nalidixic acid susceptibility testing was performed on a total of 194 mdr dt104 isolates in 4 different countries ; no resistant isolates were found . in 2001 , nalidixic acid susceptibility testing of 1,812 mdr dt104 strains in 11 countries identified 109 ( 6.0% ) resistant strains . similarly , trimethoprim resistance was found in 1.2% of the 180 mdr dt104 strains tested in 1992 , but in 6.6% of 1,855 mdr dt104 strains tested in 2001 . cephalosporin - resistant mdr dt104 remained rare , with only 0.5% resistant strains in 2001 , and no clear trend observable . figure 4 shows the overall trend of resistance to quinolone , trimethoprim , and cephalosporins from 1992 to 2001 . the increase in quinolone resistance seen in 1996 and in 1998 was caused by a general increase in quinolone - resistant mdr dt104 in scotland and an outbreak of quinolone - resistant mdr dt104 in denmark ( 14 ) , respectively . the increase in trimethoprim resistance from 1995 to 1996 was also caused by a general increase in trimethoprim - resistant mdr dt104 in scotland . proportion of multidrug - resistant salmonella enterica serovar typhimurium with additional resistance to quinolones , cephalosporin , or trimethoprim , 19922001 . the present survey was conducted to gain a better understanding of the global impact of dt104 and mdr s. typhimurium , given the severity of illness than can result from infection with s. typhimurium and mdr strains in particular . the survey 's primary findings are that during the period 19922001 , the total number of mdr s. typhimurium and s. typhimurium dt104 cases increased , while that of other types of s. typhimurium decreased the collected data also may not always accurately describe the real national incidence or be directly comparable between countries . the total number of isolates of nontyphoidal salmonellae registered at the national level decreased from 1992 to 2001 . this result may be biased because of changes in surveillance practices . since the survey indicated that surveillance systems generally improved throughout the study period , this trend is most likely correct , however . concurrent with this decrease , an overall decrease in the number of s. typhimurium isolates was observed , thus keeping the proportion of s. typhimurium cases among total salmonella cases constant . the decrease was primarily seen in europe and north america , whereas australia and new zealand saw an increase in both the number of cases of s. typhimurium and of the total number of nontyphoidal salmonella cases . in germany the total number of nontyphoidal salmonella cases is available for the whole country , but for administrative reasons data on serotypes , antimicrobial susceptibility , and phage types are based only on results from the new federal states of germany ( formerly east germany ) and the city of berlin . however , german studies have shown that serotype distribution and drug resistance are comparable between former west and east germany ( 32 ) ( w. rabsch , pers . mdr s. typhimurium has increased in the past decades in almost all the regions covered in this study . most of this increase is due to the concurrent upsurge of dt104 , whereas other phage types , such as u302 , dt120 , dt12 , and dt193 , were reported to play a smaller role in this development . a high proportion of mdr s. typhimurium was primarily observed in europe and north america . because of the low coverage of participating countries in asia , south america , and africa , the situation in these areas was difficult to assess . both australia and new zealand , however , reported high incidence of s. typhimurium , but mdr strains of s. typhimurium and dt104 were largely absent . the isolated increase in mdr s. typhimurium in australia in 2001 can be explained by a large outbreak of mdr dt104 associated with consumption of halva ( dessert made of sesame seeds ) ( 33 ) . that dt104 has not spread markedly in australia and new zealand may be explained both by geography and the very strict food and livestock import restrictions in force in these countries , which prevent any large - scale introduction and spread of foreign salmonella types in the food animal production chain ( 34 ) . although high and generally increasing levels of mdr s. typhimurium were observed in europe , the united kingdom presents a special case . it and germany had an increase in dt104 in the beginning of the 1990s , before most other countries ( 5,35 ) . in fact , dt104 was first isolated in the united kingdom in the early 1980s , years before it was isolated in other countries ( 5 ) . in the united kingdom , the incidence of dt104 peaked in 1996 and has since declined ( and this study ) . possible explanations for this finding include the management of bovine spongiform encephalitis in the united kingdom , associated general improvements in farm hygiene , and an overall decline in cattle production ( 36 ) . although multidrug - resistance and dt104 were closely linked , large country - specific differences were seen , even between neighboring countries . most pronounced was the difference between germany and the netherlands in 2001 , where 64% and 94% , respectively , of mdr isolates were dt104 . such differences probably reflect both real differences and biases resulting from , for instance , different laboratory reporting practices . for example , some of the countries in this survey ( new zealand , norway , and the united states ) performed antimicrobial susceptibility tests on all their dt104 isolates but only on a subset of non - dt104 strains . in australia and in scandinavia , where the incidence of domestically acquired salmonellosis is generally low , many cases appeared to be imported . in a recent study of australian dt104 isolates , 37% were associated with travel abroad , particularly to southeast asia ( d. lightfoot , pers . complete data on travel association of mdr s. typhimurium cases were available for norway and finland and showed that most mdr s. typhimurium patients were infected abroad . in sweden , where information on dt104 but not mdr was available , most dt104 patients were infected abroad . a special issue concerns the possibility of acquisition , with time , of resistance traits additional to the classical penta - resistant pattern . of particular concern is the additional acquisition of quinolone resistance by mdr dt104 , since fluoroquinolones are often the drugs of first choice when treating severe salmonellosis . as mentioned , several studies have now shown that multidrug resistance and quinolone resistance may be associated with particular adverse health effects . when seen in the light of the ability of dt104 to spread and establish itself in a large variety of food animal lines ( cattle , pigs , poultry ) , the increase in the number of mdr s. typhimurium strains that include quinolone resistance becomes particularly problematic . quinolone , and in particular the fluoroquinolones , have been part of human medicine since the 1980s , resulting in no or very limited resistance in salmonellae . it was not until the license of fluoroquinolones for food animal production in the early 1990s that resistant salmonella strains emerged ( 37 ) . the use of fluoroquinolones for food production animals should therefore be discontinued or at least severely restricted as quickly as possible . first , it was limited to countries with relatively sophisticated surveillance systems in place , since only countries performing phage typing or resistance testing in addition to serotyping were eligible . therefore , the survey contains no representative data on the situation in these regions . who global salm - surv seeks to enhance the capacity of countries to provide such data . second , the collection of isolates at the national level is likely to vary from country to country , depending on a number of factors such as sampling frequency at the local level , availability of laboratory reagents , and the degree to which isolates and results are forwarded to the national level . local practice , the priority given to foodborne illnesses , and financial factors will influence on how often a physician will request a fecal sample . furthermore , many countries stated that strains and information were not always routinely collected centrally , while in some of the countries strains were never forwarded from certain local laboratories . for these reasons , care should be taken in the interpretation of results of this survey ; in particular when comparing incidence rates between countries . however , these limitations are inherent to surveillance in general and do not apply only to this survey . cross - checking these survey data with available published surveillance data showed them to be in line with each other in the united states ( 38 ) , the netherlands ( 39 ) , and denmark ( 40 ) . in summary , on a global scale , only a small number of countries perform antimicrobial susceptibility testing or phage typing , although the number of countries doing so more than doubled throughout the study period . despite its limitations , the survey showed that the incidence of both mdr s. typhimurium and mdr s. typhimurium dt104 increased markedly worldwide during the 1990s , although the problem has primarily affected europe and north america . the survey implies that mdr s. typhimurium poses a serious and increasing public health problem in large parts of the world . surveillance and control programs such as the global salm - surv international network recently launched by who should therefore be reinforced .

the incidence of multidrug - resistant ( mdr ) salmonella typhimurium infections in humans , and in particular mdr definitive phage type 104 ( dt104 ) , has increased substantially in many countries in the last 2 decades , often associated with increased illness . to examine the magnitude of this problem , a survey was conducted among countries with available antimicrobial resistance or phage typing surveillance data . a total of 29 , primarily industrialized , countries participated in the survey , which covered the years 19922001 . overall , the incidence of mdr s. typhimurium and dt104 increased continuously during this period , although the problem affected primarily europe and north america . the increase appeared to have peaked in the united kingdom but not in other countries . also , the incidence of quinolone - resistant s. typhimurium was increasing . this survey implies that mdr s. typhimurium constitutes an increasing public health problem in large parts of the world and emphasizes the importance of surveillance and control programs .

Methods Participating Countries The Questionnaire Results Surveillance Phage Typing Antimicrobial Drug Resistance Discussion

the survey addressed information on antimicrobial drug resistance testing , phage typing , or both . instead , an invitation to participate in the survey was distributed through the who global salm - surv ( gss ) network ( 25 ) and directly to all enter - net ( 26 ) network countries , plus a group of large countries known or assumed to have resistance testing or phage typing as an integrated part of their national salmonella surveillance system . of these , 44 were sent directly to countries known or assumed to have resistance testing or phage typing , and 8 were sent out to member states responding to the gss invitation . of the 52 questionnaires , 32 were sent to countries in the european region , 6 to the american region , 7 to the asian region , 5 to the african region , and 2 to the oceania region . each country was requested to give information on the total annual number of laboratory - verified episodes of nontyphoidal salmonellosis , s. typhimurium , mdr s. typhimurium , and s. typhimurium dt104 from 1992 to 2001 . if only a subset of isolates were tested , the countries were asked to state the proportion of s. typhimurium strains tested and what the criteria for choosing the isolates were . in countries where only a subset of salmonella isolates had been submitted for phage typing , antimicrobial drug - resistance testing , or both , the total number of dt104 or mdr s. typhimurium isolates was extrapolated from the reported numbers of isolates and the proportion of tested isolates . the survey addressed information on antimicrobial drug resistance testing , phage typing , or both . instead , an invitation to participate in the survey was distributed through the who global salm - surv ( gss ) network ( 25 ) and directly to all enter - net ( 26 ) network countries , plus a group of large countries known or assumed to have resistance testing or phage typing as an integrated part of their national salmonella surveillance system . a total of 52 questionnaires were sent out in june and july 2002 . of these , 44 were sent directly to countries known or assumed to have resistance testing or phage typing , and 8 were sent out to member states responding to the gss invitation . of the 52 questionnaires , 32 were sent to countries in the european region , 6 to the american region , 7 to the asian region , 5 to the african region , and 2 to the oceania region . country names and names of geographic regions and subregions are used as described in the united nations classification system ( 27 ) . each country was requested to give information on the total annual number of laboratory - verified episodes of nontyphoidal salmonellosis , s. typhimurium , mdr s. typhimurium , and s. typhimurium dt104 from 1992 to 2001 . in countries where only a subset of salmonella isolates had been submitted for phage typing , antimicrobial drug - resistance testing , or both , the total number of dt104 or mdr s. typhimurium isolates was extrapolated from the reported numbers of isolates and the proportion of tested isolates . the questionnaire was sent to 52 countries , and a completed questionnaire was received from 29 , a response rate of 56% ( figure 1 ) . these countries were australia , austria , belgium , canada , denmark , england and wales , finland , germany , greece , ireland , japan , luxembourg , the netherlands , new zealand , norway , scotland , south africa , spain , sweden , and switzerland . the 8 countries and 1 regional center ( 31% response rate ) that participated in the survey that were not associated with enter - net when data were collected were brazil , the czech republic , hungary , israel , latvia , malta , republic of south korea , united states , and the caribbean epidemiology centre ( carec ) , a regional center that represents 21 countries in the caribbean ( anguilla , antigua and barbuda , aruba , bahamas , barbados , belize , bermuda , british virgin islands , cayman islands , dominica , grenada , guyana , jamaica , montserrat , netherlands antilles , st . for the purpose of this study , participating countries in the survey of multidrug - resistant salmonella enterica serotype typhimurium , 19922001 , internationally ( a ) and in europe ( b ) . throughout the study period , the proportion of s. typhimurium among nontyphoidal salmonellae has been relatively stable . in the caribbean region , south america , eastern asia , and europe , a general decrease in the proportion of s. typhimurium was observed . in north america , on the other hand , the situation has remained relatively stable , whereas both australia and new zealand have seen an increase in the number and proportion of s. typhimurium cases from 1992 to 2001 . data on s. typhimurium and incidence are based on data from the new federal states of germany and the city of berlin . in this study phage typing was carried out in 6 of the participating countries ; by 2001 , this number had risen to 22 . five countries ( brazil , japan , norway , switzerland , and the united states ) only performed phage typing when an mdr strain was found or in outbreak situations . although not all countries performed phage typing in accordance with the colindale scheme ( 28 ) , the countries using different standards provided information that allowed for comparison with results obtained using the colindale scheme ( 2931 ) . in general , the incidence and proportion of dt104 increased throughout the period . again , the incidence peaked in 1996 and then decreased . in most other european countries and north america , the relative numbers of dt104 strains had increased throughout the period . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : england and wales , scotland , ireland ; scandinavia : denmark , finland , norway , sweden ; western europe : austria , germany , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : spain and israel ; north america : canada , united states ; eastern asia : the republic of korea ; oceania : australia and new zealand . listed by frequency , the most common antimicrobial drug classes included in the test panel were ( fluoro)quinolones , broad - spectrum penicillins , phenicols , aminoglycosides , tetracyclines , cephalosporins , sulfonamides , and trimethoprim . table 2 shows the distribution of mdr s. typhimurium presented as 2-year time - bands . in most european countries and north america , mdr s. typhimurium was common and , with the exception of the united kingdom and ireland , multidrug resistance increased from the mid-1990s to the end of the study period . multidrug - resistant salmonella enterica serovar typhimurium as a percentage of all s. typhimurium in 9 world regions , 19922001 . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : scotland and ireland ; scandinavia and the baltics : denmark , finland , norway , and latvia ; western europe : austria , germany , luxembourg , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : greece , malta , and spain ; north america : canada , united states ; south america : brazil ; eastern asia : the republic of korea ; oceania : australia . mdr dt104 was a frequent subtype of mdr s. typhimurium in most of the countries . overall , the proportion of mdr dt104 of all dt104 has remained fairly stable ; 84% of dt104 were classified as mdr in 2001 , compared with 72% in 1992 . finally , we looked at trends in development of additional resistance in the classical penta - resistant phenotype ( r - type acssut ) , with focus on 3 clinically important antimicrobial drug classes : quinolones , cephalosporins , and trimethoprim . the increase in quinolone resistance seen in 1996 and in 1998 was caused by a general increase in quinolone - resistant mdr dt104 in scotland and an outbreak of quinolone - resistant mdr dt104 in denmark ( 14 ) , respectively . the increase in trimethoprim resistance from 1995 to 1996 was also caused by a general increase in trimethoprim - resistant mdr dt104 in scotland . proportion of multidrug - resistant salmonella enterica serovar typhimurium with additional resistance to quinolones , cephalosporin , or trimethoprim , 19922001 . throughout the study period , the proportion of s. typhimurium among nontyphoidal salmonellae has been relatively stable . in the caribbean region , south america , eastern asia , and europe , a general decrease in the proportion of s. typhimurium was observed . in north america , on the other hand , the situation has remained relatively stable , whereas both australia and new zealand have seen an increase in the number and proportion of s. typhimurium cases from 1992 to 2001 . , population 10 ; ist , incidence of s. typhimurium per 10 population . data on s. typhimurium and incidence are based on data from the new federal states of germany and the city of berlin . five countries ( brazil , japan , norway , switzerland , and the united states ) only performed phage typing when an mdr strain was found or in outbreak situations . although not all countries performed phage typing in accordance with the colindale scheme ( 28 ) , the countries using different standards provided information that allowed for comparison with results obtained using the colindale scheme ( 2931 ) . in general , the incidence and proportion of dt104 increased throughout the period . in 1992 , 8.7% of s. typhimurium the incidence peaked in 1996 and then decreased . in most other european countries and north america , the relative numbers of dt104 strains had increased throughout the period . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : england and wales , scotland , ireland ; scandinavia : denmark , finland , norway , sweden ; western europe : austria , germany , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : spain and israel ; north america : canada , united states ; eastern asia : the republic of korea ; oceania : australia and new zealand . in 1992 , susceptibility testing was performed in 14 of the 29 participating countries and in all but one in 2000 . listed by frequency , the most common antimicrobial drug classes included in the test panel were ( fluoro)quinolones , broad - spectrum penicillins , phenicols , aminoglycosides , tetracyclines , cephalosporins , sulfonamides , and trimethoprim . table 2 shows the distribution of mdr s. typhimurium presented as 2-year time - bands . in most european countries and north america , mdr s. typhimurium was common and , with the exception of the united kingdom and ireland , multidrug resistance increased from the mid-1990s to the end of the study period . multidrug - resistant salmonella enterica serovar typhimurium as a percentage of all s. typhimurium in 9 world regions , 19922001 . only countries that had data available for 2 or more 2-year periods are included : united kingdom and ireland : scotland and ireland ; scandinavia and the baltics : denmark , finland , norway , and latvia ; western europe : austria , germany , luxembourg , and the netherlands ; eastern europe : czech republic and hungary ; southern europe : greece , malta , and spain ; north america : canada , united states ; south america : brazil ; eastern asia : the republic of korea ; oceania : australia . mdr dt104 was a frequent subtype of mdr s. typhimurium in most of the countries . overall , the proportion of mdr dt104 of all dt104 has remained fairly stable ; 84% of dt104 were classified as mdr in 2001 , compared with 72% in 1992 . finally , we looked at trends in development of additional resistance in the classical penta - resistant phenotype ( r - type acssut ) , with focus on 3 clinically important antimicrobial drug classes : quinolones , cephalosporins , and trimethoprim . the increase in quinolone resistance seen in 1996 and in 1998 was caused by a general increase in quinolone - resistant mdr dt104 in scotland and an outbreak of quinolone - resistant mdr dt104 in denmark ( 14 ) , respectively . proportion of multidrug - resistant salmonella enterica serovar typhimurium with additional resistance to quinolones , cephalosporin , or trimethoprim , 19922001 . the present survey was conducted to gain a better understanding of the global impact of dt104 and mdr s. typhimurium , given the severity of illness than can result from infection with s. typhimurium and mdr strains in particular . the survey 's primary findings are that during the period 19922001 , the total number of mdr s. typhimurium and s. typhimurium dt104 cases increased , while that of other types of s. typhimurium decreased the collected data also may not always accurately describe the real national incidence or be directly comparable between countries . since the survey indicated that surveillance systems generally improved throughout the study period , this trend is most likely correct , however . the decrease was primarily seen in europe and north america , whereas australia and new zealand saw an increase in both the number of cases of s. typhimurium and of the total number of nontyphoidal salmonella cases . mdr s. typhimurium has increased in the past decades in almost all the regions covered in this study . a high proportion of mdr s. typhimurium was primarily observed in europe and north america . because of the low coverage of participating countries in asia , south america , and africa , the situation in these areas was difficult to assess . both australia and new zealand , however , reported high incidence of s. typhimurium , but mdr strains of s. typhimurium and dt104 were largely absent . the isolated increase in mdr s. typhimurium in australia in 2001 can be explained by a large outbreak of mdr dt104 associated with consumption of halva ( dessert made of sesame seeds ) ( 33 ) . although high and generally increasing levels of mdr s. typhimurium were observed in europe , the united kingdom presents a special case . it and germany had an increase in dt104 in the beginning of the 1990s , before most other countries ( 5,35 ) . in fact , dt104 was first isolated in the united kingdom in the early 1980s , years before it was isolated in other countries ( 5 ) . in the united kingdom , the incidence of dt104 peaked in 1996 and has since declined ( and this study ) . possible explanations for this finding include the management of bovine spongiform encephalitis in the united kingdom , associated general improvements in farm hygiene , and an overall decline in cattle production ( 36 ) . although multidrug - resistance and dt104 were closely linked , large country - specific differences were seen , even between neighboring countries . for example , some of the countries in this survey ( new zealand , norway , and the united states ) performed antimicrobial susceptibility tests on all their dt104 isolates but only on a subset of non - dt104 strains . in australia and in scandinavia , where the incidence of domestically acquired salmonellosis is generally low , many cases appeared to be imported . complete data on travel association of mdr s. typhimurium cases were available for norway and finland and showed that most mdr s. typhimurium patients were infected abroad . when seen in the light of the ability of dt104 to spread and establish itself in a large variety of food animal lines ( cattle , pigs , poultry ) , the increase in the number of mdr s. typhimurium strains that include quinolone resistance becomes particularly problematic . quinolone , and in particular the fluoroquinolones , have been part of human medicine since the 1980s , resulting in no or very limited resistance in salmonellae . first , it was limited to countries with relatively sophisticated surveillance systems in place , since only countries performing phage typing or resistance testing in addition to serotyping were eligible . therefore , the survey contains no representative data on the situation in these regions . second , the collection of isolates at the national level is likely to vary from country to country , depending on a number of factors such as sampling frequency at the local level , availability of laboratory reagents , and the degree to which isolates and results are forwarded to the national level . for these reasons , care should be taken in the interpretation of results of this survey ; in particular when comparing incidence rates between countries . cross - checking these survey data with available published surveillance data showed them to be in line with each other in the united states ( 38 ) , the netherlands ( 39 ) , and denmark ( 40 ) . in summary , on a global scale , only a small number of countries perform antimicrobial susceptibility testing or phage typing , although the number of countries doing so more than doubled throughout the study period . despite its limitations , the survey showed that the incidence of both mdr s. typhimurium and mdr s. typhimurium dt104 increased markedly worldwide during the 1990s , although the problem has primarily affected europe and north america . the survey implies that mdr s. typhimurium poses a serious and increasing public health problem in large parts of the world . surveillance and control programs such as the global salm - surv international network recently launched by who should therefore be reinforced .

the idea of fabricating synthetic molecular machines has fascinated a whole generation of supramolecular chemists . molecular switches , which undergo mechanical motion in response to chemical , photochemical , or electrochemical stimuli , represent excellent precursors for molecular machines . especially , mechanically interlocked molecules such as rotaxanes have been investigated thoroughly in this respect . in solution , molecular switches are randomly oriented , which makes the implementation of directional processes difficult . the still challenging integration of molecular switches and machines into larger ordered arrays and functional devices will help solve this problem . inspired by nature s use of membranes to generate order , chemists can employ soft interfaces such as langmuir blodgett films or hard interfaces such as metal surfaces as alternatives to membranes to fabricate ordered arrays of synthetic molecular machines . the use of metal surfaces is particularly appealing , as the generation of self - assembled monolayers , layer - by - layer self - assembly , and the fabrication of surface - bound metal organic frameworks ( surmofs ) are well understood . several seminal steps in this direction have been made , e.g. , leigh s monolayers of axle - fluorinated , light - switchable rotaxanes moving diiodomethane droplets uphill , rapenne s unidirectionally rotating surface - bound piano - stool rotors , feringa s surface - anchored overcrowded double bond motors , or stoddart s electrochemically switched cantilever - bending molecular - muscle rotaxanes . in the latter example , the finding that a cantilever can be bent , when the rotaxanes are switched , suggests that the rotaxanes deposited on the cantilever operate in a directional way . this aspect , however , has so far not been further investigated in greater detail . the aim of the present study is to provide evidence for a coupled motion of switchable interlocked molecules . the term coupled motion as we use it here refers to switching whole arrays of rotaxane shuttles that are aligned in a well - ordered state before switching and are again aligned in another well - ordered state after switching . for such motion , lateral order and the alignment of the deposited molecules are essential , and multilayers may enhance the achievable effects . therefore , our study also aims to fabricate well - ordered multilayers of rotaxanes , in which a coupled motion can be observed . here we describe novel ordered mono- and multilayers of chemically switchable amide rotaxanes on gold using either a click chemistry approach to covalently attach them to an azide - terminated self - assembled monolayer ( sam ) or a coordination - chemistry - based layer - by - layer self - assembly strategy . the rotaxane layers reversibly switch in a coupled manner between two differently ordered layer structures . the present study examines the importance of ordered arrays for coupled molecular motions in stimulus - responsive shuttle rotaxanes . starting from suitably functionalized tetralactam macrocycles mcn , a templated one - step rotaxane synthesis has been achieved by efficiently trapping diketopiperazine 1 inside the wheels to form hydrogen - bonded pseudorotaxanes protn ( scheme 1 ; for experimental details , see the supporting information ) . the sterically demanding tritylphenyl stopper groups have been attached to the axle alkynes by a cu - catalyzed click reaction with 2 . for surface deposition , alkyne and terpyridine side chains have been introduced by sonogashira and suzuki cross - coupling reactions with ( trimethylsilyl)acetylene and boronic acid pinacol ester 3 , respectively . after trimethylsilyl ( tms ) deprotection , rot3 is ready to be clicked covalently to an azide - functionalized surface . rot6 can be used for the deposition by metal - coordination - based layer - by - layer self - assembly . top : the axle threading reaction is templated by hydrogen - bond - mediated formation of pseudorotaxane protn , to which the two tritylphenyl azide stoppers are then clicked . bottom : the acetylene and terpyridine side chains are attached to the iodo - substituted precursor rotaxanes by sonogashira and suzuki cross - coupling reactions , respectively . evidence for rotaxane formation comes from significant upfield shifts ( figure 1a , b ) observed for the diketopiperazine protons h ( = 1.01 ppm ) and h ( = 0.84 ppm ) , which are located inside the wheel s cavity and experience the anisotropy of the surrounding aromatic rings . also , the triazole proton h is affected by the presence of the wheel ; the corresponding signal shifts to higher field by = 0.33 ppm . bottom : partial h nmr spectra ( 4 mm , cdcl3 , 298 k ) of ( a ) free , stoppered axle , ( b ) rotaxane rot6 before addition of 1 equiv of tetrabutylammonium chloride ( ( tba)cl ) , ( c ) rotaxane rot6 after addition of 1 equiv of ( tba)cl , and ( d ) rot6 after chloride removal by precipitation of nacl with 1 equiv of sodium tetrakis(3,5-bis(trifluoromethyl)phenyl)borate ( nabar4 ) . the large signal shifts upon chloride addition and removal agree with those expected for the wheel translating from the central diketopiperazine station to one of the triazoles . the slight peak broadening and minor shift differences in spectrum d compared to spectrum b are due to the polarity change caused by the added salts . to gain insight into the switching process before transfer to the surface , a h nmr study of the chloride - induced axle movement of rot6 has been performed in cdcl3 ( figure 1b d ) . clearly , chloride addition causes significant signal shifts , providing evidence for a wheel translocation from the central diketopiperazine to a triazole station , where the wheel binds mainly through c the downfield shifts of the signals ( i ) for the amide nh protons h ( = 0.70 ppm ) reflecting the change in hydrogen - bonding partner , ( ii ) for the triazole proton h ( = 0.44 ppm ) , which moves deeper into the wheel , but at the same time interacts with the chloride anion , and ( iii ) for the diketopiperazine proton h ( = 0.14 ppm ) , which moves somewhat away from the center of the wheel cavity , are most indicative . also , the isophthaloyl diamide protons h and h and almost all other signals are clearly affected by the axle movement . as only one set of nmr signals is observed for either one , the wheel s shuttling motion between the two triazoles and a concerted chloride flip / axle rotation both proceed fast on the nmr time scale . removal of the chloride anion by precipitation with nabar yields the same spectrum as obtained prior to the switching cycle with the exception of the additional signals for the bar anions . consequently , the stimulus - induced structural changes in the rotaxane can be fully reversed . analogous results have also been obtained with the diiodo - substituted precursor rotaxane rot5 ( supporting information , figure s14 ) . for the deposition of a monolayer of rot3 ( figure 2a ) , an azide - functionalized self - assembled monolayer ( sam ) was generated by reacting a hcl - cleaned gold substrate with a 1 mm ethanol solution of bis(11-azidoundecyl ) disulfide ( aud ) for 24 h. subsequently , the acetylene - functionalized rotaxane rot3 was attached covalently through a click reaction catalyzed by the cu / n - heterocyclic carbene complex cat . this catalyst exhibits excellent activity in dichloromethane , a solvent that needs to be used because of the limited solubility of the rotaxane in other solvents . ( a ) monolayers of covalently fixed rot3 are made by first depositing an azide - terminated sam of aud from ethanol solution onto a hcl - cleaned gold surface . ( b ) on a pst sam , first pd ions are deposited from acetonitrile and mediate the connection to the first layer of rot6 deposited from dichloromethane solution . subsequently , this monolayer can be converted into multilayers by an alternating deposition of fe ions and rot6 . similarly , a 1:3 mixed monolayer of tdt and dt can serve as a templating sam with fe ions connecting the next layer to it . figure 2b summarizes the formation of mono- and multilayers of rot6 by coordination - based layer - by - layer self - assembly on a templating , rigid pyridine - functionalized sam of ( e)-4-(pyridin-4-yl)stilbenethiol ( pst ) . a 10 min metal ion deposition from a 1 mm acetonitrile solution of [ pd(ncch3)4](bf4)2 was followed by the deposition of a monolayer of the rotaxane from a 1 mm dichloromethane solution of rot6 which was given 24 h to assemble into an ordered monolayer array . as this rot6 monolayer is again terminated by terpyridyl side chains , multilayers can be fabricated following this procedure repeatedly , now using fe ions to connect the two terpyridines of adjacent rotaxanes . alternating deposition steps with fe(bf4)26h2o as the metal ion source and rot6 thus led to a growing multilayer . alternatively , a mixed template sam prepared from a 1:3 mixture of terpyridine - terminated dodecanethiol ( tdt ) and decanethiol ( dt ) ( figure 2b ) can be used as the basis for multilayer growth ( supporting information , figures s24s27 ) . this ratio is the optimum for metal deposition , and phase segregation of the two components was ruled out earlier . the dt decyl chain is shorter by two carbon atoms as compared to the tdt dodecyl chain , resulting in well - ordered alkyl chains , yet flexible , unordered terpyridine groups that are exposed on top of the sam . the fabrication of the tdt / dt - supported rot6 multilayer follows the same procedure as that deposited on pst . as this sam bears terpyridine groups as the anchors for the next layer , fe is also used as the first metal ion layer . to allow imaging of the deposition process , not only fully covered surfaces but also surfaces structured laterally by microcontact printing ( cp ) have been produced . these surfaces have the advantage that atomic force microscopy ( afm ) can be used to determine layer thicknesses by comparing the heights of the growing multilayer with the constant height of the areas passivated by a simple alkanethiol . poly(dimethylsiloxane ) stamps patterned with 10 m wide dots spaced at 5 m distances were inked with octadecanethiol ( odt ) and then brought into contact with a freshly cleaned gold surface . afterward , the space between the dots was backfilled with pst , followed by formation of the rot6 multilayer as described above . the characterization of the surface films is achieved by combining data from x - ray photoelectron spectroscopy ( xps ) , transmission uv / vis spectroscopy , afm , time - of - flight secondary ion mass spectrometric ( tof - sims ) imaging , and near - edge x - ray absorption fine structure ( nexafs ) spectroscopy . ( a ) a comparison of the high - resolution n 1s xp spectra ( excitation energy : 500 ev ) of the aud sam before and after click deposition of rot3 reveals conversion of the azide ( green signals ) to the triazole ( red ) superimposed by the amide and diketopiperazine n atoms of the rotaxane ( blue ) . ( b ) xps c 1s / au 4f7/2 ratios increase with each deposited rot6 layer . ( c ) transmission uv / vis spectra of 010 rot6 layers on pst / semitransparent gold . ( d ) relative increase of the c 1s / au 4f7/2 ratios upon deposition of rot6 on different templating sams ( pst vs tdt / dt , 1:3 ) . afm images and height profiles of micropatterned surfaces functionalized ( a ) with odt dots between unfilled voids , ( b ) with odt dots and voids backfilled with pst , and ( c ) with odt dots and voids backfilled with pst and one rot6 layer . ( d ) tof - sims images of the surface backfilled with pst and rot6 . left : secondary ion intensities of cxhy ions indicative of odt alkyl chains . right : secondary ion intensities of cn and c3n ions indicative of the presence of pyridines . in line with earlier results , the aud sam exhibits three signals in the high - resolution n 1s xp spectrum ( figure 3a ) which can be assigned to the three azide nitrogen atoms . after the click reaction with rot3 , three signals appear shifted to those binding energies expected for the triazole nitrogen atoms . in addition , a more intense signal for the secondary amides of the macrocycle and a smaller signal for the tertiary amides of the diketopiperazine axle appear . multilayer growth of rot6 on the pst sam can be followed by transmission uv / vis , when the multilayer is deposited on a semitransparent gold surface ( figure 3c ) . all bands observed in the solution spectrum of the ( rot4)2fe control complex are also found for the rotaxanes deposited on the surface : the metal - to - ligand charge transfer ( mlct ) band at ca . 560 nm , the ligand - centered band at ca . 340 nm , and the * transition of the aromatic rings at ca . in addition , the surface plasmon band appears at around 470 nm and , as expected , shifts slightly to higher wavelengths with increasing layer thickness . a plot of the intensity at 270 nm over the rotaxane layer number clearly demonstrates a linear growth from which we conclude that the same amount of rot6 is added in each rotaxane deposition step . the carbon / gold ratio extracted from the c 1s and au 4f7/2 xps data ( figure 3b ) increases with each deposition of the rotaxane , because the number of carbon atoms in the organic layer increases and the gold signal is more attenuated at the same time caused by inelastic scattering of the photoelectrons emitted from the gold surface at the organic layers above . a comparison of the xps c 1s / au 4f7/2 ratios of the rot6 single and triple layers on pst with the same layers on tdt / dt ( 1:3 ) reveals significantly more rotaxane deposited in each layer on the pst sam ( figure 3d ) . this is in marked contrast to the formation of densely packed multilayers of mc3 on tdt / dt . the introduction of the axle with its two bulky stopper groups increases the size of rotaxane rot6 significantly compared to that of mc3 . thus , it does not match the grid of the terpyridine anchor points on the tdt / dt monolayer as nicely as mc3 . the smaller terminal pyridines of the pst sam offer a finer grid of coordination sites that allows rot6 to assemble laterally more easily and to form layers of higher density . when a micropatterned surface was examined by afm directly after deposition of the odt dots , a clear contrast between the odt - passivated dots and the void space between them was observed , which translates into a layer thickness of the odt monolayer of 1.94 nm ( figure 4a ) . after the space between the passivated dots is backfilled with one layer of rot6 on pst , the contrast reverses as the rotaxane layer is now higher than the odt layer ( figure 4b ) . the thickness of one rotaxane layer can be determined from figure 4b ( voids backfilled with pst alone ) and figure 4c ( voids backfilled with pst and rot6 ) to be ca . this value nicely corresponds to thicknesses of 1.51.8 nm determined earlier for layers of macrocycle mc3 . as the fe fe distance calculated for mc3 and rot6 oligomers is ca . 3.5 nm , the tilt angle between the rotaxane wheel and the surface can be estimated to be ca . 30. the distribution of odt and rot6 on the micropatterned surface was also imaged by tof - sims . a 25 25 m area of the surface was scanned with 256 256 pixel resolution by a focused beam of bi3 ions using the novel collimated burst alignment mode for high lateral as well as high mass resolution . in the positive mode , the cxhy secondary ions characteristic for the odt alkyl chains preferentially originate from the dot areas ( figure 4d , left ) . instead , the areas covered by the pst - based multilayer of rot6 do not contain any linear alkyl chains and produce only a very small cxhy ion count . vice versa , cn and c3n secondary ions in the negative mode confirm the presence of pyridines and thus of rot6 in the areas between the odt dots ( figure 4d , right ) . notably , these ions are absent in the odt areas , indicating an excellent specificity of binding rot6 to the pst areas . contact angle measurements upon repeated chloride addition and removal indicate a strong and reversible change of polarity of the rotaxane multilayers which is tentatively attributed to on - surface chloride - mediated switching ( figure 5a ) . when the aud rot3 monolayer and a pst rot6 multilayer are treated with a 1 mm dichloromethane ( dcm ) solution of tetrabutylammonium chloride ( ( tba)cl ) and then immersed for 10 min in pure dichloromethane to remove residual salt , the contact angles drop by almost 20. this indicates a significantly higher polarity after chloride addition . removal of the chloride with nabar4 ( 1 mm , dcm ) and subsequent washing with dcm , water , and dcm again to remove salt residues bring the contact angles back to the initial values , and switching can be repeated . one might argue that the deposition of a salt is expected to increase polarity , even if it is not specifically bound and does not switch the rotaxane . therefore , a macrocycle mc3 multilayer was used as a control , and this exhibited much smaller contact angle changes ( ca . furthermore , the contact angle changes were not affected significantly when the less lipophilic tetramethylammonium chloride ( ( tma)cl ) was applied . investigation of the on - surface switching via contact angle measurements for the aud rot3 monolayer and pst and tdt / dt ( 1:3)rot6 multilayers ( 20 layers ) over five switching steps . for comparison , an mc3 multilayer ( 20 layers ) is examined . these results are an indication of , but certainly not strict evidence for , on - surface rotaxane switching . angle - resolved nexafs experiments provide evidence for stimulus - induced structural changes within the surface : as synchrotron light is linearly polarized , linear dichroism effects are observed when the deposited molecules have a preferred orientation . the transition dipole moment vectors for the excitation of core electrons into the * ( aromatic carbons ) or c h * orbitals ( aliphatic carbons ) line up with the electric field vector of the incident light differently at different angles , and angle - dependent intensities for the * and c h * resonances result . before we discuss the nexafs results shown in figures 6 and 7 , let us briefly mention the following limitation : it would be very desirable to be able to analyze the nexafs data in much greater detail to correlate the linear dichroism effects with the details of the structures and the orientation of the rotaxanes on the surface . while this is certainly possible for simpler self - assembled monolayers , let us take the * resonance as an example , as this is the most important one in our paper : the linear dichroism depends on the orientation of aromatic rings in the mono- and multilayers . the macrocyclic wheels are rather rigid , but contain aromatic rings in many different orientations , even when the macrocycles are fixed in a certain orientation . if one includes the axle with the trityl stoppers , additional orientations of aromatic rings come into play . consequently , many different dichroism effects finally are superimposed , resulting in a net effect , which is typically not very large as we have experienced several times in the past for other surfaces . despite this limitation , the observation of such a linear dichroism clearly indicates the rotaxanes to be ordered into arrays with preferred orientations . angle - resolved nexafs spectra of ( a ) a rot3 monolayer on aud , ( b ) a rot6 monolayer on pst ( left , ( tba)cl ; right , ( tma)cl ) , and ( c ) a rot6 monolayer on tdt / dt ( 1:3 ) . in each case , the nexafs results obtained for the pristine layer , that after chloride addition , and that after chloride removal with nabar4 are shown ( top to bottom ) . red and black lines represent the nexafs spectra obtained at 30 and 90 angles of the incident synchrotron light beam , respectively . similar to the results in figure 7 , the nexafs results obtained for the pristine layer , that after chloride addition , and that after chloride removal with nabar4 are shown ( top to bottom ) . red and black lines represent the nexafs spectra obtained at 30 and 90 angles of the incident synchrotron light beam , respectively . the pristine monolayer of rot3 on aud ( figure 6a ) exhibits a linear dichroism on the * resonance after chloride addition , this angle dependence vanished almost completely . consequently , either the switched rotaxanes may be disordered after switching or the dichroism effects almost exactly cancel . chloride binding to the rotaxane thus induces a structural change , while the question of whether this is accompanied by a loss of orientation can not clearly be answered . as neither the underlying aud alkyl chains nor the tetrabutylammonium counterions contain systems and thus do not affect the * resonance , the structural change must have occurred within the rotaxane structure , and we ascribe it to the chloride - induced switching of the rotaxane . interestingly , chloride removal with nabar4 leads back to angle - resolved nexafs spectra very similar to the initial ones . consequently , switching the rotaxanes back into the chloride - free state yields an ordered structure similar to the initial one . the reversibility of the structural changes strongly supports rotaxane switching to be the origin of the linear dichroism changes . the monolayer of rot6 on pst ( figure 6b ) again exhibits a linear dichroism , indicating a preferred orientation of the rotaxanes . in contrast to the rot3 monolayer , the linear dichroism became even more pronounced upon chloride addition . clearly , this surface can be switched between two different structures , which both contain the rotaxanes in a preferred orientation . as a control , the pst the results are virtually identical indicating that the counterion is not the primary origin of the structural changes . in marked contrast , the tdt / dt rot6 monolayer exhibits only negligible linear dichroism effects before and after switching ( figure 6c ) . this observation is interesting as this surface was found to be less densely packed ( see above , figure 3d ) . even if the individual , nonordered rotaxanes deposited on the tdt / dt sam undergo chloride - induced switching , no coupled axle movement is observed that transfers an ordered structure into another ordered structure . in turn , we conclude the reversibly switchable transition of one ordered layer structure of rot6 on pst into a second , yet different ordered structure as a clear indication that the densely packed rot6 layer on pst switches in a coupled manner . finally , the triple layer of rot6 on pst again exhibits linear dichroism effects on the * resonance that change with chloride addition and removal ( figure 7 ) . the * resonance of the initial triple layer is more intense at a 30 angle of the incident light . after chloride addition , the more intense * resonance appears at 90 instead . rot6 monolayer is consequently transferable to multilayers of rot6 on pst as well . in principle , one might envisage another mechanism by which the transition between two ordered surfaces could be accomplished at least for those surfaces that are fabricated using the metal coordination approach : the rotaxanes on the surface might dissociate from the surface , switch , and redeposit in a different packing . this scenario can , however , be ruled out on the basis of the following arguments : ( i ) in those cases in which the metal ion coordination is clearly reversible ( e.g. , when zn(ii ) is used to connect two terpyridines ) , no continuous layer growth is observed . ( ii ) the mono- and multilayers of macrocycles have been tested extensively , even against alkaline edta solutions for stability , and have not shown any degradation overnight . this quite high stability agrees well with our assumption of densely packed surfaces in which lateral interactions between the rotaxanes help keep them in place . amide rotaxanes that exhibit axle translocation in the wheel between two different stations in response to the presence of chloride have been synthesized and effectively deposited on gold substrates . two approaches yielded monolayers : covalent attachment through a click reaction and a coordination - chemistry - based approach . the latter monolayer formed the basis for multilayer fabrication leading to up to 20 rotaxane layers . chloride - induced axle movement has successfully been transferred from the solution to the surface . linear dichroism effects observed in angle - resolved nexafs experiments not only provide evidence for oriented surface - deposited rotaxanes , but clearly demonstrate switching of single and triple layers between two different layer structures . a number of control experiments and the comparison of different layers allow us to ascribe the structural changes to the rotaxane switching rather than other potential sources for structural changes . this study represents a structural examination of ordered rotaxane multilayers on a solid support that exhibits coupled transitions between two different structures by stimulus - induced mechanical motion . in particular , the almost complete absence of linear dichroism effects in the switching experiments performed with the tdt / dt - based surfaces shows how important order is for a structurally well - defined switching between two different orientations . a dense packing of the rotaxanes under study in well - ordered layers results in limited degrees of freedom for the axles of each rotaxane through spatial constraints caused by the next neighbors . a perfectly simultaneous switching of all rotaxanes in a domain is unlikely , because the overall barrier would be expected to be very high . therefore , we propose a nucleation / growth mechanism for the switching process : the switching of the first rotaxane is quite unfavorable , as the unswitched state packs well , whereas the switched one does not in an environment of unswitched neighbors . once the nucleus of a small ensemble of switched rotaxanes has formed , neighboring rotaxanes will switch faster , as the switched state now packs well when embedded within switched neighbors . this leads to a quicker growth of the switched domain . a detailed understanding of the factors that govern these processes will have strong implications for the future integration of molecular machines into larger devices . also , the conversion of chemical processes on the molecular scale into macroscopic effects may be envisaged on the basis of our study , if one , for example , considers volume changes caused by rotaxane switches tilting up in a coupled fashion .

interfaces provide the structural basis for function as , for example , encountered in nature in the membrane - embedded photosystem or in technology in solar cells . synthetic functional multilayers of molecules cooperating in a coupled manner can be fabricated on surfaces through layer - by - layer self - assembly . ordered arrays of stimulus - responsive rotaxanes undergoing well - controlled axle shuttling are excellent candidates for coupled mechanical motion . such stimulus - responsive surfaces may help integrate synthetic molecular machines in larger systems exhibiting even macroscopic effects or generating mechanical work from chemical energy through cooperative action . the present work demonstrates the successful deposition of ordered mono- and multilayers of chemically switchable rotaxanes on gold surfaces . rotaxane mono- and multilayers are shown to reversibly switch in a coupled manner between two ordered states as revealed by linear dichroism effects in angle - resolved nexafs spectra . such a concerted switching process is observed only when the surfaces are well packed , while less densely packed surfaces lacking lateral order do not exhibit such effects .

Introduction Results and Discussion Conclusions

the idea of fabricating synthetic molecular machines has fascinated a whole generation of supramolecular chemists . molecular switches , which undergo mechanical motion in response to chemical , photochemical , or electrochemical stimuli , represent excellent precursors for molecular machines . the still challenging integration of molecular switches and machines into larger ordered arrays and functional devices will help solve this problem . inspired by nature s use of membranes to generate order , chemists can employ soft interfaces such as langmuir blodgett films or hard interfaces such as metal surfaces as alternatives to membranes to fabricate ordered arrays of synthetic molecular machines . the use of metal surfaces is particularly appealing , as the generation of self - assembled monolayers , layer - by - layer self - assembly , and the fabrication of surface - bound metal organic frameworks ( surmofs ) are well understood . , leigh s monolayers of axle - fluorinated , light - switchable rotaxanes moving diiodomethane droplets uphill , rapenne s unidirectionally rotating surface - bound piano - stool rotors , feringa s surface - anchored overcrowded double bond motors , or stoddart s electrochemically switched cantilever - bending molecular - muscle rotaxanes . in the latter example , the finding that a cantilever can be bent , when the rotaxanes are switched , suggests that the rotaxanes deposited on the cantilever operate in a directional way . the aim of the present study is to provide evidence for a coupled motion of switchable interlocked molecules . the term coupled motion as we use it here refers to switching whole arrays of rotaxane shuttles that are aligned in a well - ordered state before switching and are again aligned in another well - ordered state after switching . for such motion , lateral order and the alignment of the deposited molecules are essential , and multilayers may enhance the achievable effects . therefore , our study also aims to fabricate well - ordered multilayers of rotaxanes , in which a coupled motion can be observed . here we describe novel ordered mono- and multilayers of chemically switchable amide rotaxanes on gold using either a click chemistry approach to covalently attach them to an azide - terminated self - assembled monolayer ( sam ) or a coordination - chemistry - based layer - by - layer self - assembly strategy . the rotaxane layers reversibly switch in a coupled manner between two differently ordered layer structures . the present study examines the importance of ordered arrays for coupled molecular motions in stimulus - responsive shuttle rotaxanes . rot6 can be used for the deposition by metal - coordination - based layer - by - layer self - assembly . to gain insight into the switching process before transfer to the surface , a h nmr study of the chloride - induced axle movement of rot6 has been performed in cdcl3 ( figure 1b d ) . as only one set of nmr signals is observed for either one , the wheel s shuttling motion between the two triazoles and a concerted chloride flip / axle rotation both proceed fast on the nmr time scale . consequently , the stimulus - induced structural changes in the rotaxane can be fully reversed . for the deposition of a monolayer of rot3 ( figure 2a ) , an azide - functionalized self - assembled monolayer ( sam ) was generated by reacting a hcl - cleaned gold substrate with a 1 mm ethanol solution of bis(11-azidoundecyl ) disulfide ( aud ) for 24 h. subsequently , the acetylene - functionalized rotaxane rot3 was attached covalently through a click reaction catalyzed by the cu / n - heterocyclic carbene complex cat . subsequently , this monolayer can be converted into multilayers by an alternating deposition of fe ions and rot6 . figure 2b summarizes the formation of mono- and multilayers of rot6 by coordination - based layer - by - layer self - assembly on a templating , rigid pyridine - functionalized sam of ( e)-4-(pyridin-4-yl)stilbenethiol ( pst ) . a 10 min metal ion deposition from a 1 mm acetonitrile solution of [ pd(ncch3)4](bf4)2 was followed by the deposition of a monolayer of the rotaxane from a 1 mm dichloromethane solution of rot6 which was given 24 h to assemble into an ordered monolayer array . as this rot6 monolayer is again terminated by terpyridyl side chains , multilayers can be fabricated following this procedure repeatedly , now using fe ions to connect the two terpyridines of adjacent rotaxanes . alternatively , a mixed template sam prepared from a 1:3 mixture of terpyridine - terminated dodecanethiol ( tdt ) and decanethiol ( dt ) ( figure 2b ) can be used as the basis for multilayer growth ( supporting information , figures s24s27 ) . the dt decyl chain is shorter by two carbon atoms as compared to the tdt dodecyl chain , resulting in well - ordered alkyl chains , yet flexible , unordered terpyridine groups that are exposed on top of the sam . these surfaces have the advantage that atomic force microscopy ( afm ) can be used to determine layer thicknesses by comparing the heights of the growing multilayer with the constant height of the areas passivated by a simple alkanethiol . ( a ) a comparison of the high - resolution n 1s xp spectra ( excitation energy : 500 ev ) of the aud sam before and after click deposition of rot3 reveals conversion of the azide ( green signals ) to the triazole ( red ) superimposed by the amide and diketopiperazine n atoms of the rotaxane ( blue ) . ( d ) relative increase of the c 1s / au 4f7/2 ratios upon deposition of rot6 on different templating sams ( pst vs tdt / dt , 1:3 ) . in line with earlier results , the aud sam exhibits three signals in the high - resolution n 1s xp spectrum ( figure 3a ) which can be assigned to the three azide nitrogen atoms . multilayer growth of rot6 on the pst sam can be followed by transmission uv / vis , when the multilayer is deposited on a semitransparent gold surface ( figure 3c ) . all bands observed in the solution spectrum of the ( rot4)2fe control complex are also found for the rotaxanes deposited on the surface : the metal - to - ligand charge transfer ( mlct ) band at ca . the carbon / gold ratio extracted from the c 1s and au 4f7/2 xps data ( figure 3b ) increases with each deposition of the rotaxane , because the number of carbon atoms in the organic layer increases and the gold signal is more attenuated at the same time caused by inelastic scattering of the photoelectrons emitted from the gold surface at the organic layers above . this is in marked contrast to the formation of densely packed multilayers of mc3 on tdt / dt . when a micropatterned surface was examined by afm directly after deposition of the odt dots , a clear contrast between the odt - passivated dots and the void space between them was observed , which translates into a layer thickness of the odt monolayer of 1.94 nm ( figure 4a ) . the thickness of one rotaxane layer can be determined from figure 4b ( voids backfilled with pst alone ) and figure 4c ( voids backfilled with pst and rot6 ) to be ca . 3.5 nm , the tilt angle between the rotaxane wheel and the surface can be estimated to be ca . in the positive mode , the cxhy secondary ions characteristic for the odt alkyl chains preferentially originate from the dot areas ( figure 4d , left ) . instead , the areas covered by the pst - based multilayer of rot6 do not contain any linear alkyl chains and produce only a very small cxhy ion count . vice versa , cn and c3n secondary ions in the negative mode confirm the presence of pyridines and thus of rot6 in the areas between the odt dots ( figure 4d , right ) . notably , these ions are absent in the odt areas , indicating an excellent specificity of binding rot6 to the pst areas . when the aud rot3 monolayer and a pst rot6 multilayer are treated with a 1 mm dichloromethane ( dcm ) solution of tetrabutylammonium chloride ( ( tba)cl ) and then immersed for 10 min in pure dichloromethane to remove residual salt , the contact angles drop by almost 20. this indicates a significantly higher polarity after chloride addition . removal of the chloride with nabar4 ( 1 mm , dcm ) and subsequent washing with dcm , water , and dcm again to remove salt residues bring the contact angles back to the initial values , and switching can be repeated . one might argue that the deposition of a salt is expected to increase polarity , even if it is not specifically bound and does not switch the rotaxane . furthermore , the contact angle changes were not affected significantly when the less lipophilic tetramethylammonium chloride ( ( tma)cl ) was applied . angle - resolved nexafs experiments provide evidence for stimulus - induced structural changes within the surface : as synchrotron light is linearly polarized , linear dichroism effects are observed when the deposited molecules have a preferred orientation . the transition dipole moment vectors for the excitation of core electrons into the * ( aromatic carbons ) or c h * orbitals ( aliphatic carbons ) line up with the electric field vector of the incident light differently at different angles , and angle - dependent intensities for the * and c h * resonances result . before we discuss the nexafs results shown in figures 6 and 7 , let us briefly mention the following limitation : it would be very desirable to be able to analyze the nexafs data in much greater detail to correlate the linear dichroism effects with the details of the structures and the orientation of the rotaxanes on the surface . while this is certainly possible for simpler self - assembled monolayers , let us take the * resonance as an example , as this is the most important one in our paper : the linear dichroism depends on the orientation of aromatic rings in the mono- and multilayers . the macrocyclic wheels are rather rigid , but contain aromatic rings in many different orientations , even when the macrocycles are fixed in a certain orientation . consequently , many different dichroism effects finally are superimposed , resulting in a net effect , which is typically not very large as we have experienced several times in the past for other surfaces . despite this limitation , the observation of such a linear dichroism clearly indicates the rotaxanes to be ordered into arrays with preferred orientations . angle - resolved nexafs spectra of ( a ) a rot3 monolayer on aud , ( b ) a rot6 monolayer on pst ( left , ( tba)cl ; right , ( tma)cl ) , and ( c ) a rot6 monolayer on tdt / dt ( 1:3 ) . in each case , the nexafs results obtained for the pristine layer , that after chloride addition , and that after chloride removal with nabar4 are shown ( top to bottom ) . red and black lines represent the nexafs spectra obtained at 30 and 90 angles of the incident synchrotron light beam , respectively . similar to the results in figure 7 , the nexafs results obtained for the pristine layer , that after chloride addition , and that after chloride removal with nabar4 are shown ( top to bottom ) . red and black lines represent the nexafs spectra obtained at 30 and 90 angles of the incident synchrotron light beam , respectively . the pristine monolayer of rot3 on aud ( figure 6a ) exhibits a linear dichroism on the * resonance after chloride addition , this angle dependence vanished almost completely . consequently , either the switched rotaxanes may be disordered after switching or the dichroism effects almost exactly cancel . chloride binding to the rotaxane thus induces a structural change , while the question of whether this is accompanied by a loss of orientation can not clearly be answered . as neither the underlying aud alkyl chains nor the tetrabutylammonium counterions contain systems and thus do not affect the * resonance , the structural change must have occurred within the rotaxane structure , and we ascribe it to the chloride - induced switching of the rotaxane . interestingly , chloride removal with nabar4 leads back to angle - resolved nexafs spectra very similar to the initial ones . the reversibility of the structural changes strongly supports rotaxane switching to be the origin of the linear dichroism changes . the monolayer of rot6 on pst ( figure 6b ) again exhibits a linear dichroism , indicating a preferred orientation of the rotaxanes . in contrast to the rot3 monolayer , the linear dichroism became even more pronounced upon chloride addition . clearly , this surface can be switched between two different structures , which both contain the rotaxanes in a preferred orientation . as a control , the pst the results are virtually identical indicating that the counterion is not the primary origin of the structural changes . in marked contrast , the tdt / dt rot6 monolayer exhibits only negligible linear dichroism effects before and after switching ( figure 6c ) . this observation is interesting as this surface was found to be less densely packed ( see above , figure 3d ) . even if the individual , nonordered rotaxanes deposited on the tdt / dt sam undergo chloride - induced switching , no coupled axle movement is observed that transfers an ordered structure into another ordered structure . in turn , we conclude the reversibly switchable transition of one ordered layer structure of rot6 on pst into a second , yet different ordered structure as a clear indication that the densely packed rot6 layer on pst switches in a coupled manner . finally , the triple layer of rot6 on pst again exhibits linear dichroism effects on the * resonance that change with chloride addition and removal ( figure 7 ) . rot6 monolayer is consequently transferable to multilayers of rot6 on pst as well . in principle , one might envisage another mechanism by which the transition between two ordered surfaces could be accomplished at least for those surfaces that are fabricated using the metal coordination approach : the rotaxanes on the surface might dissociate from the surface , switch , and redeposit in a different packing . , when zn(ii ) is used to connect two terpyridines ) , no continuous layer growth is observed . ( ii ) the mono- and multilayers of macrocycles have been tested extensively , even against alkaline edta solutions for stability , and have not shown any degradation overnight . this quite high stability agrees well with our assumption of densely packed surfaces in which lateral interactions between the rotaxanes help keep them in place . amide rotaxanes that exhibit axle translocation in the wheel between two different stations in response to the presence of chloride have been synthesized and effectively deposited on gold substrates . the latter monolayer formed the basis for multilayer fabrication leading to up to 20 rotaxane layers . chloride - induced axle movement has successfully been transferred from the solution to the surface . linear dichroism effects observed in angle - resolved nexafs experiments not only provide evidence for oriented surface - deposited rotaxanes , but clearly demonstrate switching of single and triple layers between two different layer structures . a number of control experiments and the comparison of different layers allow us to ascribe the structural changes to the rotaxane switching rather than other potential sources for structural changes . this study represents a structural examination of ordered rotaxane multilayers on a solid support that exhibits coupled transitions between two different structures by stimulus - induced mechanical motion . in particular , the almost complete absence of linear dichroism effects in the switching experiments performed with the tdt / dt - based surfaces shows how important order is for a structurally well - defined switching between two different orientations . a dense packing of the rotaxanes under study in well - ordered layers results in limited degrees of freedom for the axles of each rotaxane through spatial constraints caused by the next neighbors . a perfectly simultaneous switching of all rotaxanes in a domain is unlikely , because the overall barrier would be expected to be very high . therefore , we propose a nucleation / growth mechanism for the switching process : the switching of the first rotaxane is quite unfavorable , as the unswitched state packs well , whereas the switched one does not in an environment of unswitched neighbors . this leads to a quicker growth of the switched domain . a detailed understanding of the factors that govern these processes will have strong implications for the future integration of molecular machines into larger devices . also , the conversion of chemical processes on the molecular scale into macroscopic effects may be envisaged on the basis of our study , if one , for example , considers volume changes caused by rotaxane switches tilting up in a coupled fashion .

mosquito collection - fourth instar anopheline larvae were collected from 13 localities , including eight in the state of roraima ( rr ) in northern amazonian brazil and five in the state of par ( pa ) in eastern amazonian brazil ( fig . 1 , supplementary data ) . if the site was positive for anophelines , collections continued for an hour to measure relative species abundance . the study was conducted over three years ( 2009 - 2011 ) during malaria transmission season in each state . the larval sites in rr were located between amajari and ecuador , roughly 500 km along the rr-174 , in 2009 and 2011 , and in pa between moj and marab , approximately 560 km along pa-475/263/150 in 2010 . the collection protocol was approved by the evandro chagas institute , par state ethical committee and the new york state department of health , institutional review board . these included biotic factors such as temperature , alkalinity , ph , conductivity , salinity and turbidity . additionally , canopy coverage was estimated by spherical densitometer ( minakawa et al . 2005 ) . ( 1946 ) and confirmed by species specific its2 restriction fragment length polymorphism ( zapata et al . 2007 ) . morphological keys do not reflect the recent differences between the colombian and venezuelan an . goeldii , samples , therefore samples were sequenced and compared to those from calado et al . ( 2008 ) using a bayesian inference ( bi ) phylogenetic approach ( data not shown ) . total genomic dna was extracted using the dneasy blue tissue kit ( qiagen , ca , usa ) and maintained at griffin laboratory at -80c . subsequent analysis of similar larval sites based upon the habitat type and type of water body sampled ( pond , pool , swamp etc . ) , examined variation in larval densities using two - way analysis of variance ( anova ) . additional analyses examining differences in habitat ecology , based on biotic factors , surrounding vegetation and canopy coverage were examined to test the hypothesis that environmental heterogeneity is positively correlated with genetic diversity . amplification and sequencing - a representative sample of one-50 mosquitoes per locality was selected for dna amplification and sequencing of the coi . subsets of up to 10 samples per locality for the white gene and up to five for its2 were amplified and sequenced . the 658-bp barcode fragment of the coi gene was amplified using the forward primer lco1490 and the reverse primer hco2198 ( folmer et al . 1994 ) . individual polymerase chain reaction ( pcr ) reactions were performed using ready - to - go rt - pcr beads ( amersham pharmacia / biotech , nj , usa ) or pcr supermix high fidelity ( gotaq , promega , ca ) run on a bio - rad ptc-100 or 200 series thermal cycler ( bio - rad inc ) , using the conditions stipulated in hebert et al . the applied genomics technology core ( wadsworth center ) carried out the sequencing on an abi prism 3700 automated dna sequencer . the forward and reverse coi sequences were aligned using sequencher 3.0 ( gene codes corps , mi , usa ) , grouped together by sight and trimmed in mega v. 3.1 ( kumar et al . triannulatus samples from each locality was sequenced following the protocol of pedro and sallum ( 2009 ) , to yield a 449-bp fragment of the 3 ' coi . high levels of intraspecific variation , deep structure and complex phylogenies based on the 3 ' end of the coi gene ( moreno et al . ( silva - do - nascimento & loureno - de - oliveira 2002 , silva - do - nascimento et al . the barcode region is more variable than the 3 ' end of the coi ( ruiz - lopez et al . 2012 ) and able to improve resolution at deeper nodes ( saunders & le gall 2010 ) . therefore , analyses herein focused on the barcode fragment and included only a subset based on the 3 ' end to allow systematic comparisons of an . nuclear genes were included to investigate the history of the nuclear genome and provide independent evidence of population divergence ( loiaza et al . additionally , nuclear genes do not suffer the short comings of mitochondrial markers ( haploid , small genome size , short coalescent time , maternal inheritance ) ( ballard & whitlock 2004 ) and are not as biased in base composition ( brower & de salle 1998 ) . an 800-bp fragment of the white gene was amplified using the w2r and wf primers , with the pcr conditions as reported in mirabello and conn ( 2008 ) . a 500-bp fragment of the ribosomal its2 was amplified using the primers 18s and 28s following the parameters in li and wilkerson ( 2005 ) . the pcr products were cleaned , sequenced and aligned creating a 570-bp fragment of the white gene including the intron . the final product size for its2 was 433 bp and 371 bp for an . haplotypes of heterozygous individuals were inferred for each set of gene sequences using phase 2.1 software ( stephens et al . unique sequences for all markers are available in genbank ( accessions kc167670-kc167816 ) . genealogical relationship - genealogical trees for each species were estimated for the concatenated ( coi + white ) data set using bi analysis performed with mrbayes v. 3.1 ( huelsenbeck & ronquist 2001 , ronquist & huelsenbeck 2003 ) . data were partitioned by gene using the model of nucleotide substitution ( hky+i and trn+g for an . goeldii ) that best fit the white gene and coi , respectively , determined by jmodeltest ( posada 2008 ) . the settings were two simultaneous , independent runs of the markov chain monte carlo for two million generations , sampling every 1,000 generations with a burnin of 25% . the outgroup anophe - les marajoara ( dq076221 and ay956296 deposited in genbank ) was chosen based upon its phylogenetic position in sallum et al . ( 2000 ) . genetic variation - genetic structure of multiple populations based on statistical parsimony groupings were examined by analysis of molecular variance ( amova ) using arlequin v. 3.1.1 ( excoffier et al . 2005 ) and spatial analysis of molecular variance ( samova ) , v. 1.0 ( dupanloup et al . 2002 ) . both algorithms were used to cluster the 600-bp barcode fragment data into genetically and in the case of samova , geographically , homogeneous populations by generating f statistics ( fsc , fst , fct ) . goeldii into groups of k = 2 - 20 , with 1,000 simulated annealing steps from each of 100 sets of initial starting conditions . triannulatus using the 449-bp fragment haplotypes of pedro and sallum ( 2009 ) ( gu445849 - 899 and af417702 ) combined with the samples in the present study , to test their hypothesis of geographical sub - structuring within the species based on five populations across brazil . population structure and demographic history - a statistical parsimony network ( spn ) estimated genealogical relationships among haplotypes with a 95% identity for each marker using tcs 1.13 ( clement et al . 2000 ) , except an . homoplasy in all networks was resolved using the algorithm estimation rules in crandall and templeton ( 1993 ) . estimates of time to coalescence were calculated for the coi fragment only and compared using s values ( watterson 1975 ) . the differentiation and polymorphism statistics for coi and white gene sequences by species and locality were computed in dnasp , v. 5.0 ( librado & rozas 2009 ) . because of the high proportion of shared haplotypes among habitats within a single locality , samples from an individual town / city were pooled to more accurately represent diversity statistics . furthermore , haplotype distributions across four ecoregions were examined for each of the seven environmental variables to test the hypothesis that climatic differences sustain ( and drive ) diversity ( hoorn et al . 2010 ) . to test this hypothesis , we considered both the availability of breeding sites and haplotype diversity as measures of gene flow . individual sites were grouped within ecoregions , based on descriptions from rosa - freitas et al . the hypothesis of strict neutrality for each species was examined using dnasp , with the statistics dt ( tajima 1989 ) and d and f ( fu 1993 ) evaluating the likelihood of background selection and fu 's fs ( fu 1997 ) and r2 ( ramos - onsins & rozas 2002 ) examining evidence of population expansion . the mismatch distributions ( simulated in arlequin ) were conducted to confirm demographic expansion using sudden and spatial models of expansion . statistically significant differences between observed and simulated distributions were evaluated with the sum of square deviations to reject the hypothesis of demographic expansion ( zarza et al . 2008 ) . dates of population expansion were estimated for the coi gene with the formula t = /2 u ( rogers & harpending 1992 ) using 10 generations per year and the mutation rate of drosophila estimated at 1 10 substitutions per site per year ( walton et al . site diversity and larval abundance - larvae were predominantly collected from the edges of naturally occurring ponds , comprising 49.19% ( n = 200 ) of the total anopheles identified . triannulatus larvae were relatively uncommon in collections from seepages , stream margins and swamps , where they comprised only 10.9% of the total . goeldii , but not an . triannulatus . a large proportion , 68.97% ( n = 140 ) , of an . goeldii larvae appeared equally likely to inhabit artificial pools ( 37.93% , n = 77 ) , ponds ( 29.55% , n = 60 ) and temporary ditches ( 25.12% , n = 51 ) . no correlations between abundance and habitat type for either species were detected by anova ( data not shown ) . goeldii were similar : the 76 sites were not differentiated by larval abundance ( t = -0.2980 , df = 42 , p = 0.3836 ) . goeldii and those negative for either species ( f = 4.89 , p = 0.032 ) that was not supported by a t test ( t = 1.179 , df = 16 , p = 0.055 ) . there are no other significant differences between environmental variables for sites unique for either species and sites either positive or negative for both ( table i ) . table icharacteristics ( 95% confidence intervals ) of habitats with anopheles triannulatus only , anopheles goeldii only , both species or neither species present an . triannulatus an . goeldii both neither collections ( n ) 12 13 17 34 ph 5.5 0.00 5.58 0.15 5.68 0.24 5.62 0.16 alkalinity ( ppm ) 35 29.40 33.85 27.50 47.06 27.91 52.35 42.58 temperature ( c ) 26.29 1.37 27.44 1.88 26.79 1.10 26.48 0.95 conductivity ( s / cm ) 35.68 16.33 39.49 19.68 34.01 12.93 26.04 9.76 salinity ( ppm ) 18.09 8.13 19.92 10.31 25.11 17.34 13.24 4.90 turbidity ( jtus ) 21.67 15.93 28.46 16.88 17.50 6.88 13.09 5.36 canopy coverage ( % ) 11.28 14.20 13.74 13.43 13.77 11.38 10.55 6.33 genealogical relationship - the concatenated ( coi + white gene with intron ) bi tree for an . triannulatus , with 50 parsimoniously informative sites , indicated two strongly supported ( 97 and 98 posterior probabilities ) distinct populations of an . the two populations correspond to the brazilian states , with the exception of samples from moj and goiansia ( 9 and 10 , respectively ) , that were included in the northern group ( rr localities ) . the close relationship between the rr samples and localities moj and goiansia was also evident in the spn . this may indicate a historical boundary separating north and south populations , with the northern limit occurring just south of the amazon river and including parts of goiansia and moj . the more derived population 1 corresponds to localities in the amazon basin , near the hypothesised population origin ( pedro & sallum 2009 ) , with the majority of population 2 occurring along one of the proposed routes of lineage expansion . within grouping 2 , smaller subdivisions , related to geography and corresponding to populations 2 and 3 , were apparent between haplotypes 9 and 3 that are separated by six mutations and two haplotypes 4 and 1 , separated by two mutations ( fig . i + white gene ) bayesian inference trees , partitioned by gene using the model of nucleotide substitution that best fit the data as determined by jmodeltest . blue : samples from goiansia , tucuru , jacund and marab [ state of par ( pa ) ] corresponding to an . triannulatus population 3 ; green : moj and goiansia samples ( pa ) in addition to an . triannulatus population 2 ; orange : localities in the state of roraima and an . black : localities in the state of roraima ; grey : moj and goiansia samples from the state of par ( pa ) ; n : number of sequences ; white : samples from goiansia , tucuru , jacund and marab ( pa ) . for an . triannulatus , black refers to population 1 , grey ( with the addition of haplotype 14 ) to population 2 and white to population 3 . the concatenated ( coi + white gene with intron ) bi tree for an . goeldii , with 32 parsimoniously informative sites , indicated two moderately supported ( 72 and 68 posterior probabilities ) populations which correspond to the brazilian states ( fig . 2 ) . unlike an . triannulatus , there does not appear to be any sub - structuring in the samples from pa or support for a separate goiansia / moj population . genetic variation - amova indicated consistently higher fst and fct fixation indices across genes and various coi groupings for an . triannulatus , i.e. , genetic structuring among and within localities rather than between groups ( table ii ) . however , there was strong genetic differentiation ( 41.01% ) also seen in the coi grouping between states , which correlates well with the groups defined by the bayesian tree . table iianalysis of molecular variance calculations for inter and intravariation among species and populations for various markers anopheles triannulatus anopheles goeldii gene source of variation variance ( % ) fixation index variance ( % ) fixation index coi between rr and pa 41.01 fct = 0.41 45.12 fct = 0.45 among localities within states 29.23 fsc = 0.50 5.29 fsc = 0.10 within a given locality 29.76 fst = 0.70 49.59 fst = 0.50 white between rr and pa -3.01 fct = -0.03 89.46 fct = 0.89 among localities within states 58.62 fsc = 0.57 0.42 fsc = 0.04 within a given locality 44.38 fst = 0.56 10.12 fst = 0.90 coi between spn populations 38.85 fct = 0.39 - - among localities 33.92 fsc = 0.55 - - within a given locality 27.23 fst = 0.73 - - a : p < 0.05 ; b : p < 0.001 ; c : not significant ; coi : cytochrome oxidase i ; pa : state of par ; rr : state of roraima ; spn : statistical parsimony network derived groupings . a : p < 0.05 ; b : p < 0.001 ; c : not significant ; coi : cytochrome oxidase i ; pa : state of par ; rr : state of roraima ; spn : statistical parsimony network derived groupings . the small genetic differentiation of an . goeldii between localities suggests panmixia with no significant barriers to gene flow within individual states . amova indicated a large proportion of variation between the two states and among samples from different breeding site types in a given locality ( table ii ) . genetic structuring in the latter may indicate environmental differences and niche partitioning as drivers of diversity . triannulatus 449-bp fragment of the coi gene ( n = 35 ) to all samples from pedro and sallum ( 2009 ) was unable to replicate the five groupings originally described across brazil . 3 ) led to the discovery of two very different demographic histories between these sympatric species , which often co - occur within the same larval habitats . the time of the beginning of lineage divergence was calculated following da/2 k , where da is the average nucleotide divergence between lineages and 2 k is the divergence rate ( heckel et al . 2000 , yang et al . 2011 ) to estimate these parameters and their significance based on the coalescence ( dnasp v. 5.10 ) ( librado & rozas 2009 ) and mega v. 5.1 ( tamura et al . 2011 ) . a parsimony network for an . triannulatus indicated that a minimum of six mutational steps separate three potential populations based on geography ( fig . the most common haplotypes were 1 ( n = 18 ) , 2 ( n = 15 ) , 3 ( n = 11 ) and 6 ( n = 9 ) ( fig . 3 ) , with seven haplotypes ( 15.6% ) shared among localities and 38 ( 84.4% ) unique ( supplementary data ) . triannulatus was consistent with a category i phylogeographic pattern ( avise 2000 ) characterised by pronounced genetic gaps between spatially circumscribed haplogroups . a high proportion of singletons ( 23 , 51.1% ) , in addition to individual populations within an . triannulatus containing star - shaped nodes ( surrounding haplotypes 2 , 3 and 5 ) , supports demographic expansions , background selection or selective sweeps ( slatkin & hudson 1991 , fu 1997 ) . based upon the 600-bp fragment , da between the northern and southern states is 0.0118 [ standard deviation ( sd ) = 0.003 ] . therefore , the estimated divergence is 437,500 - 737,500 years ago , in the pleistocene . the two pa populations had a da = 0.014 ( sd = 0.004 ) , indicating more ancestral divergence ( congruent with bayesian tree ) , approximately 500,000 - 900,000 years ago . goeldii coi is consistent with a category iii phylogeographic pattern ( avise 2000 ) where most haplotypes are closely related , yet some geographic localisation exists . the most common haplotypes were 46 ( n = 21 ) , 47 ( n = 33 ) and 48 ( n = 13 ) ( fig . 3 ) , with 14 haplotypes ( 41.2% ) shared among localities , and 20 ( 58.8% ) unique ( supplementary data ) . similar to an . triannulatus , there was a relatively high proportion of singletons ( 14 , 41.2% ) , indicating a demographic expansion , background selection or selective sweep ( slatkin & hudson 1991 , fu 1997 ) , particularly in pa ( singletons arising from haplotype 56 ) . in contrast , haplotypes from rr exhibited longer branches , missing haplotypes and a near equal distribution of shared haplotypes and single mutations , consistent with a signal of an older lineage with balancing selection ( fig . 3 ) . da between northern and southern states is 0.008 ( sd = 0.002 ) . therefore , the estimated divergence is 316,000 - 516,500 years ago ( pleistocene ) . triannulatus nuclear data ( white gene and its2 ) indicate a history of contractions and expansions . similar to the coi gene , the its2 network depicts a category i pattern whereas the white gene reveals a much more homogeneous haplotype distribution . overall , the white gene follows a category ii intraspecific pattern , with pronounced gaps between some branches and some clustering of anciently separated geographic populations ( fig . 4statistical parsimony network of white gene ( a ) and internal transcribed spacer 2 sequences ( b ) at 95% , except its2 an . black : localities in the state of roraima ; grey : moj and goiansia samples from the state of par ( pa ) ; n : number of sequences ; white : samples from goiansia , tucuru , jacund and marab ( pa ) . goeldii nuclear data ( white gene and its2 ) reveal distinct geographical populations . the white gene has a distinct category i pattern separated by five mutational steps , while the its2 haplotype clusters ( fig . 4 ) are less pronounced , but still correspond primarily to rr and pa with a few outliers . polymorphism analyses of the coi sequences found haplotype and nucleotide diversity was greater among an . genetic polymorphism analyses of the coi sequences indicated that haplotype and nucleotide diversity was greater among an . the white gene exhibited lower nucleotide diversities compared to coi , but surprisingly an . triannulatus continued to demonstrate high haplotype diversity ( supplementary data ) , which combined with relatively low nucleotide diversity may signal a rapid expansion ( avise 2000 , de jong et al . high coi variation between states and geographic populations is strongly supported by population differentiation statistics ( table iii ) , although the gst values were low , suggesting a history of gene flow ( table iii ) . table iiiinter and intrapopulation differentiation of anopheles triannulatus and anopheles goeldii populations based on statistical parsimony network comparison an . goeldii rr vs. pa three populations rr vs. pa hs 0.89 0.86 0.86 k s * 1.77 1.41 1.60 z * 7.43 7.00 7.83 snn 0.99 1.00 0.97 129 258 125.84 gst 0.06 0.09 0.045 kt 10.09 10.09 7.43 a : p < 0.0001 ; g st : genetic differentiation ; h s : genetic differentiation based on haplotype data ; k s * : differentiation based on sequence data ; k t : average number of nucleotide differences ; n : number of total individuals compared ; pa : state of par ; rr : state of roraima ; s nn : measures how often the nearest neighbours of sequences are found in the same population ; : genetic differentiation based on allele frequencies ; z * : rank statistic to analyse sequence similarity . a : p < 0.0001 ; g st : genetic differentiation ; h s : genetic differentiation based on haplotype data ; k s * : differentiation based on sequence data ; k t : average number of nucleotide differences ; n : number of total individuals compared ; pa : state of par ; rr : state of roraima ; s nn : measures how often the nearest neighbours of sequences are found in the same population ; : genetic differentiation based on allele frequencies ; z * : rank statistic to analyse sequence similarity . in general , neutrality tests were negative for both species ( table iv ) , suggesting an excess of rare polymorphisms consistent with either negative / positive selection or an increase in population size , although only one of these tests ( fs ) was significant for an . mismatch distribution for sudden expansions did not support this finding and , instead , revealed marginally significant expansion patterns for only population 1 , corresponding to rr . table ivmultiple statistical analyses evaluating the probability for a recent population expansion species n d t d f f s r 2 anopheles triannulatus 129 -0.22187 -1.28038 -0.99732 -10.329 0.08650 roraima 57 -0.37846 -1.32424 -1.17022 -1.488 0.09200 par 72 -0.47543 -0.97094 -0.93438 -5.842 0.08900 population 1 56 -0.08718 -1.11433 -0.88956 -1.209 0.10200 population 2 36 -1.06921 -0.55581 -0.85324 -3.582 0.07770 population 3 37 -1.02686 -0.40080 -0.71980 -4.809 0.07800 anopheles goeldii 133 -0.30013 -1.24177 -1.01960 -5.867 0.08260 roraima 103 0.06971 -1.6295 -1.1537 -1.677 0.0989 par 30 -0.0415 0.15645 0.11023 -3.321 0.1185 a : significance , p < 0.50 ; d : fu and li 's d ( fu 1993 ) ; d t : tajima 's d ( tajima 1989 ) ; f : fu and li 's f ( fu 1993 ) ; f s : fu 's fs ( fu 1997 ) ; n : sample size ; r 2 : ramos - onsin 's and roza 's r 2 ( ramos - onsins & rozas 2002 ) . a : significance , p < 0.50 ; d : fu and li 's d ( fu 1993 ) ; d t : tajima 's d ( tajima 1989 ) ; f : fu and li 's f ( fu 1993 ) ; f s : fu 's fs ( fu 1997 ) ; n : sample size ; r 2 : ramos - onsin 's and roza 's r 2 ( ramos - onsins & rozas 2002 ) . fig . line represents the model for all anopheles triannulatus ( a ) , all anopheles goeldii ( b ) , an . triannulatus population 2 ( c ) ( moj and goiansia samples ) and an . triannulatus population 3 ( d ) ( goiansia , tucuru and jacund samples ) . distribution patterns of mosquito species are dependent on the availability of suitable aquatic habitats ( grillet 2000 ) . overlapping geographic distributions , in addition to the absence of significant difference in species abundance across sampled habitats , suggest that an . triannulatus . the lack of any significant differentiation between sympatric habitats , to those unique to a species or absent of either , suggests these species may be more broadly adapted ( eisenberg et al however , co - occurrence of species does not imply the same ability to exploit shared habitats ( diabate et al . the combined sequence results support distinct demographic histories for the two species despite similar distribution and larval habitat co - occurrence . across molecular markers an . goeldii consistently depicts two moderately supported populations corresponding to the two brazilian states , however , an . triannulatus appears to have undergone historical fragmentation , subsequent secondary contact and homogenisation followed by more recent divergence due to allopatry . either environmental changes ( e.g. , habitat availability and climatic oscillations ) alone can not account for population divergence or differences in biology allow these two species to respond differently to climatic oscillations . goeldii strongly supports the alternative hypothesis of geographic isolation driving divergence rather than environmental changes . while often contested in the neotropics , the pleistocene refugia hypothesis ( haffer 1969 ) postulates that climatic fluctuations resulted in the fragmentation of continuous forest into refuges separated by expanses of grass - dominated savannah or desert . alternatively , the miocene marine incursion hypothesis suggests sea level changes during the late tertiary ( 23.7 - 1.8 million years ago ) created three isolated regions : the brazilian shield , the guiana shield and the eastern andean slopes ( webb 1995 , bates 2001 , hoorn et al . the last commonly explored mechanism of divergence is the riverine barrier hypothesis , where major rivers act as barriers to gene flow , thus promoting speciation between populations on either side ( wallace 1852 , aleixo 2004 , mirabello & conn 2008 ) . although no boundary was indicated by samova , this does not discount the possibility that an historical boundary existed , separating north and south populations . although one study found evidence for waterways over 4 km wide being complete barriers to gene flow ( pedro & sallum 2009 ) , others have determined that the amazon is not an effective barrier ( fairley et al . 2002 , mirabello & conn 2008 ) and that geographic distance and/or differing demographic histories may be the main forces responsible for partitioning genetic variation ( mirabello & conn 2008 ) . triannulatus rr population , but their inclusion with geographically similar pa samples in an . goeldii may be attributed to individual species ' responses to dispersal opportunities ( brouat et al . 2004 ) . the two an . triannulatus populations identified in goiansia could also be the result of a suture zone . species dispersal activity and differential selection can cause hybrid zones to appear mobile and settle as boundaries between environments or in regions of low population density ( barton & hewitt 1985 , mallet 1993 , 2010 ) . suture zones that span several species suggest divergence was driven by a common influence of environmental change ( morgan et al . 2010 ) . further evaluation of species in the goiansia area could resolve the contribution of environmental change to population diversity . overlapping geographic distributions , in addition to the absence of significant difference in species abundance across sampled habitats , implies little habitat differentiation between species and a broad ecological range . although biological differences may drive divergence uniquely in each of these sympatric species , a common cause for diversity patterns can not be ruled out . geographic boundaries / distance , variation in dispersal activity and ecological range may each contribute to the distinct patterns of divergence seen in an . as species complexes are common in anophelines , additional studies are needed to resolve the extent of divergence . continued studies using multiple markers at a local and continental scale will resolve the status of speciation and may clarify what role , if any , a given population , lineage or species has on malaria transmission , because population differences influence peak biting times , feeding preferences and vectorial capacity ( lounibos & conn 2000 ) .

to evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in anopheles triannulatus , larval habitat characteristics across the brazilian states of roraima and par and genetic sequences were examined . a comparison with anopheles goeldii was utilised to determine whether high genetic diversity was unique to an . triannulatus . student t test and analysis of variance found no differences in habitat characteristics between the species . analysis of population structure of an . triannulatus and an . goeldii revealed distinct demographic histories in a largely overlapping geographic range . cytochrome oxidase i sequence parsimony networks found geographic clustering for both species ; however nuclear marker networks depicted an . triannulatus with a more complex history of fragmentation , secondary contact and recent divergence . evidence of pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography . we hypothesise that niche partitioning is a driving force for diversity , particularly in an . triannulatus .

MATERIALS AND METHODS RESULTS DISCUSSION

mosquito collection - fourth instar anopheline larvae were collected from 13 localities , including eight in the state of roraima ( rr ) in northern amazonian brazil and five in the state of par ( pa ) in eastern amazonian brazil ( fig . morphological keys do not reflect the recent differences between the colombian and venezuelan an . subsequent analysis of similar larval sites based upon the habitat type and type of water body sampled ( pond , pool , swamp etc . ) , examined variation in larval densities using two - way analysis of variance ( anova ) . additional analyses examining differences in habitat ecology , based on biotic factors , surrounding vegetation and canopy coverage were examined to test the hypothesis that environmental heterogeneity is positively correlated with genetic diversity . the forward and reverse coi sequences were aligned using sequencher 3.0 ( gene codes corps , mi , usa ) , grouped together by sight and trimmed in mega v. 3.1 ( kumar et al . therefore , analyses herein focused on the barcode fragment and included only a subset based on the 3 ' end to allow systematic comparisons of an . nuclear genes were included to investigate the history of the nuclear genome and provide independent evidence of population divergence ( loiaza et al . the settings were two simultaneous , independent runs of the markov chain monte carlo for two million generations , sampling every 1,000 generations with a burnin of 25% . genetic variation - genetic structure of multiple populations based on statistical parsimony groupings were examined by analysis of molecular variance ( amova ) using arlequin v. 3.1.1 ( excoffier et al . 2005 ) and spatial analysis of molecular variance ( samova ) , v. 1.0 ( dupanloup et al . triannulatus using the 449-bp fragment haplotypes of pedro and sallum ( 2009 ) ( gu445849 - 899 and af417702 ) combined with the samples in the present study , to test their hypothesis of geographical sub - structuring within the species based on five populations across brazil . population structure and demographic history - a statistical parsimony network ( spn ) estimated genealogical relationships among haplotypes with a 95% identity for each marker using tcs 1.13 ( clement et al . furthermore , haplotype distributions across four ecoregions were examined for each of the seven environmental variables to test the hypothesis that climatic differences sustain ( and drive ) diversity ( hoorn et al . the hypothesis of strict neutrality for each species was examined using dnasp , with the statistics dt ( tajima 1989 ) and d and f ( fu 1993 ) evaluating the likelihood of background selection and fu 's fs ( fu 1997 ) and r2 ( ramos - onsins & rozas 2002 ) examining evidence of population expansion . dates of population expansion were estimated for the coi gene with the formula t = /2 u ( rogers & harpending 1992 ) using 10 generations per year and the mutation rate of drosophila estimated at 1 10 substitutions per site per year ( walton et al . a large proportion , 68.97% ( n = 140 ) , of an . goeldii larvae appeared equally likely to inhabit artificial pools ( 37.93% , n = 77 ) , ponds ( 29.55% , n = 60 ) and temporary ditches ( 25.12% , n = 51 ) . goeldii and those negative for either species ( f = 4.89 , p = 0.032 ) that was not supported by a t test ( t = 1.179 , df = 16 , p = 0.055 ) . there are no other significant differences between environmental variables for sites unique for either species and sites either positive or negative for both ( table i ) . table icharacteristics ( 95% confidence intervals ) of habitats with anopheles triannulatus only , anopheles goeldii only , both species or neither species present an . triannulatus , with 50 parsimoniously informative sites , indicated two strongly supported ( 97 and 98 posterior probabilities ) distinct populations of an . the two populations correspond to the brazilian states , with the exception of samples from moj and goiansia ( 9 and 10 , respectively ) , that were included in the northern group ( rr localities ) . the close relationship between the rr samples and localities moj and goiansia was also evident in the spn . this may indicate a historical boundary separating north and south populations , with the northern limit occurring just south of the amazon river and including parts of goiansia and moj . the more derived population 1 corresponds to localities in the amazon basin , near the hypothesised population origin ( pedro & sallum 2009 ) , with the majority of population 2 occurring along one of the proposed routes of lineage expansion . blue : samples from goiansia , tucuru , jacund and marab [ state of par ( pa ) ] corresponding to an . triannulatus population 3 ; green : moj and goiansia samples ( pa ) in addition to an . triannulatus population 2 ; orange : localities in the state of roraima and an . black : localities in the state of roraima ; grey : moj and goiansia samples from the state of par ( pa ) ; n : number of sequences ; white : samples from goiansia , tucuru , jacund and marab ( pa ) . triannulatus , black refers to population 1 , grey ( with the addition of haplotype 14 ) to population 2 and white to population 3 . goeldii , with 32 parsimoniously informative sites , indicated two moderately supported ( 72 and 68 posterior probabilities ) populations which correspond to the brazilian states ( fig . triannulatus , there does not appear to be any sub - structuring in the samples from pa or support for a separate goiansia / moj population . triannulatus , i.e. table iianalysis of molecular variance calculations for inter and intravariation among species and populations for various markers anopheles triannulatus anopheles goeldii gene source of variation variance ( % ) fixation index variance ( % ) fixation index coi between rr and pa 41.01 fct = 0.41 45.12 fct = 0.45 among localities within states 29.23 fsc = 0.50 5.29 fsc = 0.10 within a given locality 29.76 fst = 0.70 49.59 fst = 0.50 white between rr and pa -3.01 fct = -0.03 89.46 fct = 0.89 among localities within states 58.62 fsc = 0.57 0.42 fsc = 0.04 within a given locality 44.38 fst = 0.56 10.12 fst = 0.90 coi between spn populations 38.85 fct = 0.39 - - among localities 33.92 fsc = 0.55 - - within a given locality 27.23 fst = 0.73 - - a : p < 0.05 ; b : p < 0.001 ; c : not significant ; coi : cytochrome oxidase i ; pa : state of par ; rr : state of roraima ; spn : statistical parsimony network derived groupings . a : p < 0.05 ; b : p < 0.001 ; c : not significant ; coi : cytochrome oxidase i ; pa : state of par ; rr : state of roraima ; spn : statistical parsimony network derived groupings . the small genetic differentiation of an . amova indicated a large proportion of variation between the two states and among samples from different breeding site types in a given locality ( table ii ) . genetic structuring in the latter may indicate environmental differences and niche partitioning as drivers of diversity . 3 ) led to the discovery of two very different demographic histories between these sympatric species , which often co - occur within the same larval habitats . triannulatus was consistent with a category i phylogeographic pattern ( avise 2000 ) characterised by pronounced genetic gaps between spatially circumscribed haplogroups . based upon the 600-bp fragment , da between the northern and southern states is 0.0118 [ standard deviation ( sd ) = 0.003 ] . goeldii coi is consistent with a category iii phylogeographic pattern ( avise 2000 ) where most haplotypes are closely related , yet some geographic localisation exists . similar to an . triannulatus , there was a relatively high proportion of singletons ( 14 , 41.2% ) , indicating a demographic expansion , background selection or selective sweep ( slatkin & hudson 1991 , fu 1997 ) , particularly in pa ( singletons arising from haplotype 56 ) . in contrast , haplotypes from rr exhibited longer branches , missing haplotypes and a near equal distribution of shared haplotypes and single mutations , consistent with a signal of an older lineage with balancing selection ( fig . triannulatus nuclear data ( white gene and its2 ) indicate a history of contractions and expansions . similar to the coi gene , the its2 network depicts a category i pattern whereas the white gene reveals a much more homogeneous haplotype distribution . black : localities in the state of roraima ; grey : moj and goiansia samples from the state of par ( pa ) ; n : number of sequences ; white : samples from goiansia , tucuru , jacund and marab ( pa ) . 4 ) are less pronounced , but still correspond primarily to rr and pa with a few outliers . polymorphism analyses of the coi sequences found haplotype and nucleotide diversity was greater among an . genetic polymorphism analyses of the coi sequences indicated that haplotype and nucleotide diversity was greater among an . high coi variation between states and geographic populations is strongly supported by population differentiation statistics ( table iii ) , although the gst values were low , suggesting a history of gene flow ( table iii ) . table iiiinter and intrapopulation differentiation of anopheles triannulatus and anopheles goeldii populations based on statistical parsimony network comparison an . goeldii rr vs. pa three populations rr vs. pa hs 0.89 0.86 0.86 k s * 1.77 1.41 1.60 z * 7.43 7.00 7.83 snn 0.99 1.00 0.97 129 258 125.84 gst 0.06 0.09 0.045 kt 10.09 10.09 7.43 a : p < 0.0001 ; g st : genetic differentiation ; h s : genetic differentiation based on haplotype data ; k s * : differentiation based on sequence data ; k t : average number of nucleotide differences ; n : number of total individuals compared ; pa : state of par ; rr : state of roraima ; s nn : measures how often the nearest neighbours of sequences are found in the same population ; : genetic differentiation based on allele frequencies ; z * : rank statistic to analyse sequence similarity . a : p < 0.0001 ; g st : genetic differentiation ; h s : genetic differentiation based on haplotype data ; k s * : differentiation based on sequence data ; k t : average number of nucleotide differences ; n : number of total individuals compared ; pa : state of par ; rr : state of roraima ; s nn : measures how often the nearest neighbours of sequences are found in the same population ; : genetic differentiation based on allele frequencies ; z * : rank statistic to analyse sequence similarity . in general , neutrality tests were negative for both species ( table iv ) , suggesting an excess of rare polymorphisms consistent with either negative / positive selection or an increase in population size , although only one of these tests ( fs ) was significant for an . table ivmultiple statistical analyses evaluating the probability for a recent population expansion species n d t d f f s r 2 anopheles triannulatus 129 -0.22187 -1.28038 -0.99732 -10.329 0.08650 roraima 57 -0.37846 -1.32424 -1.17022 -1.488 0.09200 par 72 -0.47543 -0.97094 -0.93438 -5.842 0.08900 population 1 56 -0.08718 -1.11433 -0.88956 -1.209 0.10200 population 2 36 -1.06921 -0.55581 -0.85324 -3.582 0.07770 population 3 37 -1.02686 -0.40080 -0.71980 -4.809 0.07800 anopheles goeldii 133 -0.30013 -1.24177 -1.01960 -5.867 0.08260 roraima 103 0.06971 -1.6295 -1.1537 -1.677 0.0989 par 30 -0.0415 0.15645 0.11023 -3.321 0.1185 a : significance , p < 0.50 ; d : fu and li 's d ( fu 1993 ) ; d t : tajima 's d ( tajima 1989 ) ; f : fu and li 's f ( fu 1993 ) ; f s : fu 's fs ( fu 1997 ) ; n : sample size ; r 2 : ramos - onsin 's and roza 's r 2 ( ramos - onsins & rozas 2002 ) . line represents the model for all anopheles triannulatus ( a ) , all anopheles goeldii ( b ) , an . triannulatus population 2 ( c ) ( moj and goiansia samples ) and an . distribution patterns of mosquito species are dependent on the availability of suitable aquatic habitats ( grillet 2000 ) . overlapping geographic distributions , in addition to the absence of significant difference in species abundance across sampled habitats , suggest that an . the lack of any significant differentiation between sympatric habitats , to those unique to a species or absent of either , suggests these species may be more broadly adapted ( eisenberg et al however , co - occurrence of species does not imply the same ability to exploit shared habitats ( diabate et al . the combined sequence results support distinct demographic histories for the two species despite similar distribution and larval habitat co - occurrence . goeldii consistently depicts two moderately supported populations corresponding to the two brazilian states , however , an . triannulatus appears to have undergone historical fragmentation , subsequent secondary contact and homogenisation followed by more recent divergence due to allopatry . , habitat availability and climatic oscillations ) alone can not account for population divergence or differences in biology allow these two species to respond differently to climatic oscillations . alternatively , the miocene marine incursion hypothesis suggests sea level changes during the late tertiary ( 23.7 - 1.8 million years ago ) created three isolated regions : the brazilian shield , the guiana shield and the eastern andean slopes ( webb 1995 , bates 2001 , hoorn et al . 2002 , mirabello & conn 2008 ) and that geographic distance and/or differing demographic histories may be the main forces responsible for partitioning genetic variation ( mirabello & conn 2008 ) . triannulatus rr population , but their inclusion with geographically similar pa samples in an . triannulatus populations identified in goiansia could also be the result of a suture zone . overlapping geographic distributions , in addition to the absence of significant difference in species abundance across sampled habitats , implies little habitat differentiation between species and a broad ecological range . although biological differences may drive divergence uniquely in each of these sympatric species , a common cause for diversity patterns can not be ruled out . geographic boundaries / distance , variation in dispersal activity and ecological range may each contribute to the distinct patterns of divergence seen in an .

noninvasive brain stimulation ( nibs ) finds increasingly more applications in clinical neuroscience . under the term nibs , different techniques are summarized that allow a noninvasive stimulation of the brain but that are characterized by different modes of action . recent studies in the field have made clear that the response capacity to any nibs protocol is subject of a significant interindividual variability [ 25 ] . contrary to initial expectations , these studies displayed that a specified number of subjects will not show the expected effects following a given nibs protocol but showed that these subjects may show no responses or even the opposite effects . taking into consideration that nibs are currently been applied in clinical practice to treat various neuropsychiatric disorders ( e.g. , depression , schizophrenia , and stroke ) , it is necessary to better understand the response variability following nibs . thus , we decided to perform a comparative study of the efficacy and the response variability of two well - established nibs protocols with different physiological modes of action : transcranial direct current stimulation ( tdcs ) and paired - associative stimulation ( pas ) . tdcs is one of the most widespread techniques in clinical trials and experimental settings , because the application is simple and the neurophysiological and behavioural effects are discussed to be rather strong . animal studies showed that tdcs modulates spontaneous neuron activity by a tonic depolarisation ( anodal tdcs ) or hyperpolarisation ( cathodal tdcs ) of their membrane potentials resulting in consecutive long - lasting changes in the neuronal firing rates [ 68 ] . a more recent animal study conducted on motor - cortex slices from the mouse brain slices confirmed the polarity - specific effects of anodal tdcs and showed that anodal tdcs induces long - lasting synaptic potentiation outlasting the duration of stimulation . human studies confirmed these findings by showing similar polarity - dependent changes in motor - cortical excitability following anodal or cathodal tdcs . anodal tdcs led to an increase of motor - evoked potentials ( meps ) and cathodal tdcs resulted in a decrease of meps following a stimulation of 13 minutes or respective 9 minutes [ 10 , 11 ] . pharmacological challenges in healthy subject demonstrated that this modulation in motor - cortical excitability following tdcs is critically dependent on calcium homeostasis and the activity of glutamatergic nmda receptors [ 12 , 13 ] . in summary , these findings from animal and human studies suggest that the after - effects following tdcs are related to neural plasticity and to the molecular mechanisms of long - term potentiation and long - term depression . on the other hand , pas is mainly used in experimental settings because the application is more complicated than the application of tdcs . pas fulfils several criteria of a plasticity protocol , as the after - effects are long - lasting , persist the duration of the intervention , and are input - specific and nmda - dependent . however , the special characteristics are that the after - effects of pas seem to be synapse - specific following hebbian principles and that they are related to spike - dependent plasticity [ 3 , 14 , 16 ] . human studies indicate that the repeated pairing of an electric stimulus of a peripheral nerve ( somatosensory afferent ) followed by a single magnetic pulse of the contralateral motor cortex with an interstimulus interval of 25 ms ( or adjusted to the individual n20-latency plus 2 ms ) results in a long - lasting mep increase in terms of ltp - like plasticity [ 1 , 14 , 16 ] . an adjustment of the interstimulus interval to 10 ms ( or adjustment to the n20-latency minus 5 ms ) causes a reduction of mep amplitudes ( ltd - like plasticity ) [ 1 , 14 , 16 ] . the aim of this study was to compare the efficacy and response variability of two ltp - like plasticity inducing nibs protocols using the most established stimulation configurations . anodal tdcs was applied with 1 ma for 13 minutes [ 7 , 17 ] and pas25 consisted of 180 pairs of peripheral nerve stimulation followed by a magnetic pulse with an interstimulus interval of 25 ms [ 14 , 15 , 18 ] . only one previous study has yet directly compared these two ltp - like plasticity inducing protocols . this study showed the same level of excitability change and the same proportion of responders following anodal tdcs and pas25 but no mean change of motor - cortex excitability when all subjects were analysed . these findings contrast earlier reports that investigated either anodal tdcs ( for review see ) or pas ( for review see ) and showed significant group - level changes in cortical excitability . we aimed to either replicate or refute these initial findings and to extend the current knowledge of response variability following nibs . we hypothesised that both plasticity protocols will result in a mean increase in cortical excitability but that a certain proportion of subjects will not show the expected results . the study protocol was conducted in accordance with the declaration of helsinki and approved by the ethics committee of the ludwig maximilians university of munich . after giving written informed consent , 30 healthy participants , all aged between 19 and 42 years , all subjects were medication - free and screened by clinically experienced psychiatrists for psychiatric comorbidities . subject received two experimental sessions ( anodal tdcs versus pas25 ) on two different days and sessions for each subject were 7 to 8 days apart . subjects received on the first study day anodal tdcs and on the second study day pas25 . subjects were examined in half - reclined sitting position with their arms resting passively supported . electromyographic activity ( emg ) was recorded by surface electrodes on the right first dorsal interosseous muscle ( fdi ) . raw signals were amplified and bandpass - filtered ( 3 hz2 khz range ) using a digitimer d-360 amplifier setup ( digitimer ltd . , uk ) . , cambridge , uk ) controlled by signal software ( version 5 , cambridge electronic design , cambridge , uk ) . motor - evoked potentials ( mep ) were induced by tms applied to the left primary motor cortex ( m1 ) with a standard figure - of - eight magnetic coil ( outer diameter 70 mm , the magstim company ltd . , uk ) and a monophasic magstim bistim stimulator ( the magstim company ltd . , uk ) . throughout all experiments , the coil was held tangentially to the skull , with the handle pointing backwards and in a 45 angle lateral to the midline . the stimulation site that produced the largest motor - evoked potential ( mep ) at moderately suprathreshold stimulation intensities was defined as the hot spot and marked for further optimal coil positioning . rmt was recorded in the resting fdi muscle and defined as the minimum stimulator intensity that resulted in an mep amplitude of 50 v in at least 5 of 10 measurements . the stimulation intensity corresponding to mep amplitudes of 1 mv ( 0.3 mv ) ( s1mv ) was adjusted at baseline and kept unchanged throughout the experiments . single - pulse mep measurements using the s1mv intensity were conducted at baseline ( 40 stimuli ) and after stimulation ( time points 0 , 5 , 10 , 20 , and 30 minutes ; 20 stimuli at each time point ) to monitor after - effects following both plasticity protocols ( pas and tdcs ) . to test for after - effects on cortical recruitment , input - output curves ( io ) were measured at baseline and 8 minutes after stimulation using an increasing stimulus intensity order ( 90% , 110% , and 130% of rmt ) with 7 stimuli for each intensity ( figure 1 ) . single- and paired - pulse tms was applied at 0.2 hz . to assess the after - effects of the respective stimulation types on inhibitory and facilitatory intracortical networks , short - latency intracortical inhibition ( sici ) and intracortical facilitation ( icf ) were obtained at baseline and 15 minutes after stimulation using a standardized paired - pulse protocol . the conditioning stimulus was set at 80% rmt intensity and the test stimulus at s1mv ( 0.3 mv ) in the resting fdi . the intensity of the test pulse was not adjusted after the intervention for paired - pulse measures . in total , 65 randomised stimuli were applied , with 15 stimuli using the test stimulus alone and 10 stimuli for each interstimulus interval ( isi ) ( sici : 2 ms and 3 ms ; icf : 7 ms , 9 ms , and 12 ms ) . anodal tdcs was applied through saline soaked rectangular surface sponge - electrodes ( 35 cm ) using a ce - certified standard stimulator ( dc - stimulator - plus , neuroconn gmbh , ilmenau , germany ) . the anodal electrode was positioned on the left side of the skull over the representational field of the right fdi as identified by tms ; the cathodal electrode was contralaterally above the right orbit . the stimulation intensity of the tonic electrical field was set at 1 ma and applied for a total duration of 13 minutes [ 7 , 17 ] , which has been consistently shown in previous tdcs publications to be an optimal duration time for the induction of cortical excitability changes in terms of ltp - like plasticity lasting for approximately one hour following tdcs . according to foregoing publications [ 14 , 15 , 18 ] , the pas25 protocol consisted of 180 pairs of peripheral nerve stimuli followed by tms stimuli after an interstimulus interval ( isi ) of 25 ms . the peripheral nerve stimulation was applied to the right ulnaris nerve at the level of the wrist using a ce - certified ds7a peripheral nerve stimulator ( digitimer ltd . , uk ) . the stimulation intensity was set at 300% of the individual perceptual threshold , resulting in an average electrical intensity of 9.8 2.1 ma , which has been demonstrated to result in a reliable plasticity response . the tms stimuli were applied to the motor - cortical representation of the right fdi as identified in the excitability measurements . to maintain a constant level of attention during the stimulation , subjects were asked to watch their right hand , silently count the number of stimuli delivered , and report the adding number to the examiners request every 2030 stimuli ( random choice by examiner ) . all subjects mean count of the total number of paired stimuli was 176182 ( mean = 179.5 1.5 ) and did not significantly differ from the total count of 180 stimuli , indicating a sufficient level of attention [ 22 , 23 ] . for statistical analysis , spss 22 for windows was used and the level of significance was set at alpha = 0.05 . to test for differences concerning baseline parameters between the two experimental sessions paired - samples t - tests were computed for all depending variables . cortical excitability changes were expressed as increase or decrease in mean mep amplitudes before and after stimulation . as the assumption of normal data distribution was violated for most depending variables ( kolmogorov - smirnov test , p between < 0.001 and 0.043 ) , square root transformations were applied to meet the requirements to conduct rm - anovas . to test the time course of plasticity changes , a rm - anova ( 6 2 ) with the factors time course ( baseline , 0 min , 5 min , 10 min , 20 min , and 30 min ) and stimulation type ( anodal tdcs and pas ) and another rm - anova ( 2 2 ) with the factors time ( baseline , mean post - meps averaged ) and again stimulation type ( anodal tdcs and pas ) were performed . in the next step , separate rm - anovas for each stimulation type alone were conducted . to test for differences in the cortical recruitment , a rm - anova with the factors ( before and after stimulation ) and intensity ( 90% , 110% , and 130% rmt ) was conducted for both stimulation conditions separately . changes in intracortical excitability over time were analysed with rm - anovas with the factors isi ( test pulse , 2 ms , 3 ms , 7 ms , 9 ms , and 12 ms ) and time ( baseline , 15 minutes after stimulation ) . when appropriate , that is , significant interactions in the rm - anovas , student 's t - tests ( paired , two - tailed ) were performed to determine more specifically whether mep amplitudes differed before and after plasticity induction within and between conditions . in cases of lacking interactions , sphericity was tested with mauchly 's test and , if necessary ( mauchly 's test < 0.05 ) , the greenhouse - geisser correction was used . to test the individual response pattern , subjects were categorised in responders ( r ) and nonresponders ( nr ) to the respective ltp - protocol . to test for whether any of the obtained baseline parameters of cortical excitability showed a correlation with the excitability changes following anodal tdcs or pas ( relative mean post - meps ) subjects were aged between 19 and 42 years ( mean 27.4 4.8 ) , 14 were female ( 47% ) , with one exception all were right handed ( n = 29 , 94% ) , the average body - height was 176.5 9 cm , and 13 were smokers ( 43% ) with a mean fagerstrom - score of 3 ( table 1 ) . to compare baseline values in both experiments , paired - samples t - tests were computed for all depending variables : rmt , s1mv ( both single- and double - pulse ) , 1 mv mep , sici ( 2 ms , 3 ms , mean ) , icf ( 7 ms , 9 ms , and 12 ms , mean 912 ms ) , and recruitment curve ( 90% , 110% , and 130% rmt ) . none of the tested variables showed significant differences between the first and the second experimental sessions ( all p > 0.129 , table 2 ) . a repeated - measures analysis of variance ( rm - anova ) was conducted with the factors time course ( baseline , 0 min , 5 min , 10 min , 20 min , and 30 min ) and stimulation this analysis revealed a significant main effect on time course ( f5,25 = 4.412 , p = 0.001 ) but neither an effect on stimulation ( f1,29 = 2.190 , p = 0.150 ) nor an effect on the time course stimulation interaction ( f5,25 = 0.617 , p = 0.687 ) . in addition , the overall rm - anova with the factors time ( baseline , mean post - meps averaged ) and again stimulation ( anodal tdcs , pas ) showed also a significant main effect on time ( f1,29 = 12.392 , p = 0.001 ) and no effect on stimulation rm - anovas separately computed for both stimulation protocols separately showed a significant main effect on time course in the pas - group ( f5,25 = 3.963 , p = 0.002 ) but not in the tdcs - group ( f5,25 = 1.408 , p = 0.225 ) . to analyse the general excitability changes following both stimulation types , a mean value of all poststimulation time points was included into an additional rm - anova analysis , which showed a significant main effect on time for both anodal tdcs ( f1,29 = 4.534 , p = 0.042 ) and pas ( f1,29 = 16.041 , p < 0.001 ) . for anodal tdcs , paired - samples t - tests showed a significant difference comparing baseline to the mean of all time points following stimulation ( t1,29 = 2.13 , p = 0.042 ) . in the case of pas , a significant increase in mep size was found comparing baseline to the mean of all time points following stimulation ( t1,29 = 4.01 , p < 0.001 ) and at all single time points after stimulation ( all t1,29 > 2.13 , all p < 0.042 ) with the exception of 0 minutes after stimulation ( t1,29 = 1.95 , p = 0.061 ) . baseline meps did not differ between the anodal and the pas conditions ( t1,29 = 0.06 , p = 0.953 ) and also mean post - meps did not differ between the anodal and the pas conditions ( t1,29 = 1.575 , p = 0.126 ) ( figure 2 ) . to further explore the observed differences between the mep increase in tdcs and pas , we further conducted a rm - anova of the standard deviations . this analysis revealed a trend - level effect on time course ( f5,25 = 1.91 , p = 0.097 ) and no effect on stimulation ( f1,29 = 0.74 , p = 0.397 ) and no time course stimulation interaction ( f5,25 = 1.02 , p = 0.407 ) . this finding can be explained by higher standard deviations after stimulation in both conditions . a rm - anova for the io - curves with the factors time ( baseline , after stimulation ) and ( 90% , 110% , and 130% rmt ) revealed a significant main effect on intensity for both anodal tdcs ( f2,28 = 179.87 , p < 0.001 ) and pas ( f2,28 = 112.60 , p ( tdcs : f1,29 = 2.60 , p = 0.118 ; pas : f1,29 = 3.07 , p = 0.090 ) and no time intensity interaction ( tdcs : f2,28 = 0.679 , p = 0.515 ; pas : f2,28 = 2.02 , p = 0.151 ) . for paired - pulse measurements , two additional rm - anovas were conducted for both anodal tdcs and pas separately with the factors time ( test pulse , 2 ms , 3 ms , 7 ms , 9 ms , and 12 ms ) or ( test pulse , mean sici ( 2 ms , 3 ms ) , and mean icf ( 9 ms , 12 ms ) ) . in the case of anodal tdcs , the 2 6 analysis showed a significant main effect on ( f4,26 = 101.34 , p < 0.001 ) but not on time ( f1,29 = 0.03 , p = 0.871 ) or a time isi interaction ( f4,26 = 0.49 , p = 0.787 ) . for pas this rm - anova revealed both a significant isi effect ( f4,26 = 91.13 , p < 0.001 ) and a significant time effect ( f1,29 = 5.97 , p = 0.021 ) but no time isi interaction ( f4,26 = 1.65 , p = 0.150 ) . a similar pattern was obtained in the time and mean isi 2 3 rm - anova . for anodal tdcs , the analysis showed a significant mean isi effect ( f1,29 = 128.02 , p < 0.001 ) and no time effect ( f1,29 = 1.01 , p = 0.753 ) or time isi interaction ( f1,29 = 1.06 , p = 0.352 ) . for pas we found a significant mean isi effect ( f1,29 = 124.64 , p < 0.001 ) and time effect ( f1,29 = 7.768 , p = 0.009 ) and a time isi interaction ( f1,29 = 3.99 , p = 0.024 ) . in the pas experiments , subsequent dependent samples t - tests were conducted to compare paired - pulse measures before and after stimulation . these analyses showed an increase in all tested variables following pas with significant differences for sici at 2 ms isi ( t1,29 = 2.61 , p = 0.014 ) and mean sici ( t1,29 = 2.42 , p = 0.022 ) and the test pulse ( t1,29 = 3.96 , p < 0.001 ) . thus , the significant effects reported from the rm - anova are very likely to be a consequence of the increase in the test pulse after stimulation . due to the lacking time effect , no further t - tests were conducted for the anodal experiments . to obtain an overview over the individual response patterns of all subjects these cut - offs defined response as an mep size increase following the respective stimulation types over a cut - off of > 100% , > 110% , and 150% relative to the individual baseline ( figures 3 and 4 ) . chi - square ( chi ) tests were computed to compare the stimulation protocol and the respective individual response pattern . these analyses revealed trend - level differences between anodal and pas responders at > 110% ( p = 0.052 ) and > 150% ( p = 0.058 ) cut - off ranges but not at > 100% ( 0.243 ) in favour of the pas stimulation . defining a decrease of sici and an increase in icf following ltp - protocols as response , we again defined three different cut - off ranges : > 100% , > 110% , and > 150% increase of the respective relative mean values ( post / pre : sici(2 - 3 ms ) ; icf(912 ms ) ) . chi tests were used to compare the distribution of responders between both experiments . for sici decrease no significant difference in the distribution of responders was found ( > 100% : p = 1.000 ; > 110% : p = 0.795 ; > 150% : p = 1.000 ) . in comparison , the analysis for icf increase revealed a significant difference in the distribution of responders in all of the three ranges ( > 100% : p = 0.009 ; > 110% : p = 0.002 ; > 150% : p = 0.005 ) in favour of anodal tdcs . in order to explore whether gender affected the mep increase following stimulation , chi tests were obtained from both experiments comparing distribution of response and gender . for all cut - off ranges , this analysis did not reveal any significant differences between gender and anodal tdcs ( 100% : p = 0.796 ; 110% : p = 0.491 ; 150% : p = 0.273 ) or pas ( 100% : p = 0.855 ; 110% : p = 0.855 ; 150% : p = 0.261 ) . pearson correlation coefficients were used to examine the relationship between relative baseline values ( age ; standard deviation of meps ; sici 2 ms , sici 3 ms , icf 7 ms , icf 9 ms , and icf 12 ms ) and the relative mean mep values following stimulation in both experiments . for pas these analyses revealed a positive trend - level correlation between age and relative mean poststimulation meps ( r = 0.345 , p = 0.062 ) , which was not observed after anodal tdcs ( r = 0.010 , p = 0.959 ) . in addition , we observed for anodal tdcs a positive correlation between the relative icf values at baseline ( 12 ms isi ) and the relative mean poststimulation mep values ( r = 0.557 , p = 0.001 ) . concerning all other variables no significant correlations to further investigate the impact of the observed icf - correlation in the anodal experiments , we compared the relative baseline icf values ( 12 ms ) between responders and nonresponders in the anodal condition . these analyses revealed a trend - level difference in the case of the > 100% cut - off range ( t1,28 = 1.87 , p = 0.072 ) but significantly higher relative baseline 12 ms icf values for both > 110% ( t1,28 = 2.15 , p = 0.041 ) and > 150% ( t1,28 = 3.55 , p = 0.0014 ) in responders compared to nonresponders . the present results reveal a differential response pattern following two well - established ltp - protocols in a large sample of healthy controls . both ltp - protocols ( anodal tdcs and pas25 ) induced a significant increase of meps for the observation period of 30 minutes . in the pas25 experiments this mep increase could be observed for nearly all poststimulation time points , whereas in the anodal tdcs experiments the increase of meps could only be shown when all time points were averaged . the likelihood to develop a meaningful mep response was higher in the pas25 compared to the anodal tdcs experiments , but the likelihood for a response in terms of an icf - modulation showed the opposite pattern . in the anodal tdcs experiments , the baseline values for icf at 12 ms showed a positive correlation with the increase of meps after stimulation and this value differed significantly between responders and nonresponders . in summary , our results indicate a sufficient increase of cortical excitability following anodal tdcs and pas25 in terms but also demonstrate less individual variability than reported in previous studies . considering the particular importance of interindividual response differences surprisingly limited data comparing nibs protocols with different modes of action is available . one study compared the efficacy of three different ltp - protocols ( intermittent theta - burst stimulation ( tbs ) , anodal tdcs , and pas25 ) in 56 healthy controls and the authors were not able to show significant effects on excitatory or inhibitory circuits when all subjects were analysed as a group . this response pattern resembles the results of another study conducted on 18 subjects showing in the group - level analyses no increase in meps following intermittent tbs and pas25 but a decrease in mep amplitudes following continuous tbs . for tdcs , a recently published study could not observe a modulation of mep amplitudes following anodal or cathodal tdcs in 53 healthy subjects . the fact that this study used an intensity of 2 ma for tdcs whereas most tdcs studies used 1 ma [ 7 , 25 ] should be taken into account . recent evidence indicates that the increase in the tdcs intensity is not related to the efficacy of stimulation and that homeostatic mechanisms could counteract the efficacy of high stimulation intensity . for tbs , one study showed in 56 healthy subjects that neither excitatory nor inhibitory tbs resulted in a change in mep sizes after stimulation and that only 25% of all subjects showed the expected responses . the lacking group - level response following different tbs protocols has now been reported from different groups ( e.g. , [ 2730 ] ) . for pas25 , similar results with lacking increase in mep amplitudes and a response rate in 14 out of 27 subjects were shown in one study . this high response variability following standard pas protocols was confirmed in other studies with limited sample sizes [ 31 , 32 ] . the reason most frequently discussed for not being able to induce a general change in motor - cortical excitability in the presented studies with sufficient sample sizes is the high response variability across subjects [ 2 , 4 , 5 ] . our findings deviate from the aforementioned reports . in our study , both plasticity protocols were effective on a group - level analysis and our results are in the range of the initial reports for pas and tdcs [ 10 , 11 , 17 ] . furthermore , the likelihood to develop a mep response was higher in the pas25 experiments . remarkably , only 23% responded to anodal tdcs , but 47% responded to pas using the 150% cut - off . comparing the mean mep amplitudes after stimulation , a numeric but not statistical significant difference between the pas25 and tdcs experiments could be observed . thus , it may be assumed that pas25 was more effective to increase mep amplitudes . on the other hand , furthermore , baseline icf correlated positively with an increase in mep amplitudes and mep responders had significantly higher icf baseline values compared to the nonresponders in the anodal experiment . it should be noted that a decrease in sici and an increase in icf have been reported following anodal tdcs but not following pas25 ( for review see [ 1 , 16 ] ) . therefore , paired - pulse measures may not be suitable as outcome parameters for pas25 but may be helpful to detect responders to anodal tdcs . in one study baseline sici correlated with the pas25 response and another study showed that the pas25 response correlated with sici measured with a threshold tracking method . differences between these studies and our work may be the timing of the paired - pulse assessment and the configuration of the conditioning pulse . our novel observation of an icf - associated efficacy of anodal tdcs may allow hypothesizing that the after - effects of anodal tdcs appear to be located in facilitatory interneuron networks and may be dependent on synaptic modulation . the reservation must be made that our design does not allow a mechanistic explanation of results due to the lack of pharmacological interventions . we aimed to identify further baseline characteristics ( other paired - pulse measures , age , gender , and variability in baseline meps expressed by the standard deviation ) that may predict the response to anodal tdcs or pas25 , but no other factors were detected . we found a trend for a positive correlation between age and the mep changes following pas25 . this is in contrast to previous reports of a negative correlation between age and the response to pas . however , our age range is outside the age range of an expected age - dependent decrease in cortical plasticity . we report the plasticity effects following nibs in a single - session design and thus can not rule out that repetitive sessions ( as used in the clinical context ) would have resulted in another distribution of plasticity . furthermore , we focussed on two established ltp - protocols and as it is possible to modulate various parameters using tdcs ( e.g. , current intensity , current density ) and pas ( e.g. , isi , individualised pas , and target peripheral nerve ) it may be possible that other configurations would have resulted in different response patterns . regarding the paired - pulse measures , the lacking assessment of a paired - pulse response curve at different time points limits the generalizability of our sici / icf discussion . furthermore , we did not readjust the test pulse intensity after the intervention in the paired - pulse paradigms . on the one hand evidence is available that the intensity of the test pulse affects the percent sici / icf , but on the other hand this is not found to be the case in the range of mep sizes presented in our experiments [ 36 , 37 ] . the mep sizes following the test pulse of the paired - pulse paradigms were 1.04 0.31 before and 1.23 0.76 after anodal tdcs and , respectively , 1.11 0.43 before and 1.64 0.86 after pas25 . further limitations are the lacking sham condition in our experiments and the fact that we did not use randomized ordered session of anodal tdcs and pas25 . thus , we can not rule out that the order of sessions had an impact on our results . for pas , we used the ulnar nerve for our peripheral nerve stimulation and the fdi as target muscle . most published pas studies stimulated the median nerve and used the abductor pollicis brevis muscle as target ( for review see ) . however , different studies reported comparable changes in meps following both ulnar and median nerve stimulation , in the context of pas protocols ( for review see ) . one important confounding factor could be attention . in our pas experiment we asked the subjects to count the tms pulse and the reported values are within a range ( 179.5 1.5 ) [ 22 , 23 ] indicating a sufficient level of attention in our experiments . during anodal tdcs no count of stimuli is conducted , possibly leading to different attention states compared with those in the pas setup . thus , the reported higher efficacy of pas25 could also be led back to attention rather than to physiological differences between the protocols . despite our large sample , thus , replication studies with larger sample sizes are needed to confirm some of our findings . however , in line with previous research , our sample also comprised a certain degree of nonresponders . the likelihood to be a nonresponder was dependent on the kind of stimulation protocol , on the defined thresholds and on the used target items . our new observation that subject had a higher likelihood to be a responder using the high 150% mep threshold in the pas25 protocol may be explained by the fact that this protocol was more individualised ( electrical and motor thresholds ) and controlled for attention compared to anodal tdcs . moreover , we were for the first time able to identify a new baseline parameter ( icf at 12 ms ) that may predict the response to anodal tdcs . future prospective studies need to independently confirm this parameter before it can be used as individual response predictor . it is reassuring that our response rates using the lowest threshold were much higher than those reported in other trials , but the response rates using the highest threshold are less optimistic .

interindividual response variability to various motor - cortex stimulation protocols has been recently reported . comparative data of stimulation protocols with different modes of action is lacking . we aimed to compare the efficacy and response variability of two ltp - inducing stimulation protocols in the human motor cortex : anodal transcranial direct current stimulation ( a - tdcs ) and paired - associative stimulation ( pas25 ) . in two experiments 30 subjects received 1ma a - tdcs and pas25 . data analysis focused on motor - cortex excitability change and response defined as increase in mep applying different cut - offs . furthermore , the predictive pattern of baseline characteristics was explored . both protocols induced a significant increase in motor - cortical excitability . in the pas25 experiments the likelihood to develop a mep response was higher compared to a - tdcs , whereas for intracortical facilitation ( icf ) the likelihood for a response was higher in the a - tdcs experiments . baseline icf ( 12 ms ) correlated positively with an increase in meps only following a - tdcs and responders had significantly higher icf baseline values . contrary to recent studies , we showed significant group - level efficacy following both stimulation protocols confirming older studies . however , we also observed a remarkable amount of nonresponders . our findings highlight the need to define sufficient physiological read - outs for a given plasticity protocol and to develop predictive markers for targeted stimulation .

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

under the term nibs , different techniques are summarized that allow a noninvasive stimulation of the brain but that are characterized by different modes of action . recent studies in the field have made clear that the response capacity to any nibs protocol is subject of a significant interindividual variability [ 25 ] . contrary to initial expectations , these studies displayed that a specified number of subjects will not show the expected effects following a given nibs protocol but showed that these subjects may show no responses or even the opposite effects . thus , we decided to perform a comparative study of the efficacy and the response variability of two well - established nibs protocols with different physiological modes of action : transcranial direct current stimulation ( tdcs ) and paired - associative stimulation ( pas ) . a more recent animal study conducted on motor - cortex slices from the mouse brain slices confirmed the polarity - specific effects of anodal tdcs and showed that anodal tdcs induces long - lasting synaptic potentiation outlasting the duration of stimulation . human studies confirmed these findings by showing similar polarity - dependent changes in motor - cortical excitability following anodal or cathodal tdcs . anodal tdcs led to an increase of motor - evoked potentials ( meps ) and cathodal tdcs resulted in a decrease of meps following a stimulation of 13 minutes or respective 9 minutes [ 10 , 11 ] . pharmacological challenges in healthy subject demonstrated that this modulation in motor - cortical excitability following tdcs is critically dependent on calcium homeostasis and the activity of glutamatergic nmda receptors [ 12 , 13 ] . however , the special characteristics are that the after - effects of pas seem to be synapse - specific following hebbian principles and that they are related to spike - dependent plasticity [ 3 , 14 , 16 ] . human studies indicate that the repeated pairing of an electric stimulus of a peripheral nerve ( somatosensory afferent ) followed by a single magnetic pulse of the contralateral motor cortex with an interstimulus interval of 25 ms ( or adjusted to the individual n20-latency plus 2 ms ) results in a long - lasting mep increase in terms of ltp - like plasticity [ 1 , 14 , 16 ] . the aim of this study was to compare the efficacy and response variability of two ltp - like plasticity inducing nibs protocols using the most established stimulation configurations . only one previous study has yet directly compared these two ltp - like plasticity inducing protocols . this study showed the same level of excitability change and the same proportion of responders following anodal tdcs and pas25 but no mean change of motor - cortex excitability when all subjects were analysed . these findings contrast earlier reports that investigated either anodal tdcs ( for review see ) or pas ( for review see ) and showed significant group - level changes in cortical excitability . we aimed to either replicate or refute these initial findings and to extend the current knowledge of response variability following nibs . we hypothesised that both plasticity protocols will result in a mean increase in cortical excitability but that a certain proportion of subjects will not show the expected results . subjects received on the first study day anodal tdcs and on the second study day pas25 . motor - evoked potentials ( mep ) were induced by tms applied to the left primary motor cortex ( m1 ) with a standard figure - of - eight magnetic coil ( outer diameter 70 mm , the magstim company ltd . rmt was recorded in the resting fdi muscle and defined as the minimum stimulator intensity that resulted in an mep amplitude of 50 v in at least 5 of 10 measurements . single - pulse mep measurements using the s1mv intensity were conducted at baseline ( 40 stimuli ) and after stimulation ( time points 0 , 5 , 10 , 20 , and 30 minutes ; 20 stimuli at each time point ) to monitor after - effects following both plasticity protocols ( pas and tdcs ) . to assess the after - effects of the respective stimulation types on inhibitory and facilitatory intracortical networks , short - latency intracortical inhibition ( sici ) and intracortical facilitation ( icf ) were obtained at baseline and 15 minutes after stimulation using a standardized paired - pulse protocol . in total , 65 randomised stimuli were applied , with 15 stimuli using the test stimulus alone and 10 stimuli for each interstimulus interval ( isi ) ( sici : 2 ms and 3 ms ; icf : 7 ms , 9 ms , and 12 ms ) . the stimulation intensity of the tonic electrical field was set at 1 ma and applied for a total duration of 13 minutes [ 7 , 17 ] , which has been consistently shown in previous tdcs publications to be an optimal duration time for the induction of cortical excitability changes in terms of ltp - like plasticity lasting for approximately one hour following tdcs . the tms stimuli were applied to the motor - cortical representation of the right fdi as identified in the excitability measurements . to test for differences in the cortical recruitment , a rm - anova with the factors ( before and after stimulation ) and intensity ( 90% , 110% , and 130% rmt ) was conducted for both stimulation conditions separately . changes in intracortical excitability over time were analysed with rm - anovas with the factors isi ( test pulse , 2 ms , 3 ms , 7 ms , 9 ms , and 12 ms ) and time ( baseline , 15 minutes after stimulation ) . to test the individual response pattern , subjects were categorised in responders ( r ) and nonresponders ( nr ) to the respective ltp - protocol . to compare baseline values in both experiments , paired - samples t - tests were computed for all depending variables : rmt , s1mv ( both single- and double - pulse ) , 1 mv mep , sici ( 2 ms , 3 ms , mean ) , icf ( 7 ms , 9 ms , and 12 ms , mean 912 ms ) , and recruitment curve ( 90% , 110% , and 130% rmt ) . a repeated - measures analysis of variance ( rm - anova ) was conducted with the factors time course ( baseline , 0 min , 5 min , 10 min , 20 min , and 30 min ) and stimulation this analysis revealed a significant main effect on time course ( f5,25 = 4.412 , p = 0.001 ) but neither an effect on stimulation ( f1,29 = 2.190 , p = 0.150 ) nor an effect on the time course stimulation interaction ( f5,25 = 0.617 , p = 0.687 ) . in addition , the overall rm - anova with the factors time ( baseline , mean post - meps averaged ) and again stimulation ( anodal tdcs , pas ) showed also a significant main effect on time ( f1,29 = 12.392 , p = 0.001 ) and no effect on stimulation rm - anovas separately computed for both stimulation protocols separately showed a significant main effect on time course in the pas - group ( f5,25 = 3.963 , p = 0.002 ) but not in the tdcs - group ( f5,25 = 1.408 , p = 0.225 ) . to analyse the general excitability changes following both stimulation types , a mean value of all poststimulation time points was included into an additional rm - anova analysis , which showed a significant main effect on time for both anodal tdcs ( f1,29 = 4.534 , p = 0.042 ) and pas ( f1,29 = 16.041 , p < 0.001 ) . for anodal tdcs , paired - samples t - tests showed a significant difference comparing baseline to the mean of all time points following stimulation ( t1,29 = 2.13 , p = 0.042 ) . in the case of pas , a significant increase in mep size was found comparing baseline to the mean of all time points following stimulation ( t1,29 = 4.01 , p < 0.001 ) and at all single time points after stimulation ( all t1,29 > 2.13 , all p < 0.042 ) with the exception of 0 minutes after stimulation ( t1,29 = 1.95 , p = 0.061 ) . to further explore the observed differences between the mep increase in tdcs and pas , we further conducted a rm - anova of the standard deviations . this analysis revealed a trend - level effect on time course ( f5,25 = 1.91 , p = 0.097 ) and no effect on stimulation ( f1,29 = 0.74 , p = 0.397 ) and no time course stimulation interaction ( f5,25 = 1.02 , p = 0.407 ) . a rm - anova for the io - curves with the factors time ( baseline , after stimulation ) and ( 90% , 110% , and 130% rmt ) revealed a significant main effect on intensity for both anodal tdcs ( f2,28 = 179.87 , p < 0.001 ) and pas ( f2,28 = 112.60 , p ( tdcs : f1,29 = 2.60 , p = 0.118 ; pas : f1,29 = 3.07 , p = 0.090 ) and no time intensity interaction ( tdcs : f2,28 = 0.679 , p = 0.515 ; pas : f2,28 = 2.02 , p = 0.151 ) . for paired - pulse measurements , two additional rm - anovas were conducted for both anodal tdcs and pas separately with the factors time ( test pulse , 2 ms , 3 ms , 7 ms , 9 ms , and 12 ms ) or ( test pulse , mean sici ( 2 ms , 3 ms ) , and mean icf ( 9 ms , 12 ms ) ) . in the case of anodal tdcs , the 2 6 analysis showed a significant main effect on ( f4,26 = 101.34 , p < 0.001 ) but not on time ( f1,29 = 0.03 , p = 0.871 ) or a time isi interaction ( f4,26 = 0.49 , p = 0.787 ) . for pas this rm - anova revealed both a significant isi effect ( f4,26 = 91.13 , p < 0.001 ) and a significant time effect ( f1,29 = 5.97 , p = 0.021 ) but no time isi interaction ( f4,26 = 1.65 , p = 0.150 ) . for anodal tdcs , the analysis showed a significant mean isi effect ( f1,29 = 128.02 , p < 0.001 ) and no time effect ( f1,29 = 1.01 , p = 0.753 ) or time isi interaction ( f1,29 = 1.06 , p = 0.352 ) . for pas we found a significant mean isi effect ( f1,29 = 124.64 , p < 0.001 ) and time effect ( f1,29 = 7.768 , p = 0.009 ) and a time isi interaction ( f1,29 = 3.99 , p = 0.024 ) . in the pas experiments , subsequent dependent samples t - tests were conducted to compare paired - pulse measures before and after stimulation . these analyses showed an increase in all tested variables following pas with significant differences for sici at 2 ms isi ( t1,29 = 2.61 , p = 0.014 ) and mean sici ( t1,29 = 2.42 , p = 0.022 ) and the test pulse ( t1,29 = 3.96 , p < 0.001 ) . thus , the significant effects reported from the rm - anova are very likely to be a consequence of the increase in the test pulse after stimulation . to obtain an overview over the individual response patterns of all subjects these cut - offs defined response as an mep size increase following the respective stimulation types over a cut - off of > 100% , > 110% , and 150% relative to the individual baseline ( figures 3 and 4 ) . chi - square ( chi ) tests were computed to compare the stimulation protocol and the respective individual response pattern . these analyses revealed trend - level differences between anodal and pas responders at > 110% ( p = 0.052 ) and > 150% ( p = 0.058 ) cut - off ranges but not at > 100% ( 0.243 ) in favour of the pas stimulation . defining a decrease of sici and an increase in icf following ltp - protocols as response , we again defined three different cut - off ranges : > 100% , > 110% , and > 150% increase of the respective relative mean values ( post / pre : sici(2 - 3 ms ) ; icf(912 ms ) ) . in comparison , the analysis for icf increase revealed a significant difference in the distribution of responders in all of the three ranges ( > 100% : p = 0.009 ; > 110% : p = 0.002 ; > 150% : p = 0.005 ) in favour of anodal tdcs . pearson correlation coefficients were used to examine the relationship between relative baseline values ( age ; standard deviation of meps ; sici 2 ms , sici 3 ms , icf 7 ms , icf 9 ms , and icf 12 ms ) and the relative mean mep values following stimulation in both experiments . in addition , we observed for anodal tdcs a positive correlation between the relative icf values at baseline ( 12 ms isi ) and the relative mean poststimulation mep values ( r = 0.557 , p = 0.001 ) . concerning all other variables no significant correlations to further investigate the impact of the observed icf - correlation in the anodal experiments , we compared the relative baseline icf values ( 12 ms ) between responders and nonresponders in the anodal condition . these analyses revealed a trend - level difference in the case of the > 100% cut - off range ( t1,28 = 1.87 , p = 0.072 ) but significantly higher relative baseline 12 ms icf values for both > 110% ( t1,28 = 2.15 , p = 0.041 ) and > 150% ( t1,28 = 3.55 , p = 0.0014 ) in responders compared to nonresponders . the present results reveal a differential response pattern following two well - established ltp - protocols in a large sample of healthy controls . both ltp - protocols ( anodal tdcs and pas25 ) induced a significant increase of meps for the observation period of 30 minutes . in the pas25 experiments this mep increase could be observed for nearly all poststimulation time points , whereas in the anodal tdcs experiments the increase of meps could only be shown when all time points were averaged . the likelihood to develop a meaningful mep response was higher in the pas25 compared to the anodal tdcs experiments , but the likelihood for a response in terms of an icf - modulation showed the opposite pattern . in the anodal tdcs experiments , the baseline values for icf at 12 ms showed a positive correlation with the increase of meps after stimulation and this value differed significantly between responders and nonresponders . in summary , our results indicate a sufficient increase of cortical excitability following anodal tdcs and pas25 in terms but also demonstrate less individual variability than reported in previous studies . considering the particular importance of interindividual response differences surprisingly limited data comparing nibs protocols with different modes of action is available . one study compared the efficacy of three different ltp - protocols ( intermittent theta - burst stimulation ( tbs ) , anodal tdcs , and pas25 ) in 56 healthy controls and the authors were not able to show significant effects on excitatory or inhibitory circuits when all subjects were analysed as a group . this response pattern resembles the results of another study conducted on 18 subjects showing in the group - level analyses no increase in meps following intermittent tbs and pas25 but a decrease in mep amplitudes following continuous tbs . recent evidence indicates that the increase in the tdcs intensity is not related to the efficacy of stimulation and that homeostatic mechanisms could counteract the efficacy of high stimulation intensity . the lacking group - level response following different tbs protocols has now been reported from different groups ( e.g. for pas25 , similar results with lacking increase in mep amplitudes and a response rate in 14 out of 27 subjects were shown in one study . the reason most frequently discussed for not being able to induce a general change in motor - cortical excitability in the presented studies with sufficient sample sizes is the high response variability across subjects [ 2 , 4 , 5 ] . in our study , both plasticity protocols were effective on a group - level analysis and our results are in the range of the initial reports for pas and tdcs [ 10 , 11 , 17 ] . furthermore , the likelihood to develop a mep response was higher in the pas25 experiments . comparing the mean mep amplitudes after stimulation , a numeric but not statistical significant difference between the pas25 and tdcs experiments could be observed . on the other hand , furthermore , baseline icf correlated positively with an increase in mep amplitudes and mep responders had significantly higher icf baseline values compared to the nonresponders in the anodal experiment . we aimed to identify further baseline characteristics ( other paired - pulse measures , age , gender , and variability in baseline meps expressed by the standard deviation ) that may predict the response to anodal tdcs or pas25 , but no other factors were detected . furthermore , we focussed on two established ltp - protocols and as it is possible to modulate various parameters using tdcs ( e.g. regarding the paired - pulse measures , the lacking assessment of a paired - pulse response curve at different time points limits the generalizability of our sici / icf discussion . furthermore , we did not readjust the test pulse intensity after the intervention in the paired - pulse paradigms . however , different studies reported comparable changes in meps following both ulnar and median nerve stimulation , in the context of pas protocols ( for review see ) . however , in line with previous research , our sample also comprised a certain degree of nonresponders . the likelihood to be a nonresponder was dependent on the kind of stimulation protocol , on the defined thresholds and on the used target items . our new observation that subject had a higher likelihood to be a responder using the high 150% mep threshold in the pas25 protocol may be explained by the fact that this protocol was more individualised ( electrical and motor thresholds ) and controlled for attention compared to anodal tdcs . moreover , we were for the first time able to identify a new baseline parameter ( icf at 12 ms ) that may predict the response to anodal tdcs .

the online version of this article ( doi:10.1007/s40801 - 015 - 0009 - 6 ) contains supplementary material , which is available to authorized users . the amounts of intravenous immunoglobulin ( ivig ) used for short- and long - term treatment of immune thrombocytopenia ( itp ) are forecasted to be approximately 5,000 and 11,000 g per year , respectively , up to 2018 at these two tertiary care centers.the estimated provincial cost of ivig use in itp may be as high as $ 5 million annually.physicians and policy makers should reflect on the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve patient quality of life and decrease economic costs . immune thrombocytopenia ( itp ) is a heterogeneous autoimmune disorder characterized by the presence of platelet autoantibodies , low platelet counts , and an increased risk of bleeding [ 1 , 2 ] . some patients will present with asymptomatic thrombocytopenia , while others will experience bleeding complications , which range in severity from skin bruises to fatal intracranial hemorrhage . itp can be classified by duration as newly diagnosed ( acute ) , persistent ( lasting 312 months ) , or chronic ( lasting 12 months ) . treatments for patients with itp are aimed at preventing serious bleeding , improving quality of life , and achieving a safe platelet count . for newly diagnosed patients , treatment with a brief course of corticosteroids , intravenous anti - d , or intravenous immunoglobulin ( ivig ) results in rapid yet transient responses . durable remissions of more than 6 months occur in fewer than 20 % of patients following first - line therapy with prednisone [ 5 , 6 ] . higher durable response rates from 59 to 75 % may occur with high - dose dexamethasone , depending on the age of the patients studied and the duration of follow - up [ 79 ] . chronic maintenance therapy with corticosteroids is limited by toxicities , such as neuropsychiatric symptoms , glucose intolerance , and osteoporosis ; hence , other therapies are often used in relapsed or refractory itp patients to achieve a hemostatic platelet count , thereby preventing or minimizing bleeding complications . treatment choices include thrombopoietin receptor agonists , immunosuppressive agents , and ivig [ 1 , 2 ] . ivig , a blood product manufactured from pooled human plasma , is a therapeutic option in the acute and chronic management of patients with itp . patients with itp are among the highest users of ivig in canada , representing 1017 % of utilization for all indications [ 10 , 11 ] . ivig administered at a dose of 12 g / kg causes rapid transient increases in platelet counts in over 80 % of patients ; however , platelet counts generally return to pretreatment levels within 4 weeks . while repeated infusions of ivig at regular intervals may be useful as maintenance therapy for patients with itp who require ongoing treatment because of bleeding , where a reasonable alternative exists , persistent use should be limited because of finite supplies and high associated costs [ 1315 ] . further , ivig is associated with bothersome side effects , including headache , nausea , flushing , fevers , chills , fatigue , and diarrhea . less commonly , it may result in more serious complications , including anaphylaxis , hemolysis , thrombosis , renal failure , and aseptic meningitis [ 1 , 4 , 16 , 17 ] . over the past 5 years , in the province of ontario , ivig utilization for all indications has risen to 1.56 million units per year at a cost of $ 97.9 million in 2010/2011 , representing an increase of 44 % in the number of units and a 53 % increase in costs . given the growth in the utilization of ivig in various clinical conditions , it is forecasted that the supply may not be able to meet the demand . in 2006 , the ontario ministry of health and long term care ( mohltc ) identified the unsustainable increases in ivig utilization as a key priority ; however , there is currently no provincial mechanism for routinely tracking and accurately quantifying and characterizing ivig use . this study aimed to assess the utilization of ivig in patients with itp at two large tertiary care centers ; determine the extent of short- and long - term utilization ; assess the proportion of ivig usage for itp compared with all indications ; compare utilization between these two unique centers ; and forecast future demand . a retrospective analysis of all adult patients who received ivig for the treatment of itp at these two participating centers during the study period was performed . patients were eligible for inclusion if they met the following criteria : age 18 years or older ; a diagnosis of itp ; and receipt of at least one dose of ivig for the treatment of itp between january 1 , 2003 , and september 30 , 2012 , at either of two large tertiary care centers , hamilton health sciences ( hhs ) and london health sciences centre ( lhsc ) . data collected at both centers included the date of birth , sex , weight ( if available ) , date of ivig infusion , and amount of ivig administered . at hhs , potential itp patients were identified and cases were confirmed by chart review . the transfusion registry for utilization , surveillance and tracking ( trust ) database , developed by the mcmaster transfusion research program ( mtrp ) , was used as the primary source of data extraction in hamilton . trust comprises data primarily from two sources of electronic data capture : meditech ( meditech circle , westwood , ma , usa ) , and the discharge abstract database ( dad ) . meditech is a laboratory information system used at hhs , which houses laboratory values and transfusion / infusion product information . the dad is the electronic database at both institutions that collects clinical data for the canadian institute for health information ( cihi ) . it was used to identify patients with a diagnosis of itp , extract patient information , and determine the indication for ivig , etc . to confirm the diagnosis , information was obtained from patients clinic charts and electronic medical records . patient diagnoses are coded using the cihi international classification of diseases and related health problems , 10th revision , canada ( icd-10-ca ) . during the study period , two of the three hamilton hospitals issued ivig from the transfusion medicine laboratory ; hence , the information on ivig disposition was in the laboratory information system ( meditech ) and had been captured in trust . the third hospital ( at the mcmaster site ) issued ivig from the transfusion medicine laboratory between 2009 and 2012 ; however , from 2002 to 2009 , the ivig product was issued from the pharmacy and recorded manually . the information on these paper logs was entered into a spreadsheet and cross - linked with the dad data to identify all potential itp patients . a chart review was then performed on all potential itp patients to confirm their diagnosis and eligibility for final inclusion in the analysis . at lhsc , the transfusion medicine laboratory information system contained all infusion episodes and data on ivig utilized within the study period , including all inpatient and outpatient ivig utilization at all hospital sites ( i.e. , university hospital and victoria hospital of lhsc , and st . this database did not identify icd-10 codes , but all ivig requested through the transfusion medicine laboratory required an ivig request form , which documented the indications . further , one of the investigators ( ch ) retrospectively reviewed the clinical data from the recipients patient electronic records to differentiate and confirm the diagnosis of itp versus an error in coding due to another cause of thrombocytopenia . where electronic records were not available , paper charts were reviewed . the de - identified data were submitted to the mtrp , where the data from the two sites were combined and analyzed . the primary outcome of this study was a description of the proportion of patients using ivig in the short - term and long - term chronic itp settings , and the corresponding durations of therapy . secondary outcomes included the number of ivig infusions administered ; number of ivig infusions administered per patient ; average total amount administered per infusion , per course , and per treatment period ; average number of infusions and courses given per treatment period ; days between courses ; percentage of total ivig use ( for all indications ) that was used for itp ; estimated ivig product usage cost per year ; and future forecasts of ivig utilization from 2012 to 2018 . to determine the primary outcome of quantifying short- and long - term utilization , definitions of acute , short - term and long - term treatments were developed on the basis of clinical judgment by experts in the treatment of itp , both individually and by consensus as part of an advisory board meeting sponsored by glaxosmithkline inc . infusions of ivig were grouped to form courses , and courses were grouped to form treatment periods , which were then classified as acute , short - term , or long - term ( fig . 1 ) . course was defined as the number of ivig infusions administered within a 5-day period . this definition allowed for variable practice patterns , as physicians often order ivig 12 g / kg divided over 1 , 2 , or 5 days . a treatment period was defined as a collection of courses where consecutive courses were given within a 6-month ( 182-day ) time frame of each other . thus , a single treatment period could last for years if consecutive courses during that treatment period were given within 6 months of each other . consecutive courses that were given more than 6 months apart were considered to be in separate treatment periods ( i.e. , a patient may have been treated in multiple treatment periods ) . an acute treatment was defined as involving only one course in the treatment period . clinically , for example , this could correspond to using ivig once for emergency treatment of bleeding associated with low platelet counts . a short - term treatment contained 25 courses , possibly representing a longer bridging treatment until another therapy started to work , and a long - term treatment was defined as containing six or more courses in that treatment period , perhaps denoting maintenance therapy.fig . 1summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods lhsc data were sent to hhs for centralized analysis . a data dictionary was developed for the data from each site , and variable names were mapped to standardize data coding . sas 9.3 software ( sas institute , cary , nc , usa ) was used to perform this cross - mapping and for data analysis . descriptive statistics were used to determine the proportion of ivig being used for patients with itp compared with all other indications . time series forecasting utilization for 20122018 was performed using a stepwise autoregressive method ( sas 9.3 , proc forecast ) with the history data from 2003 to 2011 . the stepwise autoregressive method ( stepar ) that was used combines a time trend regression with an autoregressive model for departures from the trend . a retrospective analysis of all adult patients who received ivig for the treatment of itp at these two participating centers during the study period was performed . patients were eligible for inclusion if they met the following criteria : age 18 years or older ; a diagnosis of itp ; and receipt of at least one dose of ivig for the treatment of itp between january 1 , 2003 , and september 30 , 2012 , at either of two large tertiary care centers , hamilton health sciences ( hhs ) and london health sciences centre ( lhsc ) . data collected at both centers included the date of birth , sex , weight ( if available ) , date of ivig infusion , and amount of ivig administered . at hhs , potential itp patients were identified and cases were confirmed by chart review . the transfusion registry for utilization , surveillance and tracking ( trust ) database , developed by the mcmaster transfusion research program ( mtrp ) , was used as the primary source of data extraction in hamilton . trust comprises data primarily from two sources of electronic data capture : meditech ( meditech circle , westwood , ma , usa ) , and the discharge abstract database ( dad ) . meditech is a laboratory information system used at hhs , which houses laboratory values and transfusion / infusion product information . the dad is the electronic database at both institutions that collects clinical data for the canadian institute for health information ( cihi ) . it was used to identify patients with a diagnosis of itp , extract patient information , and determine the indication for ivig , etc . to confirm the diagnosis , patient diagnoses are coded using the cihi international classification of diseases and related health problems , 10th revision , canada ( icd-10-ca ) . during the study period , two of the three hamilton hospitals issued ivig from the transfusion medicine laboratory ; hence , the information on ivig disposition was in the laboratory information system ( meditech ) and had been captured in trust . the third hospital ( at the mcmaster site ) issued ivig from the transfusion medicine laboratory between 2009 and 2012 ; however , from 2002 to 2009 , the ivig product was issued from the pharmacy and recorded manually . the information on these paper logs was entered into a spreadsheet and cross - linked with the dad data to identify all potential itp patients . a chart review was then performed on all potential itp patients to confirm their diagnosis and eligibility for final inclusion in the analysis . at lhsc , the transfusion medicine laboratory information system contained all infusion episodes and data on ivig utilized within the study period , including all inpatient and outpatient ivig utilization at all hospital sites ( i.e. , university hospital and victoria hospital of lhsc , and st . this database did not identify icd-10 codes , but all ivig requested through the transfusion medicine laboratory required an ivig request form , which documented the indications . further , one of the investigators ( ch ) retrospectively reviewed the clinical data from the recipients patient electronic records to differentiate and confirm the diagnosis of itp versus an error in coding due to another cause of thrombocytopenia . where electronic records were not available , paper charts were reviewed . the de - identified data were submitted to the mtrp , where the data from the two sites were combined and analyzed . the primary outcome of this study was a description of the proportion of patients using ivig in the short - term and long - term chronic itp settings , and the corresponding durations of therapy . secondary outcomes included the number of ivig infusions administered ; number of ivig infusions administered per patient ; average total amount administered per infusion , per course , and per treatment period ; average number of infusions and courses given per treatment period ; days between courses ; percentage of total ivig use ( for all indications ) that was used for itp ; estimated ivig product usage cost per year ; and future forecasts of ivig utilization from 2012 to 2018 . to determine the primary outcome of quantifying short- and long - term utilization , definitions of acute , short - term and long - term treatments were developed on the basis of clinical judgment by experts in the treatment of itp , both individually and by consensus as part of an advisory board meeting sponsored by glaxosmithkline inc . infusions of ivig were grouped to form courses , and courses were grouped to form treatment periods , which were then classified as acute , short - term , or long - term ( fig . 1 ) . course was defined as the number of ivig infusions administered within a 5-day period . this definition allowed for variable practice patterns , as physicians often order ivig 12 g / kg divided over 1 , 2 , or 5 days . a treatment period was defined as a collection of courses where consecutive courses were given within a 6-month ( 182-day ) time frame of each other . thus , a single treatment period could last for years if consecutive courses during that treatment period were given within 6 months of each other . consecutive courses that were given more than 6 months apart were considered to be in separate treatment periods ( i.e. , a patient may have been treated in multiple treatment periods ) . an acute treatment was defined as involving only one course in the treatment period . clinically , for example , this could correspond to using ivig once for emergency treatment of bleeding associated with low platelet counts . a short - term treatment contained 25 courses , possibly representing a longer bridging treatment until another therapy started to work , and a long - term treatment was defined as containing six or more courses in that treatment period , perhaps denoting maintenance therapy.fig . 1summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods a data dictionary was developed for the data from each site , and variable names were mapped to standardize data coding . sas 9.3 software ( sas institute , cary , nc , usa ) was used to perform this cross - mapping and for data analysis . descriptive statistics were used to determine the proportion of ivig being used for patients with itp compared with all other indications . time series forecasting utilization for 20122018 was performed using a stepwise autoregressive method ( sas 9.3 , proc forecast ) with the history data from 2003 to 2011 . the stepwise autoregressive method ( stepar ) that was used combines a time trend regression with an autoregressive model for departures from the trend . there were 383 adult itp patients who received a total of 2,098 ivig infusions at the two major ontario tertiary care centers participating in this retrospective study between january 1 , 2003 , and september 30 , 2012 ( tables 1 , 2 ) . comparing the two centers , in london , 547 infusions were given to 150 patients , and in hamilton , 1,551 infusions were given to 233 patients ( table 2 ) . despite this difference , the proportions of male and female recipients were similar at the two sites . the mean age of the patients at the first ivig infusion was 51.3 years , with a range of ages from 18 to 96 years of age ( table 1 ) . the mean weight ( 80.4 kg ) was available and calculated from 195 recipients ( london 81/150 , 54 % ; hamilton 114/233 , 49 % ) and was also similar at the two centers ( table 1 ) . patients received , on average , 5.5 ivig infusions ( london 3.6 , hamilton 6.7 ; range 1196 ) at 0.96 g / kg per infusion ( table 2).table 1demographic characteristics by site and overallcharacteristiclondon , n = 150hamilton , n = 233total , n = 383male sex [ n ( % ) ] 56 ( 37.3)88 ( 37.8)144 ( 37.6)age at the first ivig infusion [ years ; mean / sd ] 54.0/20.0449.5/19.1651.3/19.60patients with available weight data [ n]81114195average weight at time of first infusion [ kg ; mean / sd]80.1/18.2380.6/20.1080.4/19.30 ivig intravenous immunoglobulin , sd standard deviation age was calculated using the birth year of each patient , so for some patients the age may have been overcalculated by 1 yeartable 2intravenous immunoglobulin ( ivig ) usage : infusions and courses by site and overalllondon , n = 150hamilton , n = 233total , n = 383total number of ivig infusions 5471,5512,098number of ivig infusions per patient [ mean / sd]3.6/6.796.7/15.655.5/13.00amount per infusion [ g / kg ; mean / sd]0.91/0.19020.98/0.21810.96/0.2121total number of ivig courses4281,1751,603number of ivig courses per patient [ mean / sd]2.9/6.395.0/9.624.2/8.56number of infusions per course [ mean / sd ] 1.3/0.501.3/0.491.3/0.49total grams of ivig38,980121,096160,076 sd standard deviation infusions occurring on the same day were counted as one infusion demographic characteristics by site and overall ivig intravenous immunoglobulin , sd standard deviation age was calculated using the birth year of each patient , so for some patients the age may have been overcalculated by 1 year intravenous immunoglobulin ( ivig ) usage : infusions and courses by site and overall sd standard deviation infusions occurring on the same day were counted as one infusion when ivig infusions were combined into courses , there were a total of 1,603 courses of therapy given ( london 428 courses ; hamilton 1,175 courses ) , with a mean of 4.2 courses given per patient and 1.3 infusions given per course ( table 2 ) . the majority of patients received one or two infusions per course , which was similar at the two centers . further , the majority of patients receiving infusions at both centers received approximately 1 or 2 g / kg per course , with 1 g / kg being the most commonly prescribed amount , followed by 2 g / kg . when grouped into treatment periods ( as defined earlier ) , there were a total of 492 treatment periods ( london 172 , hamilton 320 ) with 264 ( 53.7 % ) defined as acute , 172 ( 35.0 % ) short - term , and 56 ( 11.4 % ) long - term treatments ( tables 3 , 4 ) . the majority of patients , 306 ( 79.9 % ) , received all of their ivig infusions in one treatment period , but some patients had up to six treatment periods . the average number of courses given in a treatment period was 3.3 ( london 2.5 , hamilton 3.7 ; range 199 ) . this correlated with an average 4.3 ivig infusions given per treatment period ( london 3.2 , hamilton 4.8 ; range 1196 ) [ table 3 ] . the number of days between consecutive courses within these treatment periods was 29.5 days ( london 32.2 , hamilton 28.7 ) , and the number of days between consecutive treatment periods , for those patients who received ivig in more than one treatment period , was 636 days and was similar at the two centers ( london 632 , hamilton 637).table 3intravenous immunoglobulin ( ivig ) usage categorized by treatment type by site and overalllondon , n = 150hamilton , n = 233total , n = 383total number of ivig treatment periods ( % ) 172320492 acute treatment ( % ) 101 ( 58.7)163 ( 50.9)264 ( 53.7 ) short - term treatment ( % ) 60 ( 34.9)112 ( 35.0)172 ( 35.0 ) long - term treatment ( % ) 11 ( 6.4)45 ( 14.1)56 ( 11.4)number of ivig treatment periods received per patient [ mean / sd]1.1/0.441.4/0.781.3/0.68courses per treatment period [ mean / sd]2.5/5.863.7/7.793.3/7.19infusions per treatment period [ mean / sd]3.2/6.134.8/12.854.3/11.00total grams per treatment period [ mean / sd]226.6/475.08378.4/1,121.75325.4/949.41 sd standard deviationtable 4intravenous immunoglobulin ( ivig ) utilization analysis by treatment type ( overall population)key findingslong - term treatment : 6 or more coursesshort - term treatment : 25 coursesacute treatment : 1 coursenumber of classified treatment periods ( % ) 56 ( 11.4)172 ( 35.0)264 ( 53.7)patients [ n]52152228total ivig amount per treatment type [ g ; mean / sd]1,559.5/2,482.91281.6/136.2092.1/36.11average number of infusions per treatment period [ mean / sd]19.9/27.903.8/1.661.3/0.50days of therapy per treatment period [ mean / sd]427/385.1884.2/79.44 sd standard deviation intravenous immunoglobulin ( ivig ) usage categorized by treatment type by site and overall sd standard deviation intravenous immunoglobulin ( ivig ) utilization analysis by treatment type ( overall population ) sd standard deviation the number of grams of ivig used in london and hamilton for all indications at both sites was seen to be trending upward from 144,605 g in 2003 to 245,763 g in 2012 ( see table a1 in the electronic supplementary material ) . utilization for itp also increased , but , relative to all other indications , the proportion of ivig used for adult itp patients remained relatively stable ( see table a1 ) . the actual and forecasted ivig usage in grams is provided in table 5 . the proportion of ivig given to adult itp patients ranged from 5.6 to 9.1 % and was generally lower in london than in hamilton ( see table a2 in the electronic supplementary material ) . in total , 160,076 g of ivig was administered in 2,098 infusions for patients with itp , representing 7.9 % of the overall amount of ivig usage for all indications , which differed between the centers ( london 4.6 % , hamilton 10.3 % ) [ see table a1].table 5annual actual and forecasted intravenous immunoglobulin ( ivig ) short- and long - term usage in patients with immune thrombocytopeniayearamount of ivig used [ g]londonhamiltontotalshort - term treatmentlong - term treatmentshort - term treatmentlong - term treatmentshort - term treatmentlong - term treatment20031,4606002,9506,0904,4106,69020041401,2603,46711,3533,60712,61320051,18004,4227,8385,6027,83820062,3971,2603,2485,6955,6456,95520071,3001,0203,50311,1374,80312,15720081,2959602,1656,5863,4607,54620091,9771,6001,8106,5003,7878,10020102,3952,7854,5802,3906,9755,17520111,8052,9802,19012,5153,99515,4952012 2,2702,8012,6487,5424,91810,3432013 2,4143,0852,5487,4924,96210,5772014 2,5583,3682,4477,4425,00510,8102015 2,7023,6512,3477,3935,04911,0442016 2,8463,9342,2477,3435,09311,2772017 2,9904,2182,1477,2945,13711,5122018 3,1344,5012,0477,2445,18111,745 the 20122018 data were forecasted using the complete annual data from 2003 to 2011 annual actual and forecasted intravenous immunoglobulin ( ivig ) short- and long - term usage in patients with immune thrombocytopenia the 20122018 data were forecasted using the complete annual data from 2003 to 2011 this 10-year , retrospective study at two tertiary care centers analyzed data from both sites to provide comprehensive information on ivig utilization in adult itp patients . these two large centers represent 19.9 % of the total ivig utilization ( for all indications ) in the province of ontario . during the study period , there were 383 adult itp patients who received a total of 160,076 g of ivig in 2,098 infusions . this represented 7.9 % of the total ivig utilization for all indications somewhat lower than the 1017 % previously reported in atlantic canada [ 10 , 11 ] . on average , there were roughly 1.5 times more women than men who received ivig , which is in keeping with the higher prevalence of itp in women than in men , with estimated female to male ratios of 1.2:1 and 1.7:1 being previously reported [ 20 , 21 ] . the range of patient ages ( 1896 years ) was broad , reflecting the diverse patient population who develop itp and require treatment . ivig utilization patterns were generally similar at the two study centers , including the age of the patients treated , the proportions of male and female recipients , and the amount of ivig infused . however , there were more ivig infusions given to more itp patients in hamilton than in london . these two sites are independent of one another and have dissimilar catchment areas and referral practices . hamilton has a very specialized dedicated itp practice , with referrals from other tertiary care centers to its itp clinics , reflecting a potentially sicker , usually more heavily pretreated population , who may require longer and perhaps more intensive treatments . london has a more community - based referral pattern , where patients are typically seen by a specialist for the first time after diagnosis by a family physician or emergency room physician . despite the clinical heterogeneity in the two patient populations , the majority of patients received approximately 1 or 2 g / kg per course , with 1 g / kg being the most commonly prescribed dose at both centers , followed by 2 g / kg . this is in keeping with infusions used in clinical practice and guidelines [ 1 , 2 ] . the recently updated american society of hematology evidence - based guidelines for itp recommend 1 g / kg as an initial dose . however , it is noted that patients who fail to respond to 1 g / kg may respond to a higher dose of 2 g / kg . at both centers , the majority of ivig was given for acute treatment of itp ( as per our definition ) . less ivig was used for short - term treatment , and it was infrequently used for long - term treatment . it is possible that acute treatments were given for itp patients who required therapy for active bleeding , a bridge to a procedure or surgery with an increased bleeding risk ( i.e. , splenectomy ) , or a trial to assess platelet response . compared with the london site , hamilton treated a larger cohort of itp patients with slightly more ivig infusions per patient , gave more courses per patient , had a higher proportion of itp patients relative to those with other indications , and administered more grams of ivig for itp relative to other indications , as would be expected with its highly specialized itp referral pattern . unlike acute treatments , short - term and long - term therapy with ivig may be provided to patients with the intent of using it as the sole maintenance therapy or as a longer bridging solution until another therapy works to control itp . from the present data , we can predict the impact of ivig utilization in adult itp patients requiring short- and long - term treatments ( table 5 ) . the total amount of ivig used for long - term treatment of itp at these two centers from 2012 to 2018 is forecasted to remain at approximately 11,000 g per year , at an estimated cost of $ 693,000 per year , based on a unit price of approximately $ 63 per gram in 2011 , published in bloody easy 3 by callum et al . . however , short - term ivig treatments make up an additional 172 ( 35 % ) of the total number of ivig treatment periods . with 281.6 g administered per short - term treatment period , this could contribute to an estimated additional 5,000 g of ivig being used , costing $ 315,000 per year , for a total of roughly $ 1 million per year for both short- and long - term treated patients . the estimated costs from these two centers represent 19.9 % of the overall provincial utilization . assuming that the patient populations and practice patterns are similar across ontario , the overall cost in the province for short- and long - term treatments of itp may be upward of fivefold greater , or $ 5 million per year . it was not feasible to include any kind of in - depth resource utilization discussion , since various components , such as nursing costs , chair time , and monitoring for reactions , were not included as part of this study . thus , a complete and accurate cost analysis of all components was not performed , and the true cost impact of ivig to the health care system , based only on the product cost of ivig , is underestimated . as forecasted by our data , ivig usage for itp will remain a substantial burden on the public health care budget and will continue to increase by approximately 2 % per year from 2013 to 2018 . ivig usage for itp should be reduced for several reasons other than its high cost . while ivig safety and toxicity were not studied in this review , ivig is associated with numerous potential side effects . bothersome side effects include headache , nausea , flushing , fevers , chills , fatigue , and diarrhea ( provan et al . ) . less commonly , it may result in serious complications , including anaphylaxis , hemolysis , thrombosis , renal failure , and aseptic meningitis [ 1 , 4 , 16 , 17 ] . further , ivig is a limited resource , which is widely utilized and can impact the quality of life of patients , including the need for travel to an infusion clinic , the need to sit for several hours during the infusion , and anxiety over possible reactions . limiting the use of ivig for itp is one of the items highlighted by choosing wisely canada and the canadian hematology society . in the last 5 years , newer therapies , such as thrombopoietic mimetics , have become available to patients with itp , and the role of these agents as an alternative to ivig , particularly for short- and long - term treatments , requires consideration . however , most of the data came from the laboratory information system at each site , which is considered the gold - standard data repository for blood product utilization . while these two large centers represent 19.9 % ( london 6.5 % , hamilton 13.4 % ) of total ivig utilization ( for all indications ) in the province of ontario , it is not clear if the findings are representative of the province as a whole ( i.e. , community / rural settings ) . the thrombopoietin mimetics were approved for itp treatments in the past few years and may have impacted the utilization of ivig as reflected in this retrospective analysis . further , clinical trials with these new medications at the hamilton site , and a provincial ivig audit , may have reduced the enthusiasm for using ivig during the past few years and may have affected our estimates . this retrospective review of comprehensive data at the london and hamilton sites has helped to characterize ivig utilization in adult itp patients and may improve the understanding of its impact on provincial utilization and inform future clinical practice and policy decisions . with the current practice pattern , short- and long - term ivig utilization for physicians treating itp and policy makers should consider the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve both patient quality of life and economic impact . below is the link to the electronic supplementary material . supplementary material 1 ( docx 20 kb ) supplementary material 1 ( docx 20 kb ) cyrus c. hsia , neerav monga , julia elia - pacitti , and nancy heddle conceived the study . cyrus c. hsia , yang liu , kathleen eckert , and nancy heddle were involved in the collection of data . cyrus c. hsia drafted the manuscript , and all authors provided comments on the drafts and have read and approved the final version submitted for publication .

introductionintravenous immunoglobulin ( ivig ) is an immune thrombocytopenia ( itp ) therapy , which is associated with toxicities , limited availability , increasing utilization , and high cost . this study aimed to assess short- and long - term ivig utilization in patients with itp at two tertiary care centers in ontario , canada , to determine the proportion of ivig used in itp compared with all usage , and to forecast ivig demand in itp.methodsrecords from all adult itp patients who received ivig between january 1 , 2003 , and september 30 , 2012 , at hamilton health sciences and london health sciences centre were reviewed retrospectively.resultsduring the study period , 383 adult itp patients ( mean age 51.3 years ) received a total of 2,098 ivig infusions ( london 547 infusions in 150 patients ; hamilton 1,551 infusions in 233 patients ) . itp accounted for 5.6 and 9.1 % of all ivig usage in london and hamilton , respectively . the treatments included 264 ( 53.7 % ) acute , 172 ( 35.0 % ) short - term , and 56 ( 11.4 % ) long - term treatments . the amounts of ivig used for short- and long - term treatment of itp are forecasted to be approximately 5,000 and 11,000 g per year , respectively , up to 2018 . together , these two centers represent 19.9 % of the provincial ivig utilization . assuming similar patient populations and practice patterns in ontario , the overall provincial cost of ivig use in itp may be as high as $ 5 million annually.conclusionshort- and long - term ivig utilization for itp will remain an expensive resource within the ontario provincial health care system . physicians and policy makers should reflect on the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve patient quality of life and decrease economic costs.electronic supplementary materialthe online version of this article ( doi:10.1007/s40801 - 015 - 0009 - 6 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Key Points Introduction Methods Study Design Study Population Data Collection Definitions Statistical Analysis Results Discussion Conclusion Electronic supplementary material Author contributions Funding

the online version of this article ( doi:10.1007/s40801 - 015 - 0009 - 6 ) contains supplementary material , which is available to authorized users . the amounts of intravenous immunoglobulin ( ivig ) used for short- and long - term treatment of immune thrombocytopenia ( itp ) are forecasted to be approximately 5,000 and 11,000 g per year , respectively , up to 2018 at these two tertiary care centers.the estimated provincial cost of ivig use in itp may be as high as $ 5 million annually.physicians and policy makers should reflect on the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve patient quality of life and decrease economic costs . immune thrombocytopenia ( itp ) is a heterogeneous autoimmune disorder characterized by the presence of platelet autoantibodies , low platelet counts , and an increased risk of bleeding [ 1 , 2 ] . patients with itp are among the highest users of ivig in canada , representing 1017 % of utilization for all indications [ 10 , 11 ] . this study aimed to assess the utilization of ivig in patients with itp at two large tertiary care centers ; determine the extent of short- and long - term utilization ; assess the proportion of ivig usage for itp compared with all indications ; compare utilization between these two unique centers ; and forecast future demand . a retrospective analysis of all adult patients who received ivig for the treatment of itp at these two participating centers during the study period was performed . patients were eligible for inclusion if they met the following criteria : age 18 years or older ; a diagnosis of itp ; and receipt of at least one dose of ivig for the treatment of itp between january 1 , 2003 , and september 30 , 2012 , at either of two large tertiary care centers , hamilton health sciences ( hhs ) and london health sciences centre ( lhsc ) . the primary outcome of this study was a description of the proportion of patients using ivig in the short - term and long - term chronic itp settings , and the corresponding durations of therapy . secondary outcomes included the number of ivig infusions administered ; number of ivig infusions administered per patient ; average total amount administered per infusion , per course , and per treatment period ; average number of infusions and courses given per treatment period ; days between courses ; percentage of total ivig use ( for all indications ) that was used for itp ; estimated ivig product usage cost per year ; and future forecasts of ivig utilization from 2012 to 2018 . to determine the primary outcome of quantifying short- and long - term utilization , definitions of acute , short - term and long - term treatments were developed on the basis of clinical judgment by experts in the treatment of itp , both individually and by consensus as part of an advisory board meeting sponsored by glaxosmithkline inc . infusions of ivig were grouped to form courses , and courses were grouped to form treatment periods , which were then classified as acute , short - term , or long - term ( fig . 1summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods lhsc data were sent to hhs for centralized analysis . descriptive statistics were used to determine the proportion of ivig being used for patients with itp compared with all other indications . a retrospective analysis of all adult patients who received ivig for the treatment of itp at these two participating centers during the study period was performed . patients were eligible for inclusion if they met the following criteria : age 18 years or older ; a diagnosis of itp ; and receipt of at least one dose of ivig for the treatment of itp between january 1 , 2003 , and september 30 , 2012 , at either of two large tertiary care centers , hamilton health sciences ( hhs ) and london health sciences centre ( lhsc ) . the primary outcome of this study was a description of the proportion of patients using ivig in the short - term and long - term chronic itp settings , and the corresponding durations of therapy . secondary outcomes included the number of ivig infusions administered ; number of ivig infusions administered per patient ; average total amount administered per infusion , per course , and per treatment period ; average number of infusions and courses given per treatment period ; days between courses ; percentage of total ivig use ( for all indications ) that was used for itp ; estimated ivig product usage cost per year ; and future forecasts of ivig utilization from 2012 to 2018 . to determine the primary outcome of quantifying short- and long - term utilization , definitions of acute , short - term and long - term treatments were developed on the basis of clinical judgment by experts in the treatment of itp , both individually and by consensus as part of an advisory board meeting sponsored by glaxosmithkline inc . infusions of ivig were grouped to form courses , and courses were grouped to form treatment periods , which were then classified as acute , short - term , or long - term ( fig . 1summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods a data dictionary was developed for the data from each site , and variable names were mapped to standardize data coding . descriptive statistics were used to determine the proportion of ivig being used for patients with itp compared with all other indications . there were 383 adult itp patients who received a total of 2,098 ivig infusions at the two major ontario tertiary care centers participating in this retrospective study between january 1 , 2003 , and september 30 , 2012 ( tables 1 , 2 ) . comparing the two centers , in london , 547 infusions were given to 150 patients , and in hamilton , 1,551 infusions were given to 233 patients ( table 2 ) . patients received , on average , 5.5 ivig infusions ( london 3.6 , hamilton 6.7 ; range 1196 ) at 0.96 g / kg per infusion ( table 2).table 1demographic characteristics by site and overallcharacteristiclondon , n = 150hamilton , n = 233total , n = 383male sex [ n ( % ) ] 56 ( 37.3)88 ( 37.8)144 ( 37.6)age at the first ivig infusion [ years ; mean / sd ] 54.0/20.0449.5/19.1651.3/19.60patients with available weight data [ n]81114195average weight at time of first infusion [ kg ; mean / sd]80.1/18.2380.6/20.1080.4/19.30 ivig intravenous immunoglobulin , sd standard deviation age was calculated using the birth year of each patient , so for some patients the age may have been overcalculated by 1 yeartable 2intravenous immunoglobulin ( ivig ) usage : infusions and courses by site and overalllondon , n = 150hamilton , n = 233total , n = 383total number of ivig infusions 5471,5512,098number of ivig infusions per patient [ mean / sd]3.6/6.796.7/15.655.5/13.00amount per infusion [ g / kg ; mean / sd]0.91/0.19020.98/0.21810.96/0.2121total number of ivig courses4281,1751,603number of ivig courses per patient [ mean / sd]2.9/6.395.0/9.624.2/8.56number of infusions per course [ mean / sd ] 1.3/0.501.3/0.491.3/0.49total grams of ivig38,980121,096160,076 sd standard deviation infusions occurring on the same day were counted as one infusion demographic characteristics by site and overall ivig intravenous immunoglobulin , sd standard deviation age was calculated using the birth year of each patient , so for some patients the age may have been overcalculated by 1 year intravenous immunoglobulin ( ivig ) usage : infusions and courses by site and overall sd standard deviation infusions occurring on the same day were counted as one infusion when ivig infusions were combined into courses , there were a total of 1,603 courses of therapy given ( london 428 courses ; hamilton 1,175 courses ) , with a mean of 4.2 courses given per patient and 1.3 infusions given per course ( table 2 ) . when grouped into treatment periods ( as defined earlier ) , there were a total of 492 treatment periods ( london 172 , hamilton 320 ) with 264 ( 53.7 % ) defined as acute , 172 ( 35.0 % ) short - term , and 56 ( 11.4 % ) long - term treatments ( tables 3 , 4 ) . the number of days between consecutive courses within these treatment periods was 29.5 days ( london 32.2 , hamilton 28.7 ) , and the number of days between consecutive treatment periods , for those patients who received ivig in more than one treatment period , was 636 days and was similar at the two centers ( london 632 , hamilton 637).table 3intravenous immunoglobulin ( ivig ) usage categorized by treatment type by site and overalllondon , n = 150hamilton , n = 233total , n = 383total number of ivig treatment periods ( % ) 172320492 acute treatment ( % ) 101 ( 58.7)163 ( 50.9)264 ( 53.7 ) short - term treatment ( % ) 60 ( 34.9)112 ( 35.0)172 ( 35.0 ) long - term treatment ( % ) 11 ( 6.4)45 ( 14.1)56 ( 11.4)number of ivig treatment periods received per patient [ mean / sd]1.1/0.441.4/0.781.3/0.68courses per treatment period [ mean / sd]2.5/5.863.7/7.793.3/7.19infusions per treatment period [ mean / sd]3.2/6.134.8/12.854.3/11.00total grams per treatment period [ mean / sd]226.6/475.08378.4/1,121.75325.4/949.41 sd standard deviationtable 4intravenous immunoglobulin ( ivig ) utilization analysis by treatment type ( overall population)key findingslong - term treatment : 6 or more coursesshort - term treatment : 25 coursesacute treatment : 1 coursenumber of classified treatment periods ( % ) 56 ( 11.4)172 ( 35.0)264 ( 53.7)patients [ n]52152228total ivig amount per treatment type [ g ; mean / sd]1,559.5/2,482.91281.6/136.2092.1/36.11average number of infusions per treatment period [ mean / sd]19.9/27.903.8/1.661.3/0.50days of therapy per treatment period [ mean / sd]427/385.1884.2/79.44 sd standard deviation intravenous immunoglobulin ( ivig ) usage categorized by treatment type by site and overall sd standard deviation intravenous immunoglobulin ( ivig ) utilization analysis by treatment type ( overall population ) sd standard deviation the number of grams of ivig used in london and hamilton for all indications at both sites was seen to be trending upward from 144,605 g in 2003 to 245,763 g in 2012 ( see table a1 in the electronic supplementary material ) . utilization for itp also increased , but , relative to all other indications , the proportion of ivig used for adult itp patients remained relatively stable ( see table a1 ) . the proportion of ivig given to adult itp patients ranged from 5.6 to 9.1 % and was generally lower in london than in hamilton ( see table a2 in the electronic supplementary material ) . in total , 160,076 g of ivig was administered in 2,098 infusions for patients with itp , representing 7.9 % of the overall amount of ivig usage for all indications , which differed between the centers ( london 4.6 % , hamilton 10.3 % ) [ see table a1].table 5annual actual and forecasted intravenous immunoglobulin ( ivig ) short- and long - term usage in patients with immune thrombocytopeniayearamount of ivig used [ g]londonhamiltontotalshort - term treatmentlong - term treatmentshort - term treatmentlong - term treatmentshort - term treatmentlong - term treatment20031,4606002,9506,0904,4106,69020041401,2603,46711,3533,60712,61320051,18004,4227,8385,6027,83820062,3971,2603,2485,6955,6456,95520071,3001,0203,50311,1374,80312,15720081,2959602,1656,5863,4607,54620091,9771,6001,8106,5003,7878,10020102,3952,7854,5802,3906,9755,17520111,8052,9802,19012,5153,99515,4952012 2,2702,8012,6487,5424,91810,3432013 2,4143,0852,5487,4924,96210,5772014 2,5583,3682,4477,4425,00510,8102015 2,7023,6512,3477,3935,04911,0442016 2,8463,9342,2477,3435,09311,2772017 2,9904,2182,1477,2945,13711,5122018 3,1344,5012,0477,2445,18111,745 the 20122018 data were forecasted using the complete annual data from 2003 to 2011 annual actual and forecasted intravenous immunoglobulin ( ivig ) short- and long - term usage in patients with immune thrombocytopenia the 20122018 data were forecasted using the complete annual data from 2003 to 2011 this 10-year , retrospective study at two tertiary care centers analyzed data from both sites to provide comprehensive information on ivig utilization in adult itp patients . these two large centers represent 19.9 % of the total ivig utilization ( for all indications ) in the province of ontario . during the study period , there were 383 adult itp patients who received a total of 160,076 g of ivig in 2,098 infusions . compared with the london site , hamilton treated a larger cohort of itp patients with slightly more ivig infusions per patient , gave more courses per patient , had a higher proportion of itp patients relative to those with other indications , and administered more grams of ivig for itp relative to other indications , as would be expected with its highly specialized itp referral pattern . unlike acute treatments , short - term and long - term therapy with ivig may be provided to patients with the intent of using it as the sole maintenance therapy or as a longer bridging solution until another therapy works to control itp . from the present data , we can predict the impact of ivig utilization in adult itp patients requiring short- and long - term treatments ( table 5 ) . the total amount of ivig used for long - term treatment of itp at these two centers from 2012 to 2018 is forecasted to remain at approximately 11,000 g per year , at an estimated cost of $ 693,000 per year , based on a unit price of approximately $ 63 per gram in 2011 , published in bloody easy 3 by callum et al . however , short - term ivig treatments make up an additional 172 ( 35 % ) of the total number of ivig treatment periods . with 281.6 g administered per short - term treatment period , this could contribute to an estimated additional 5,000 g of ivig being used , costing $ 315,000 per year , for a total of roughly $ 1 million per year for both short- and long - term treated patients . the estimated costs from these two centers represent 19.9 % of the overall provincial utilization . assuming that the patient populations and practice patterns are similar across ontario , the overall cost in the province for short- and long - term treatments of itp may be upward of fivefold greater , or $ 5 million per year . thus , a complete and accurate cost analysis of all components was not performed , and the true cost impact of ivig to the health care system , based only on the product cost of ivig , is underestimated . as forecasted by our data , ivig usage for itp will remain a substantial burden on the public health care budget and will continue to increase by approximately 2 % per year from 2013 to 2018 . in the last 5 years , newer therapies , such as thrombopoietic mimetics , have become available to patients with itp , and the role of these agents as an alternative to ivig , particularly for short- and long - term treatments , requires consideration . while these two large centers represent 19.9 % ( london 6.5 % , hamilton 13.4 % ) of total ivig utilization ( for all indications ) in the province of ontario , it is not clear if the findings are representative of the province as a whole ( i.e. this retrospective review of comprehensive data at the london and hamilton sites has helped to characterize ivig utilization in adult itp patients and may improve the understanding of its impact on provincial utilization and inform future clinical practice and policy decisions . with the current practice pattern , short- and long - term ivig utilization for physicians treating itp and policy makers should consider the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve both patient quality of life and economic impact .

over the past two decades , it has become increasingly common for people to travel overseas in order to access medical procedures and services , including fertility treatment . people travel to receive fertility services for a broad range of push and pull reasons . some travel to receive procedures that are illegal or unavailable to them at home ; others are in search of better care , shorter waiting times , greater privacy or lower costs . the united states , for example , is a destination for some reproductive travellers , including europeans and australians seeking surrogacy or sex selection . at the same time , us citizens have travelled to india and mexico in order to access cheaper surrogacy arrangements . diasporic travellers might travel to their country of origin in order to have their cultural or religious needs met , or to access ethnically matched donors . for example , thailand banned foreigners from accessing surrogacy in 2015 , and india restricted surrogacy to heterosexual married couples in 2013 and is in the process of implementing a ban on all foreigners , except those of indian descent . nepal and mexico initially appeared to be alternative low - cost destinations , before restrictions upon foreigners access to surrogacy were enacted in 2015 and 2016 , respectively . it is impossible to tell how many people travel in order to access fertility services . in 2010 , shenfield et al estimated that there were about 2430,000 cycles of cross border fertility treatment within europe each year , involving 1114,000 patients . there is , however , no systematic collection of data , so the prevalence and outcomes of reproductive travel are unknown . while the picture of cross - border reproduction ( cbr ) is simultaneously incomplete and extraordinarily complicated , one certainty is that states can no longer assume that their citizens will seek fertility treatment and services in local , easily - regulated clinics . every country therefore faces the question of what the appropriate regulatory response , if any , should be to this dynamic and widespread ( but ultimately unquantifiable ) bypassing of domestic healthcare services and regulations . our focus in this article is on domestic responses to cbr , but it is also worth noting that there is increasing interest in the development of cross - national minimum standards . these might take the form of a hague convention on surrogacy , in order both to protect the surrogate s welfare and to avoid the creation of stateless and parentless children , or it might simply involve collaboration among members of the international federation of fertility societies in order to develop uniform clinical and safety standards. research emerging from the uk and australia suggests a number of common threads , which make them useful comparators when developing responses to cbr . in both the uk and australia , people who travel abroad for reproductive treatment may have multiple reasons for doing so , but as both countries provide high quality ivf treatment ( with varying degrees of state subsidy ) and can broadly be characterised as liberal in their access , our residents may be less likely to be motivated primarily by treatment exclusions or cost considerations . in the uk , in their qualitative study of 51 interviewees in 200910 , culley et al found that those utilising cross - border treatment were motivated by avoiding long waiting lists ; the prospect of higher success rates abroad ; the hope of receiving treatment in a less stressful environment ; and by dissatisfaction with the treatment that they had received in the uk . in australia and new zealand , a survey study in 2014 of 137 respondents ( 105 from australia and 32 from new zealand ) by rodino et al identified unavailability of treatment , treatment not permitted , long waiting lists for donor gametes and limited choice of donors as the most common motivations . in this article , we contribute to an emerging body of research into the experiences of reproductive travellers as part of our qualitative study examining cbr . in this project we are interviewing reproductive travellers from australia , as well as regulators , agencies and clinicians both within and outside australia , in order to better understand the motivations for , and experiences of cbr . at the time of writing , mid - way through a four year project , we had undertaken 54 interviews with 55 interviewees , of whom 28 had utilised cross border reproductive processes , as well as 11 facilitators and four medical practitioners engaged in cross border treatment . we are publishing these early findings in order to contribute to the development of a more nuanced analysis of cbr that explores both risks and benefits , attending to the perspectives of those who undertake it . although the research is not yet finalised , the themes that we discuss here are significant and have the potential to contribute to legal and policy developments currently being debated in australia , the uk , and elsewhere . the premise of this qualitative study , and of our argument here , is that regulators , clinicians , policymakers , and law - reformers can and should learn from the lived experiences of those who cross borders in search of reproductive treatments and services . we suggest that patients subjective experiences of risk , care , and legality differ markedly from the assumptions about patient behaviour that have tended to inform regulation in this area . we tease out this claim , and its implications for responsive regulation , across four preliminary observations . further , this division of practices is not experienced as meaningful by many participants in cbr , and is openly rejected by some . secondly , the status of the law in cbr is profoundly equivocal . even countries with extra - territorial criminal prohibitions against commercial surrogacy in practice often facilitate these prohibited arrangements through citizenship and parentage provisions . this ambiguity builds an experience of law as both there and not - there for reproductive travellers . in our interviews we have noticed that some people accessing cbr simultaneously acknowledge the presence of legal provisions ( such as prohibitions in the criminal law ; rules regarding legal parentage , or the likely unenforceability of surrogacy or egg donation contracts ) , at the same time as regarding these as abstract technicalities . time and again our participants downplayed the significance of law , as compared with real life , or a well - trodden path , in which others before them , and they themselves , have returned home with children despite the existence of restrictive legal provisions , in australia and the country of treatment . thirdly , self - sourced information , from the internet and more specifically social media such as facebook , is now the principal source of information and peer support for reproductive travellers . clinics and agencies have their own facebook pages , and there are facebook groups for different cohorts of reproductive travellers : gay dads , egg donors , egg recipients , surrogate mothers , and intended parents . personal recommendations and anecdotal evidence about the quality of care appear to matter more to patients than doctors or regulators might expect . given that many patients self - refer with the help of internet forums and facebook , reproductive travellers may be bypassing what has conventionally been a crucial source of information and support when undergoing complicated , stressful and invasive medical treatment , namely local healthcare professionals . drawing on these observations , we suggest that finding a way to ensure that as many reproductive travellers as possible access accurate , balanced information before they depart in order to make an informed assessment of overseas ( and domestic ) treatment options should be an important , but by no means straightforward , regulatory objective . we need to learn more about why some citizens of countries that pride themselves upon having among the safest and the best regulated fertility services in the world are actively bypassing those services , and seeking out treatment in apparently less well - regulated environments , often on the basis of facebook recommendations . consumer choice in cbr , or that a reform agenda should be shaped to the expectations of those who utilise cbr , especially if there is clear evidence of harmful practices or outcomes . however , we do argue that the experiences of those who travel for reproduction offer important insights that complicate common assumptions about cbr . for instance , as we shall show below , some women found the idea of altruistic surrogacy or egg donation to be more morally problematic than compensated surrogacy and egg donation . commercial involvement in assisted reproduction . to inform a more nuanced approach to the provision and regulation of fertility treatment , we must attend to the subjective experience of risk , quality , and care in cbr , especially when this involves what angela campbell calls morally ambiguous or even ostensibly self - injurious choices . in this article , we suggest that it is impossible to properly evaluate the role of law in cbr without attending to its impact upon participants lived experiences , and that , in the light of a dramatic mismatch between law s goals and reproductive travellers experiences of law , there may be grounds for some form of realignment . in many countries , including the uk and australia , a sharp distinction between altruistic and commercial arrangements has shaped the legal response to surrogacy and assisted reproduction for the past 30 years . in australia , commercial surrogacy is a criminal offence for all participants , as is trading in human gametes . in the uk , professional involvement in commercial surrogacy , egg donors in the uk have been able to receive up to 750 per cycle of donation , to include all expenses , an amount which is not intended to incentivize donation , but which is instead , in the words of the then hfea chair , lisa jardine , a level of compensation which will not deter those interested in donation but will retain donors already in the system , without attracting those who are merely financially motivated. an assumption underpinning much of the reaction to cbr in the uk and australia is that travellers go to commercial jurisdictions in order to avoid , directly or by implication , the constraints of altruistic regimes ; that is to access a more immediate or wider range of reproductive contributors who are more plentiful ( and who may also be less powerful negotiators ) because they are motivated primarily or solely by financial gain . we suggest that this flat characterisation fails to take account of the realities of payment to whom and for what , in both domestic and cross - border arrangements . in contrast , a common theme among our interviewees is a rejection of the assumption that altruistic surrogacy is morally superior to commercial surrogacy because there are fewer financial incentives . indeed , it was noted that there are aspects of altruistic surrogacy that might be described as coercive . for example , beth had undergone a radical hysterectomy as part of her treatment for cervical cancer and , after an unsuccessful surrogacy arrangement in australia , she travelled to california for an arrangement involving an egg donor and a surrogate mother . beth found the assistance of the agency in california to be vital to the whole process . she was strongly critical of the australian system and did not accept that altruistic surrogacy was less coercive than the commercial arrangements available in california : yes , but you d have a bit of hinting going on , would nt you ? if it s altruistic here and it s in - family there s a lot of hinting ; aunties are talking to sisters , friends are saying would you do it for them , what about them , why do nt you help them , or whatever . in america she s just receiving a whole stack of applications , there s no previous connection . undoubtedly the role of the agency . their role is paramount , is vital , because not only does their reputation rest on this , they have to protect the surrogate before they protect the intended parents , because if the surrogate does nt have a good experience , the surrogate s going to tell other people . there are going to be other surrogates that are wishing to go through the process to be selected . even though you think in america they re paying for them , surely they ve got loads of women , oh no , it s only a certain breed of lady that does this . so it s not just a whole line of ladies waiting around the block , it s just a very small amount of certain special ladies . yes , but you d have a bit of hinting going on , would nt you ? if it s altruistic here and it s in - family there s a lot of hinting ; aunties are talking to sisters , friends are saying would you do it for them , what about them , why do nt you help them , or whatever . in america she s just receiving a whole stack of applications , there s no previous connection . undoubtedly the role of the agency . their role is paramount , is vital , because not only does their reputation rest on this , they have to protect the surrogate before they protect the intended parents , because if the surrogate does nt have a good experience , the surrogate s going to tell other people . there are going to be other surrogates that are wishing to go through the process to be selected . even though you think in america they re paying for them , surely they ve got loads of women , oh no , it s only a certain breed of lady that does this . so it s not just a whole line of ladies waiting around the block , it s just a very small amount of certain special ladies . the distinction between regimes , and practices , characterised as altruistic and those characterised as commercial is contestable both within and across different jurisdictions . altruistic , because under canadian law the surrogate can not be paid more than her expenses . nevertheless , australians travel to canada for surrogacy ( and not , it appears , the other way around ) , and they do so in order to access paid brokering services , even though such services would be criminalised as commercial if they were operating within australia . incoherence is also present in australia s approach to the payment of egg donors , brokers involved in egg procurement and agencies who run egg so for example , eggs imported into australia from us - based services such as the world egg bank involve a cost of $ 20,000 usd to the patient for six eggs , comprising a payment of approximately $ 3000 usd to the egg donor and $ 17,000 in fees to intermediaries ( described as administrative and transport costs ) . in comparison , one us - based broker we interviewed , who matched donors and recipients and organised treatment in eight overseas destinations , reported that the egg providers in her service were paid $ 1500 usd as a price , with $ 100 spending money and $ 50 per day meal allowance on top of their travel expenses , while an additional administrative fee of $ 4500 usd was charged by her to patients ( leading to a total cost to patients of less than $ 10,000 ) . bizarrely , the first of these examples is regarded as altruistic donation under australian law , while the latter is not . there is also , as will be discussed below , an illogical and widening gulf between the active domestic medical facilitation of overseas ( compensated ) egg donation and the prohibition of domestic medical involvement in overseas ( commercial ) surrogacy . in our cbr study , we have found that the historical stigma attached to the commercialisation of reproductive contributors is not shared by intended parents . for many participants , the lack of payment to the surrogate or egg donor in domestic arrangements was believed to be unfair to her , as she was then , effectively , the only volunteer surrounded by a number of professional participants including doctors , counsellors and lawyers all of whom were acting for profit . some altruistic surrogacy arrangements ended up with an overall cost to the parents that was roughly similar to the cost of a commercial arrangement overseas . so , for example , lachlan , an interviewee with two children born through surrogacy in australia , noted that the cost of their arrangements had been about $ 80,000 for the first child and $ 50,000 for the second child , all of which went to the medical and legal professions rather than to the women who had helped them : the whole debate in terms of commercial surrogacy arrangements , if it s ever spoken about as advocates against it , it s always the quotation from someone in the legal or medical profession because they re getting sizeable rents . you almost expect to pay double in terms of the process as well it would be cheaper for us to go to india , for argument s sake , than going here in australia everybody gets paid in this , apart from the women . yes and [ our surrogate ] veronica had an old fridge and the seal was nt working , i just wanted to go out and buy a new fridge for the family . there was a decision then of okay , would that be considered a material item and will that be considered [ commercial payment when we are ] going to the courts ? so we made a decision well no , we wo nt say anything.so that s a cause of frustration . the whole debate in terms of commercial surrogacy arrangements , if it s ever spoken about as advocates against it , it s always the quotation from someone in the legal or medical profession because they re getting sizeable rents . you almost expect to pay double in terms of the process as well it would be cheaper for us to go to india , for argument s sake , than going here in australia everybody gets paid in this , apart from the women . yes and [ our surrogate ] veronica had an old fridge and the seal was nt working , i just wanted to go out and buy a new fridge for the family . there was a decision then of okay , would that be considered a material item and will that be considered [ commercial payment when we are ] going to the courts ? like lachlan , lauren , another interviewee involved in a surrogacy arrangement in australia , expressed a desire to pay her surrogate , and an anxiety about the fuzzy definition of expenses in australia : i think also a con of the altruistic system in general is that a really sort of fuzzy line of what can and ca nt be considered a surrogacy expense . so you re always sort of worrying like oh am i breaking the law by reimbursing this . there s no real sort of set list of what you can and ca nt pay for and i think that causes anxiety for surrogates as well . i think also a con of the altruistic system in general is that a really sort of fuzzy line of what can and ca nt be considered a surrogacy expense . so you re always sort of worrying like oh am i breaking the law by reimbursing this . there s no real sort of set list of what you can and ca nt pay for and i think that causes anxiety for surrogates as well . lauren also expressed a real sense of discontent about not being able to compensate her surrogate , saying:[t]here s such an inequality for giving . i find that for someone to give their body for the sake of creating another family is just i ca nt think of any greater gesture and so to not be able to return that in some way sort of makes me feel , what s the word , inadequate in a way i guess . i ll find other ways to be giving , giving with my heart , giving with my friendship and giving with my love . but if i could i d just i d give everything but yep not allowed . i would nt want to see it turn into a commercial operation but i would love a token amount just to sort of absorb some of those costs that surrogates are nt comfortable with sharing with their intended parents but also just to lighten the load on them a little bit . maybe $ 10,000 or $ 20,000 like not a huge amount of money just not as an incentive to do it but just to kind of sorry my brain s just gone dead . not to attract people for commercial reasons but more just to lighten the load on the families a little bit . so i d really love to see that happen [ t]here s such an inequality for giving . i find that for someone to give their body for the sake of creating another family is just i ca nt think of any greater gesture and so to not be able to return that in some way sort of makes me feel , what s the word , inadequate in a way i guess . i ll find other ways to be giving , giving with my heart , giving with my friendship and giving with my love . but if i could i d just i d give everything but yep not allowed . i would nt want to see it turn into a commercial operation but i would love a token amount just to sort of absorb some of those costs that surrogates are nt comfortable with sharing with their intended parents but also just to lighten the load on them a little bit . maybe $ 10,000 or $ 20,000 like not a huge amount of money just not as an incentive to do it but just to kind of sorry my brain s just gone dead . not to attract people for commercial reasons but more just to lighten the load on the families a little bit . so i d really love to see that happen lauren s preference for modest compensation , which would not be enough to attract people for commercial reasons is consistent with the findings of a recent uk survey of surrogates and intended parents , which found that the mean average of 10,00015,000 represents compensation , not payment. indeed , there is evidence that this level of compensatory payment is simply waved through in the magistrates courts in the uk ( which deal with parental orders for uk surrogacy ) , without any need for it to refer to itemized expenses . for many of our participants , being able to pay surrogates modest compensation was fairer and hence more morally satisfactory for them than asking another woman to carry a pregnancy without receiving any compensation for her time and inconvenience . for example , cybil s three - year - old son was born with the help of traditional surrogacy in western australia . cybil explained that she would have preferred to be able to pay her child s surrogate . her central rationale was that payment would create clearer boundaries , in contrast to the current system in which the definition of reasonable expenses is unclear : yeah , it would be much clearer for everyone what the boundaries are . i know that because you ca nt offer not only compensation but even gifts , like technically you ca nt even give them a bunch of flowers . i know that because you ca nt offer not only compensation but even gifts , like technically you ca nt even give them a bunch of flowers . some participants were also clear that they valued the service provision of commercial providers , not only to themselves , but also to the surrogate or egg donor . gerry , who had used a surrogacy agency in canada said : i think i just really liked the way i think we have covered this off before as well , but the agency is very respectful to the surrogate in what they call the fourth trimester , meaning , dealing with her effectively and caring in a caring way about the fact that how s she s going to feel post separation after the birth . i think for us , it s really important to have a sense that we re doing the right thing and that we re not exploiting anyone . i think i just really liked the way i think we have covered this off before as well , but the agency is very respectful to the surrogate in what they call the fourth trimester , meaning , dealing with her effectively and caring in a caring way about the fact that how s she s going to feel post separation after the birth . i think for us , it s really important to have a sense that we re doing the right thing and that we re not exploiting anyone . one of the canadian agencies we spoke to assigned a full - time support worker to each surrogate and a different employee to support each set of intended parents . the agent , sally , explained that these workers played an invaluable role in resolving issues and ensuring that disputes did not arise in the course of the relationship between the parties . in contrast , some of the intended parents who undertook unpaid surrogacy within australia felt that , after the clinic s initial counselling session , they were left on their own. for example , lachlan describes the limited service provided by the australian clinic that he and his wife attended : canberra was a funny situation where the hospital that runs the clinic , there was one lady there involved and she only dealt with surrogacy on tuesday . so if you called up on monday she would nt respond until the tuesday . if you sent an email on the wednesday you had to wait the whole week until the tuesday . as a result , it was very hard to get hold of her because there were a number of parents obviously wanting to get stuff and only tuesday . so that became in itself but you say to yourself okay , i need to call her tomorrow , type thing . canberra was a funny situation where the hospital that runs the clinic , there was one lady there involved and she only dealt with surrogacy on tuesday . so if you called up on monday she would nt respond until the tuesday . if you sent an email on the wednesday you had to wait the whole week until the tuesday . as a result , it was very hard to get hold of her because there were a number of parents obviously wanting to get stuff and only tuesday . so that became in itself but you say to yourself okay , i need to call her tomorrow , type thing . while of course not all offshore providers necessarily commit much , or any , of their commercial fee into the level of service provision offered by sally s canadian agency ( and many of our participants undertook egg donation and surrogacy abroad with no preparatory or follow up counselling ) , we draw on this contrast between a canadian agency and an australian clinic to illustrate that it is not the fee itself which determines whether practices are fair and non - exploitative yet , the legality of surrogacy arrangements are determined solely by reference to this factor . a more responsive approach for law would be to ask : what practices are beneficial and how might they be facilitated ( and perhaps also paid for ) by a regime that seeks to avoid improper inducement or impaired consent ? in the uk s altruistic only system , it is an offence for anyone other than the surrogate and the intended parents to negotiate a surrogacy arrangement on a commercial basis , and it is a criminal offence for intended parents , surrogates and agencies to advertise their willingness to participate in or facilitate surrogacy . as a result , as mcfarlane j explained in re g ( surrogacy : foreign domicile ) , the role of facilitating surrogacy arrangements has traditionally been left to groups of well - meaning amateurs. if the mischief to which the ban on commercial involvement is directed is the prevention of exploitation , the evidence is by no means clear that this is best achieved by discouraging professional agencies involvement in surrogacy . on the contrary , as natalie gamble has explained : the offer of payment does not necessarily preclude an informed choice ; and nor does the absence of payment ensure it . a much more sophisticated approach is to require regulated intermediaries to ensure that surrogates and parents are given good quality information about the risks and offered counselling to reflect on the long term commitment involved before they proceed . the offer of payment does not necessarily preclude an informed choice ; and nor does the absence of payment ensure it . a much more sophisticated approach is to require regulated intermediaries to ensure that surrogates and parents are given good quality information about the risks and offered counselling to reflect on the long term commitment involved before they proceed . if prohibitions on commercial surrogacy can be readily avoided by buying an airline ticket , with , in practice , few or no penalties for doing so and active facilitation of parenthood by the courts and immigration services , what is the status of those prohibitions ? in the uk , the law has prevented the development of commercial surrogacy brokers , but it does not treat intended parents who engage in commercial surrogacy as criminals . australia s prohibitions on the payment of gamete providers and surrogates are stricter , and in addition three australian states and territories criminalise participation in commercial surrogacy even if it occurs elsewhere . the national health and medical research council s guidelines , which apply to all fertility practitioners in australia , further state that it is ethically unacceptable to undertake or facilitate surrogate pregnancy for commercial purposes. yet prosecution , disciplinary or licensing action in response to breach or evasion of these prohibitions has been extremely rare , and uniformly unsuccessful . some of our interviewees from australian jurisdictions where criminal prohibitions on commercial surrogacy have extra - territorial effect took a calculated gamble . they understood that they were breaking the law but believed that , because so many other families had not been punished or detected , that they too would be unaffected : one is i knew that technically by the law of new south wales , we were breaking that law . [ another parent ] kind of put my feelings in that regard at ease in saying " well , if they arrest you for it , they re going to arrest hundreds of other people who have done exactly the same thing that you re thinking of doing " , which made me feel better about being more open about it . one is i knew that technically by the law of new south wales , we were breaking that law . [ another parent ] kind of put my feelings in that regard at ease in saying " well , if they arrest you for it , they re going to arrest hundreds of other people who have done exactly the same thing that you re thinking of doing " , which made me feel better about being more open about it . others who were more concerned about breaking the law undertook a variety of evasion strategies : some sought out surrogacy in canada on the basis that it too was seen as an altruistic jurisdiction ; others moved to a different australian state , like victoria , from where it is not illegal to travel for commercial surrogacy ; some simply falsified documentation in order to appear that they had done so . dian was born without a uterus and , after a negative experience in india that we come back to later , had had a child through a commercial surrogacy arrangement in the usa . she and her husband wayne were very concerned about the new south wales ban on overseas commercial surrogacy when they returned from the usa with their daughter . at the time of the interview a year later wayne was still worried that they had acted against the law , but dian was becoming less anxious : well , we did nt want to do something that broke the law . both [ our sets of our ] parents did nt know anything about it . [ we ] kept them from that ; because of that reason as well , we did nt want to worry them . wayne s brother i do nt know whether he s currently a lawyer , but he was a lawyer at one stage , and he knew what we were doing . he was extremely worried for us , but supportive , like he did nt say do nt do it. i guess i did nt want to implicate i did nt want to implicate other people . [ we ] kept them from that ; because of that reason as well , we did nt want to worry them . wayne s brother i do nt know whether he s currently a lawyer , but he was a lawyer at one stage , and he knew what we were doing . he was extremely worried for us , but supportive , like he did nt say do nt do it. i guess i did nt want to implicate i did nt want to implicate other people . participants also experienced the law of the treating country as highly ambiguous . in some respects , this is understandable , as some of the jurisdictions in which international commercial surrogacy have flourished in recent years are precisely those with a lack of clear regulation . however , we argue that this went further , to actually imbue the whole cbr process as one of law and not - law , often involving an active process of double - think . harry , a gay man who went to thailand with his partner and undertook two surrogacy arrangements simultaneously with embryos created from the same egg donor and each man s sperm , said : i mean thailand did nt have specific laws at the time . we were certainly aware that there [ were ] draft laws on the table , but at the time they did nt have those laws . however through just common practice , we actually felt that the laws were quite protective of us , of doing surrogacy . we were certainly aware that there [ were ] draft laws on the table , but at the time they did nt have those laws . however through just common practice , we actually felt that the laws were quite protective of us , of doing surrogacy . here , harry is speaking of then - current thai laws , in which the surrogate ( and her husband , if any ) were the legal parents of any child born ; a male genetic parent not married to the mother could generally not apply for custody before the child was seven years old , and any form of payment for surrogacy was unlawful . quite protective of him as an intended parent , who had no genetic relationship to one of the children , in comparison with the draft laws , passed in haste in 2015 , which expressly criminalised all paid surrogacy , and limited unpaid surrogacy to domestic arrangements involving relatives of the surrogate . protected by contracts which provided that surrogates were obligated to surrender babies to them , in contrast to domestic arrangements where she might change her mind and decide to keep it. yet , when pressed , they would acknowledge that such a contract was not likely to be enforceable . as harry explained : no , we never had a lawyer take us through contracts . so the contracts were , you know , we went through the contracts with the agent and they were in english and in thai but again , i do nt think there would be much to be gained by going through that with a lawyer because i m not sure we were happy with what was actually written in the contract , but some of it i think would nt actually be legally binding if it was actually tested in court and things like that , because of the fact that it was quite a legal grey area i think you needed to i think the important thing for intended parents is to understand what environment they were operating in , but i think because there was nt really explicit laws to refer to , that s why i think engaging with lawyers either here or there was less useful . so the contracts were , you know , we went through the contracts with the agent and they were in english and in thai but again , i do nt think there would be much to be gained by going through that with a lawyer because i m not sure we were happy with what was actually written in the contract , but some of it i think would nt actually be legally binding if it was actually tested in court and things like that , because of the fact that it was quite a legal grey area i think you needed to i think the important thing for intended parents is to understand what environment they were operating in , but i think because there was nt really explicit laws to refer to , that s why i think engaging with lawyers either here or there was less useful . likewise tom who undertook surrogacy as a single gay man in india said of the contract he signed : look , i was following what people were saying about contracts and to get the contract looked at i think some of the advice that people have shared was it costs a lot of money to have your contracts looked at and it s not actually legal or legally viable in australia anyway . so i did nt actually then i decided not to seek legal advice because i just thought it was almost pointless . look , i was following what people were saying about contracts and to get the contract looked at i think some of the advice that people have shared was it costs a lot of money to have your contracts looked at and it s not actually legal or legally viable in australia anyway . so i did nt actually then i decided not to seek legal advice because i just thought it was almost pointless . essentially then , legal advice is almost pointless for an unviable contract , which nevertheless is capable of going some way to protect the rights and liabilities of the parties throughout the arrangement . one of the preconditions for the granting of a parental order to intended parents through surrogacy in both the uk and australia is that no payment should have been made other than expenses reasonably incurred. in the uk , the courts have the power to retrospectively authorise payments made in excess of reasonable expenses . because the child s welfare is the paramount consideration when deciding whether to make a parental order , the uk courts are effectively presented with a fait accompli : if the child s settled home is with the intended parents , a parental order will invariably be in his or her best interests . thus , uk courts routinely authorise payments made to the surrogate mother , even when they vastly exceed any plausible expenses . at the same time , the statutory prohibition on payments may discourage some intended parents , concerned about having their financial arrangements scrutinised , from applying for parental orders . the legislation therefore fails to stop payments , while failing to provide legal certainty about what is and is not allowed , and also potentially deterring the acquisition of legal parenthood . in contrast , in australia there is no discretion in the surrogacy parentage transfer regimes controlled by state and territory courts . thus intended parents in commercial arrangements are not legal parents , and can never have parentage transferred . excluded from this process , parents can nevertheless approach the federal family court seeking parental responsibility orders ( a lesser form of recognition than legal parentage ) and , in a handful of these cases , the court has also perversely , this means that for some male genetic parents in commercial surrogacy their legal parentage has been secured more readily than it has been for intended parents in domestic unpaid arrangements . to add to the confusion , other judges of the court have refuted this approach as a misreading of the legislation , leading to an as yet unresolved judicial lottery . in a similar vein , in both the uk and australia , as with canada and many other altruistic jurisdictions , the state has actively acquiesced in facilitating the acquisition of citizenship for children born as a result of overseas commercial surrogacy arrangements . this is the case in australia even when the intended parents are resident in a state in which they are subject to extra - territorial criminalisation . in a parallel vein , potentially unlawful payments by australian parents to egg providers abroad are not examined as part of state processes granting citizenship or parental responsibility in surrogacy arrangements . gabriel , a gay man who undertook surrogacy in mexico said : it s obvious the australian government s allowing it to happen . if they started doing that then they re really saying , no you re not allowed to do it at all because it s illegal. but the government s not doing that , they re letting it happen . if they started doing that then they re really saying , no you re not allowed to do it at all because it s illegal. but the government s not doing that , they re letting it happen . in this way , as discussed later , intended parents understood themselves as both legal parents and not - legal parents under australian law : their children were granted passports and in practice they could use their overseas birth certificates , at the same time as those birth certificates do not record parentage for the purposes of australian law and their parental relationship had not been legally formalised . if it is possible to establish a working parental relationship in the absence of a parental order , many struggle to see the point of going through yet more expensive red - tape. indeed , some australian facilitators , such as alec , actively discouraged parents from doing so because , unlike the uk , the result was not guaranteed:if we had a system here where it was a guaranteed process and a simple process , of course i 'd be recommending everybody get them , but 95 per cent [ do nt ] get them , because we get by without it . why would we want to spend a year and a half in the court system and spend $ 30,000 on this stuff ? if we had a system here where it was a guaranteed process and a simple process , of course i 'd be recommending everybody get them , but 95 per cent [ do nt ] get them , because we get by without it . why would we want to spend a year and a half in the court system and spend $ 30,000 on this stuff ? the practical consequence of people bypassing the formal transfer of parenthood may be that the courts have to step in at a later point , perhaps following parental separation or death , in order to resolve some of the difficulties that may arise when a child s social parents are not also her legal parents . in practice , de facto tolerance of the evasion of reproductive travellers may be inevitable ; as one of culley et al s respondents put it : what are you going to do , confiscate their passports? but whether characterised as a pluralistic safety value or as out - and - out hypocrisy , there is at the very least a mixed message being sent about the status of the extra - territorial prohibition of international commercial surrogacy . there is considerable evidence that the internet is by far the most important source of information for reproductive travellers , and that healthcare professionals in their home country are very rarely people s primary source of information and advice . in the uk , hanefeld at al interviewed 77 outbound patients and found that most had identified a specific clinic or provider through facilitators in the uk or online forums . in australia , hammarberg et al s study found that those travelling abroad for surrogacy sourced most of their information online and from other parents through surrogacy. our preliminary findings strongly reflect this trend , with patients actively amassing information from a variety of internet sources . the quality and accuracy of online material is decidedly variable , and much of it is unverifiable , yet our participants regularly referred to this process as research. potential travellers visit clinics websites , but more significantly , seek advice and information from online forums , social media , and several layers of intermediaries variously described as facilitators , brokers , consultants and agencies . many of these are run by people who have previously undergone fertility treatment abroad , who go on to set - up businesses through which they can share , and make a living from , their personal experience and knowledge of overseas fertility services . in our study , potential reproductive travellers had sought peer - to - peer information , advice and support from fellow members of internet forums and facebook groups . this often involves users requesting information about other people s experiences at specific overseas clinics or agencies , which fellow forum - users will answer . as tom they were really popular in terms of people communicating and providing stories and getting updated information and asking questions , in mostly a respectful manner most of the time so it was really good well , now i guess the facebook groups have taken over the yahoo groups . some of the facebook groups are country - specific , so there s one or two for nepal , or there used to be one for thailand they were really popular in terms of people communicating and providing stories and getting updated information and asking questions , in mostly a respectful manner most of the time so it was really good well , now i guess the facebook groups have taken over the yahoo groups . some of the facebook groups are country - specific , so there s one or two for nepal , or there used to be one for thailand once people have decided to travel abroad for treatment , they often rely upon personal recommendations in order to select a particular clinic . in our cbr study , olivia chose a us - based clinic for egg donation based upon the recommendation of a real life friend : because obviously going to another country i had absolutely no idea . you do nt know whether the websites are actually legitimate , i mean there is so much fraud online and the amount of money that you re talking about with doing ivf overseas . even having a skype conversation or a telephone conversation you ve got no guarantee . so i felt i would not have gone overseas unless i knew someone who had been at that clinic . you do nt know whether the websites are actually legitimate , i mean there is so much fraud online and the amount of money that you re talking about with doing ivf overseas . even having a skype conversation or a telephone conversation you ve got no guarantee . so i felt i would not have gone overseas unless i knew someone who had been at that clinic . in one respect , olivia s experience is unusual : while all of our interviewees had , like olivia , relied upon personal recommendations , most of these had been from cyber friends . cheryl , who travelled to india for surrogacy noted that all of these clinics , american , indian , greek , they ve all got slick websites and you they all sound amazing. cheryl said that what tipped the balance for her was a very honest forum , even though it was hosted by the clinic itself : i did an awful lot of research . you get a very , very honest account of the experiences that lots and lots and lots of people had had using this clinic . you could send them private messages , so being able to message them and say i m from australia as well and i ve got a question about this that was something that made me well it tipped my decision in their favour . you get a very , very honest account of the experiences that lots and lots and lots of people had had using this clinic . you could send them private messages , so being able to message them and say i m from australia as well and i ve got a question about this that was something that made me the central role played by informal online networks and patient testimonies highlights , as hanefeld et al put it , the importance of hard information to patients. the information that patients are seeking online thus appears to be qualitatively different from what doctors and regulators might expect . success rates and professional accreditations matter , but patients are often more interested in what treatment will feel like : the attitude of staff , the clinic environment , the gut feelings other patients had had about their treatment . whether it feels safe to be treated somewhere is not the result of evaluating technical data from the clinic or local regulator , but comes instead from personal recommendations and first person narratives , largely gathered online . you get on the websites and you get on the blogs and all that sort of stuff and that provides you with far more of an education , be it right information or not far more education and reassurance than any doctor s given me ; because you re talking to other women that have been there , done that [ y]ou do your own research . you get on the websites and you get on the blogs and all that sort of stuff and that provides you with far more of an education , be it right information or not far more education and reassurance than any doctor s given me ; because you re talking to other women that have been there , done that although the internet is the primary source of soft information about the experience of fertility treatment , it is also worth noting the growing popularity of fertility and surrogacy fairs or roadshows , which are huge exhibitions in which visitors can gather information from regulated clinics and the local regulator , while also meeting people from overseas clinics and alternative therapists . discussions about one s need for fertility services have emerged from the strict confidentiality of the doctor - patient relationship to become instead a marketing opportunity for private providers . but while these fairs provide clinics with the chance to sell their services , potential patients also value the opportunity to find out if they feel a connection with the clinic staff and their approach . social media websites like facebook also play an ambiguous role as sources of information and support . facebook friends with an overseas agent or clinic , and gain considerable reassurance from the posts of other patients or clients . the distinction between peer support and viral marketing from commercial brokers in such a setting is not always clear . in the context of travel for cosmetic surgery , holliday et al have pointed out that , to agents , facebook is an important marketing tool , whereas patients did not recognise ( or refused ) the marketing definition of agents facebook pages and saw them instead as open forums for discussion. the familiarity of facebook allows it to be both a source of peer support and authentic advice ( from the point of view of patients ) , and a staggeringly successful ( because invisible ) advertising mechanism for agents . in our study , there were also instances where facebook was used by intended parents to breach the privacy of egg donors or surrogates , for example to identify and approach these women privately , or to seek and store information about them without permission . umar and gabriel were at the beginning of a surrogacy and egg donor arrangement in mexico . although egg donation is anonymous in mexico , umar had found their egg donor s facebook profile because the clinic had given them the egg donor s name , without her knowledge . gabriel said : yeah , umar has already cut out the pictures and cropped them . like i said we ll probably inform the egg donor or just message her on facebook to say thank you and this is what you ve done for us . like i said we ll probably inform the egg donor or just message her on facebook to say thank you and this is what you ve done for us . one response to the issues raised by overseas travel is to try to educate people about the implications of undertaking cbr , by providing information about clinical standards of care and the legal status of children born from such arrangements . counsellors and patient support group representatives interviewed by culley et al in the uk , for example , thought the only feasible response to reproductive travel is to educate people , and ensure that they go into it with their eyes open and fully aware of the implications. yet , it is hard to intervene in order to provide high quality information when people self - refer to overseas clinics , on the basis of facebook recommendations . in the uk , considering fertility treatment abroad : issues and risks. this politely suggests a number of issues that people should proven record on quality and standards. there is , of course , no guarantee that anyone contemplating treatment outside the uk will read this page , or follow its advice . in the australian system with seven jurisdictions and only two official regulators , varta and the wa reproductive technology council , official information is even less widely available than in the uk , leaving australians even less able to access accurate and reliable advice . there is also a mismatch between the view that healthcare professionals have a key role to play in educating people about possible risks , and the fact that healthcare professionals are seldom the first port of call for information . if people seek out information via google , facebook , and internet chatrooms , there may be little opportunity for clinicians to educate them about risks and potential pitfalls , a problem which is , as we see in the following section , exacerbated by domestic laws which criminalise cbr , and hence deter patients from incriminating themselves in front of healthcare professionals . the reordering of sources of information about fertility treatment from the medical profession to the internet is significant . it suggests that patients are increasingly willing to bypass local healthcare professionals in order to take matters into their own hands. even if doctors are still trusted sources of information , there are other factors which make the internet an attractive source of information . when people are seeking out information about treatments that may be unlawful at home , or which are stigmatised restrictive legal provisions then actively contribute to the bypassing of medical professionals , concerned about their professional registration , as a source of advice and support . shenfield et al note in the eshre good practice guide to cross border reproductive care that , [ c]ollaboration between the home practitioner and the receiving center offers the best chance of optimal care for the cross border patient , but add that this may pose a problem where it is forbidden for doctors to give information about alternatives that are not legal in the country of residence of the patient. indeed , laws which inhibit doctors from offering advice and assistance to patients who are contemplating fertility treatment overseas create a professional conflict of interest in which doctors must choose between making the care of their patient their first concern , which would militate in favour of providing advice and support , and not being seen to endorse or support illegal behaviour , which might instead prompt them to leave patients to their own devices in hammarberg et al s survey study of australians travelling abroad for surrogacy , fewer than half of the 249 intended parents who responded had sought information from australian ivf professionals and of those who did , around one - third reported a negative reaction . cheryl said : the main ivf doctor that i saw here in sydney was very against offshore surrogacy . oh that s terrible . these are women that are terribly exploited and you ll go over there and you ll get a disease and you ll be in some terrible baby factory and what not. anyway , she said those things and then i just shut down that dialogue with her . the main ivf doctor that i saw here in sydney was very against offshore surrogacy . these are women that are terribly exploited and you ll go over there and you ll get a disease and you ll be in some terrible baby factory and what not. anyway , she said those things and then i just shut down that dialogue with her . some reported that fertility doctors were unwilling to provide any form of information at all . dian , for instance , said : then [ my partner ] wayne mentioned the word the phrase it s illegal , its basically , the door was shut at that point here in australia . facilitating commercial surrogacy has prevented fertility experts in australia from engaging in even basic information - giving to their patients , such as what is involved in safe egg stimulation and embryo transfer protocols , or the risks of departing from these protocols . it has also prevented the provision of basic fertility testing or preparatory care ( such as checking hormone levels , ovarian reserve , or sperm counts ) for patients who are planning to undertake treatment abroad . for dian this meant that she had travelled to india twice and undergone egg retrieval , despite the fact that a simple hormone test could have told her in advance that ivf using her own eggs would be unlikely to work.so i got there by myself . the second or third day , while i maybe the second day after arriving in new delhi i had a consultation with dr i ( india ) . she said that my [ hormone ] level was too high ; too high or too low , i ca nt remember , but as far as she s concerned it would just be a complete waste of time to do an ivf treatment on me.so i was quite devastated , but then she said that we should just go ahead with an egg donor . at this stage [ wayne ] was nt there , and she wanted me to make the decision right there and then . so i got there by myself . the second or third day , while i maybe the second day after arriving in new delhi she said that my [ hormone ] level was too high ; too high or too low , i ca nt remember , but as far as she s concerned it would just be a complete waste of time to do an ivf treatment on me . so i was quite devastated , but then she said that we should just go ahead with an egg donor . at this stage [ wayne ] was nt there , and she wanted me to make the decision right there and then . both clinically and emotionally , we suggest that this was an adverse experience that could have been avoided if dian had undergone basic preliminary investigations and preparatory care at home , before travelling to india . professionals within australia have also expressed the concern that media coverage of cross border surrogacy , coupled with difficulties in accessing frank advice from domestic healthcare professionals once commercial surrogacy is mentioned , has meant that some women are travelling abroad for surrogacy as a fertility cure , when they are , in fact , capable of carrying a pregnancy . in contrast to surrogacy , the australian prohibitions on commercial trading in gametes are far more specifically worded , criminalising only the giving and receipt of valuable consideration , rather than potentially implicating anyone involved in facilitating the practice . this has meant that some fertility doctors are willing to recommend overseas egg donation and we found that some even facilitate shared care , with the provision of scans and tests domestically before the woman travels , as well as follow up care . thus , if dian had told her fertility doctor that she was travelling to receive paid egg donation rather than to pursue commercial surrogacy , she would be likely to have received domestic medical assistance and advice . in a parallel vein , general practitioners ( who are not covered by the same ethical guidelines as fertility specialists ) were reported by our interviewees to be assisting patients with blood and semen tests in preparation for overseas surrogacy , as well as with prescription medications and blood tests in advance and pregnancy tests post - travel for those receiving egg donation abroad . after years of unsuccessful ivf in australia , leah travelled to greece to undergo ivf and egg donation . she explained that her gp in australia was helping her : so how we coordinate it is that he tells me what i need . so i just have to make sure that he s written down for me the correct spelling of the medication that i need , what it s for . then i just tell him a little bit about the background to why we re doing it and then he ll write me an australian script for it , so that s how we re coordinating it at the moment . so i just have to make sure that he s written down for me the correct spelling of the medication that i need , what it s for . then i just tell him a little bit about the background to why we re doing it and then he ll write me an australian script for it , so that s how we re coordinating it at the moment . the implications of this finding need further exploration , but at a minimum suggest that access to local medical care and advice for reproductive travellers is often filtered through general practitioners rather than ivf clinicians , and is more effectively obtained for australian women who are seeking egg donation compared to those seeking surrogacy . it would be possible to regard reproductive travel as an aberration , relied upon in extremis by people who are prevented , either by law or de facto , from accessing reproductive services at home . in response to the increasing numbers of people travelling for reproductive purposes , enabling more people to access local fertility services but while we would support measures to improve access to services , not least because these might also meet the needs of those who can not afford to travel , we would like to suggest that we should also be interested in what local fertility providers and regulators can learn from the experiences of reproductive travellers . as discussed in this article , our preliminary fieldwork has thrown up four themes : that the legal distinction between altruistic and commercial gamete donation and surrogacy is increasingly unsustainable ; that role of the law in cbr is profoundly equivocal ; that facebook is now the principal source of information and peer support for reproductive travellers and lastly , that domestic reproductive service providers are often sidestepped . each theme suggests that the cross - border reproductive traveller does not conform to regulators assumptions about patient behaviour . implicit here is the premise that the law will best protect patients through discouraging international travel . here , the patient experience diverges . it is clear that patients paying for treatment overseas feel as though they are more in control of their treatment , and that , in contrast to their experience of domestic fertility services , they do not have to be grateful for what they receive . indeed , in many of our interviews patients have praised the standard of care they received overseas , considering it superior to that available at home . opting out of local , regulated services is not necessarily always an unwelcome last resort then , but may have positive advantages for some patients . if this is the case , we should be interested in listening to what patients say is overseas clinics may offer more support and more contact time with clinicians and nursing staff , as well as a greater choice of donors or surrogates . overseas clinics also make excellent use of social media to contact patients and to facilitate a high quality care experience. nicky hudson and lorraine culley , for example , found that treatment overseas gave their interviewees active involvement in deciding on treatment protocols , choice about donors , control over the timing of treatment and good access to the clinician leading their care. we should be interested in this apparent disjunction between what matters most to would - be parents and what matters most to doctors , regulators and legislators . in van hoof et al s study of internet forums , in which dutch patients shared their experiences of having received ivf treatment in belgium , respect for the person behind the patient was identified as the main reason for the patients belief that the quality of care was higher in belgium . van hoof et al comment that patient centred care is generally seen as a dimension of care that has nothing to do with effectiveness and efficiency , whereas forum users considered that the central position of the patient [ was ] key for every dimension of good quality of care. this is a revealing illustration of the gap between the normal markers for success , as judged objectively , and the marker of high - quality care for patients , in which patient - centred care is not just a desirable extra , but is central to every aspect of what matters to them . for example , rosalind , who travelled to greece to use donated eggs observed : weve spent nearly 60,000 dollars in australia and not one of my doctors have called and said , hey how are you or let s do this now or anything like that.so that s why we chose him because he just took this real personal approach with us and it was him that was in contact with us . we ve spent nearly 60,000 dollars in australia and not one of my doctors have called and said , hey how are you or let s do this now or anything like that.so that s why we chose him because he just took this real personal approach with us and it was him that was in contact with us . clinicians , regulators and politicians have tended to assume that patients choose clinics on the basis of their success rates and the costs of treatment . like to be treated there with scepticism because , by definition , anecdotal evidence is not evidence at all . but while it may not be statistically significant , in practice , the anecdotal clearly matters to patients . patients want to receive treatment from caring clinicians , and they want to feel a connection with their donor or surrogate . in our interviews , we note how frequently the language of intimate relationships is invoked in relation to donors and surrogates . clinicians might assume that patients are principally interested in the health and screening results of potential donors and/or surrogates , whereas patients may be looking for an emotional bond with them . the most striking example of this from our cbr study was an intended mother who explained that she and her partner had fallen in love with an egg donor , and that , for them , this trumped the discovery that she was a carrier of the tay sachs gene . a gap between what matters to experts and what matters to patients is also evident in relation to the law , including but not limited to criminal prohibitions , citizenship and the rules of legal parentage . for example , in our study , it is striking how few intended parents of children born through surrogacy had sought legal formalisation of their relationship . tom , who had undertaken surrogacy in india said : im not going to the family court [ to seek parental responsibility ] . i m fine with [ the indian birth certificate ] as being a formal document . because my name is on it i feel that gives me the parental rights that i need even though legally i know i m not actually the parent in australia . that s the weird thing is that in india i m the legal parent and the surrogate has no parental rights and i come back to australia and i do nt have any parental rights but the surrogate has got all the parental rights , and she s not even actually living in the country . ( emphasis added ) i m not going to the family court [ to seek parental responsibility ] . i m fine with [ the indian birth certificate ] as being a formal document . because my name is on it i feel that gives me the parental rights that i need even though legally i know i m not actually the parent in australia . that s the weird thing is that in india i m the legal parent and the surrogate has no parental rights and i come back to australia and i do nt have any parental rights but the surrogate has got all the parental rights , and she s not even actually living in the country . ( emphasis added ) many interviewees appeared unconcerned about , or actively avoided knowing about the risks to which their family was exposed as a result of their lack of legal parentage . what mattered instead was the lived experience of the practicalities of new parenthood : being able to get back into australia , obtain a medicare card to access medical treatment for the child , and later to enrol him or her in school . this was particularly the case for those whose names appeared on the foreign - issued birth certificate , who had encountered a no - questions - asked response from authorities . charlotte , a parent through surrogacy and egg donation in the us , described her decision not to seek legal advice as i just assumed everything would be fine because i m on their birth certificate. in our interviews , it was common for genetic and non - genetic parents to take such a view , relying upon a document that they acknowledge , when pushed , would not be likely to stand up to challenge . like the unenforced or ambiguous laws of the treating country , and the blind - eye approach of domestic regulators , the birth certificate both is , and is not , legal protection. we suggest that any attempt to further reform or refine laws relating to cbr must attend to the subjective and lived experience of law , which like the personal experience of treatment , may stand in contradiction to policy maker s understandings of participants motivations or of law s effect upon behaviour . these experiences can usefully be explored to complicate , for example , the dominant assumption that payment in reproductive endeavours is exploitative and inevitably impairs informed consent . our interviews explore both cross - border compensated surrogacy and egg donation and domestic uncompensated arrangements . though we are still at the beginning of our work , it is already clear that the picture is far more complex than might have been expected , both morally and legally . indeed , as this preliminary snapshot from our interviews has shown , some participants chose compensated arrangements precisely because they considered an uncompensated arrangement to be ethically questionable . perhaps more importantly , our research has begun to demonstrate that the incentive of legal parentage under australian law , or threat of its denial , will not drive intended parents away from paid surrogacy or egg donation . the law , as it currently stands , works in obstructive and confounding ways that push people outside its reach or encourage them to ignore its limits and this is ultimately counter - productive and likely to cause harm to the participants and their future children . there is a still a great deal to learn about cross - border reproductive treatment . large - scale bypassing of domestic fertility services is a relatively new phenomenon and we have no reliable way of recording its outcomes . in order to ensure that the law is capable of minimising the risk of harm to all participants , we argue that it is necessary to learn from the experience of those undertaking cross - border reproductive treatment , at and outside the limits of the law .

abstractdrawing upon the preliminary findings of an australian empirical project on cross - border reproduction ( cbr ) , this article argues that regulators and policymakers could learn from the experiences of those who travel overseas in order to access fertility treatment and surrogacy . it makes four principal observations . first , the distinction between so - called altruistic and commercial gamete donation and surrogacy is increasingly unsustainable and is not experienced as meaningful by many participants in cbr . secondly , the status of the law in cbr is profoundly equivocal ; for participants it is often there and not there at the same time . thirdly , self - sourced information , from the internet and more specifically social media such as facebook , is now the principal source of information and peer support for reproductive travellers . fourthly , and relatedly , domestic reproductive services providers are often sidestepped . if one of the goals of regulation is to minimise the risk of harm to participants , it is not clear that it is currently achieving this aim , and this article argues that any reforms will only work if they are more responsive to the reality of cbr .

I. Introduction II. ALTRUISTIC OR COMMERCIAL: AN UNTENABLE DISTINCTION? III. EVASION, TOLERATION, AND AMBIGUITY IN LAW IV. INTERNET-ASSISTED REPRODUCTION AND THE UBIQUITY OF FACEBOOK V. THE ROLE OF DOMESTIC REPRODUCTIVE SERVICE PROVIDERS VI. CONCLUSION: LEARNING LESSONS FROM THE EXPERIENCE OF CROSS-BORDER REPRODUCTIVE TREATMENT

over the past two decades , it has become increasingly common for people to travel overseas in order to access medical procedures and services , including fertility treatment . at the same time , us citizens have travelled to india and mexico in order to access cheaper surrogacy arrangements . diasporic travellers might travel to their country of origin in order to have their cultural or religious needs met , or to access ethnically matched donors . it is impossible to tell how many people travel in order to access fertility services . while the picture of cross - border reproduction ( cbr ) is simultaneously incomplete and extraordinarily complicated , one certainty is that states can no longer assume that their citizens will seek fertility treatment and services in local , easily - regulated clinics . our focus in this article is on domestic responses to cbr , but it is also worth noting that there is increasing interest in the development of cross - national minimum standards . these might take the form of a hague convention on surrogacy , in order both to protect the surrogate s welfare and to avoid the creation of stateless and parentless children , or it might simply involve collaboration among members of the international federation of fertility societies in order to develop uniform clinical and safety standards. in this article , we contribute to an emerging body of research into the experiences of reproductive travellers as part of our qualitative study examining cbr . in this project we are interviewing reproductive travellers from australia , as well as regulators , agencies and clinicians both within and outside australia , in order to better understand the motivations for , and experiences of cbr . we are publishing these early findings in order to contribute to the development of a more nuanced analysis of cbr that explores both risks and benefits , attending to the perspectives of those who undertake it . although the research is not yet finalised , the themes that we discuss here are significant and have the potential to contribute to legal and policy developments currently being debated in australia , the uk , and elsewhere . the premise of this qualitative study , and of our argument here , is that regulators , clinicians , policymakers , and law - reformers can and should learn from the lived experiences of those who cross borders in search of reproductive treatments and services . we suggest that patients subjective experiences of risk , care , and legality differ markedly from the assumptions about patient behaviour that have tended to inform regulation in this area . further , this division of practices is not experienced as meaningful by many participants in cbr , and is openly rejected by some . secondly , the status of the law in cbr is profoundly equivocal . this ambiguity builds an experience of law as both there and not - there for reproductive travellers . in our interviews we have noticed that some people accessing cbr simultaneously acknowledge the presence of legal provisions ( such as prohibitions in the criminal law ; rules regarding legal parentage , or the likely unenforceability of surrogacy or egg donation contracts ) , at the same time as regarding these as abstract technicalities . thirdly , self - sourced information , from the internet and more specifically social media such as facebook , is now the principal source of information and peer support for reproductive travellers . given that many patients self - refer with the help of internet forums and facebook , reproductive travellers may be bypassing what has conventionally been a crucial source of information and support when undergoing complicated , stressful and invasive medical treatment , namely local healthcare professionals . drawing on these observations , we suggest that finding a way to ensure that as many reproductive travellers as possible access accurate , balanced information before they depart in order to make an informed assessment of overseas ( and domestic ) treatment options should be an important , but by no means straightforward , regulatory objective . consumer choice in cbr , or that a reform agenda should be shaped to the expectations of those who utilise cbr , especially if there is clear evidence of harmful practices or outcomes . however , we do argue that the experiences of those who travel for reproduction offer important insights that complicate common assumptions about cbr . to inform a more nuanced approach to the provision and regulation of fertility treatment , we must attend to the subjective experience of risk , quality , and care in cbr , especially when this involves what angela campbell calls morally ambiguous or even ostensibly self - injurious choices . in this article , we suggest that it is impossible to properly evaluate the role of law in cbr without attending to its impact upon participants lived experiences , and that , in the light of a dramatic mismatch between law s goals and reproductive travellers experiences of law , there may be grounds for some form of realignment . in many countries , including the uk and australia , a sharp distinction between altruistic and commercial arrangements has shaped the legal response to surrogacy and assisted reproduction for the past 30 years . in the uk , professional involvement in commercial surrogacy , egg donors in the uk have been able to receive up to 750 per cycle of donation , to include all expenses , an amount which is not intended to incentivize donation , but which is instead , in the words of the then hfea chair , lisa jardine , a level of compensation which will not deter those interested in donation but will retain donors already in the system , without attracting those who are merely financially motivated. an assumption underpinning much of the reaction to cbr in the uk and australia is that travellers go to commercial jurisdictions in order to avoid , directly or by implication , the constraints of altruistic regimes ; that is to access a more immediate or wider range of reproductive contributors who are more plentiful ( and who may also be less powerful negotiators ) because they are motivated primarily or solely by financial gain . we suggest that this flat characterisation fails to take account of the realities of payment to whom and for what , in both domestic and cross - border arrangements . the distinction between regimes , and practices , characterised as altruistic and those characterised as commercial is contestable both within and across different jurisdictions . nevertheless , australians travel to canada for surrogacy ( and not , it appears , the other way around ) , and they do so in order to access paid brokering services , even though such services would be criminalised as commercial if they were operating within australia . for many participants , the lack of payment to the surrogate or egg donor in domestic arrangements was believed to be unfair to her , as she was then , effectively , the only volunteer surrounded by a number of professional participants including doctors , counsellors and lawyers all of whom were acting for profit . some participants were also clear that they valued the service provision of commercial providers , not only to themselves , but also to the surrogate or egg donor . while of course not all offshore providers necessarily commit much , or any , of their commercial fee into the level of service provision offered by sally s canadian agency ( and many of our participants undertook egg donation and surrogacy abroad with no preparatory or follow up counselling ) , we draw on this contrast between a canadian agency and an australian clinic to illustrate that it is not the fee itself which determines whether practices are fair and non - exploitative yet , the legality of surrogacy arrangements are determined solely by reference to this factor . in the uk s altruistic only system , it is an offence for anyone other than the surrogate and the intended parents to negotiate a surrogacy arrangement on a commercial basis , and it is a criminal offence for intended parents , surrogates and agencies to advertise their willingness to participate in or facilitate surrogacy . if prohibitions on commercial surrogacy can be readily avoided by buying an airline ticket , with , in practice , few or no penalties for doing so and active facilitation of parenthood by the courts and immigration services , what is the status of those prohibitions ? others who were more concerned about breaking the law undertook a variety of evasion strategies : some sought out surrogacy in canada on the basis that it too was seen as an altruistic jurisdiction ; others moved to a different australian state , like victoria , from where it is not illegal to travel for commercial surrogacy ; some simply falsified documentation in order to appear that they had done so . at the time of the interview a year later wayne was still worried that they had acted against the law , but dian was becoming less anxious : well , we did nt want to do something that broke the law . in some respects , this is understandable , as some of the jurisdictions in which international commercial surrogacy have flourished in recent years are precisely those with a lack of clear regulation . harry , a gay man who went to thailand with his partner and undertook two surrogacy arrangements simultaneously with embryos created from the same egg donor and each man s sperm , said : i mean thailand did nt have specific laws at the time . quite protective of him as an intended parent , who had no genetic relationship to one of the children , in comparison with the draft laws , passed in haste in 2015 , which expressly criminalised all paid surrogacy , and limited unpaid surrogacy to domestic arrangements involving relatives of the surrogate . so the contracts were , you know , we went through the contracts with the agent and they were in english and in thai but again , i do nt think there would be much to be gained by going through that with a lawyer because i m not sure we were happy with what was actually written in the contract , but some of it i think would nt actually be legally binding if it was actually tested in court and things like that , because of the fact that it was quite a legal grey area i think you needed to i think the important thing for intended parents is to understand what environment they were operating in , but i think because there was nt really explicit laws to refer to , that s why i think engaging with lawyers either here or there was less useful . so the contracts were , you know , we went through the contracts with the agent and they were in english and in thai but again , i do nt think there would be much to be gained by going through that with a lawyer because i m not sure we were happy with what was actually written in the contract , but some of it i think would nt actually be legally binding if it was actually tested in court and things like that , because of the fact that it was quite a legal grey area i think you needed to i think the important thing for intended parents is to understand what environment they were operating in , but i think because there was nt really explicit laws to refer to , that s why i think engaging with lawyers either here or there was less useful . one of the preconditions for the granting of a parental order to intended parents through surrogacy in both the uk and australia is that no payment should have been made other than expenses reasonably incurred. at the same time , the statutory prohibition on payments may discourage some intended parents , concerned about having their financial arrangements scrutinised , from applying for parental orders . the legislation therefore fails to stop payments , while failing to provide legal certainty about what is and is not allowed , and also potentially deterring the acquisition of legal parenthood . excluded from this process , parents can nevertheless approach the federal family court seeking parental responsibility orders ( a lesser form of recognition than legal parentage ) and , in a handful of these cases , the court has also perversely , this means that for some male genetic parents in commercial surrogacy their legal parentage has been secured more readily than it has been for intended parents in domestic unpaid arrangements . in this way , as discussed later , intended parents understood themselves as both legal parents and not - legal parents under australian law : their children were granted passports and in practice they could use their overseas birth certificates , at the same time as those birth certificates do not record parentage for the purposes of australian law and their parental relationship had not been legally formalised . the practical consequence of people bypassing the formal transfer of parenthood may be that the courts have to step in at a later point , perhaps following parental separation or death , in order to resolve some of the difficulties that may arise when a child s social parents are not also her legal parents . in practice , de facto tolerance of the evasion of reproductive travellers may be inevitable ; as one of culley et al s respondents put it : what are you going to do , confiscate their passports? but whether characterised as a pluralistic safety value or as out - and - out hypocrisy , there is at the very least a mixed message being sent about the status of the extra - territorial prohibition of international commercial surrogacy . there is considerable evidence that the internet is by far the most important source of information for reproductive travellers , and that healthcare professionals in their home country are very rarely people s primary source of information and advice . some of the facebook groups are country - specific , so there s one or two for nepal , or there used to be one for thailand they were really popular in terms of people communicating and providing stories and getting updated information and asking questions , in mostly a respectful manner most of the time so it was really good well , now i guess the facebook groups have taken over the yahoo groups . some of the facebook groups are country - specific , so there s one or two for nepal , or there used to be one for thailand once people have decided to travel abroad for treatment , they often rely upon personal recommendations in order to select a particular clinic . you get on the websites and you get on the blogs and all that sort of stuff and that provides you with far more of an education , be it right information or not far more education and reassurance than any doctor s given me ; because you re talking to other women that have been there , done that although the internet is the primary source of soft information about the experience of fertility treatment , it is also worth noting the growing popularity of fertility and surrogacy fairs or roadshows , which are huge exhibitions in which visitors can gather information from regulated clinics and the local regulator , while also meeting people from overseas clinics and alternative therapists . social media websites like facebook also play an ambiguous role as sources of information and support . the distinction between peer support and viral marketing from commercial brokers in such a setting is not always clear . the familiarity of facebook allows it to be both a source of peer support and authentic advice ( from the point of view of patients ) , and a staggeringly successful ( because invisible ) advertising mechanism for agents . one response to the issues raised by overseas travel is to try to educate people about the implications of undertaking cbr , by providing information about clinical standards of care and the legal status of children born from such arrangements . counsellors and patient support group representatives interviewed by culley et al in the uk , for example , thought the only feasible response to reproductive travel is to educate people , and ensure that they go into it with their eyes open and fully aware of the implications. yet , it is hard to intervene in order to provide high quality information when people self - refer to overseas clinics , on the basis of facebook recommendations . the reordering of sources of information about fertility treatment from the medical profession to the internet is significant . even if doctors are still trusted sources of information , there are other factors which make the internet an attractive source of information . when people are seeking out information about treatments that may be unlawful at home , or which are stigmatised restrictive legal provisions then actively contribute to the bypassing of medical professionals , concerned about their professional registration , as a source of advice and support . indeed , laws which inhibit doctors from offering advice and assistance to patients who are contemplating fertility treatment overseas create a professional conflict of interest in which doctors must choose between making the care of their patient their first concern , which would militate in favour of providing advice and support , and not being seen to endorse or support illegal behaviour , which might instead prompt them to leave patients to their own devices in hammarberg et al s survey study of australians travelling abroad for surrogacy , fewer than half of the 249 intended parents who responded had sought information from australian ivf professionals and of those who did , around one - third reported a negative reaction . in contrast to surrogacy , the australian prohibitions on commercial trading in gametes are far more specifically worded , criminalising only the giving and receipt of valuable consideration , rather than potentially implicating anyone involved in facilitating the practice . then i just tell him a little bit about the background to why we re doing it and then he ll write me an australian script for it , so that s how we re coordinating it at the moment . then i just tell him a little bit about the background to why we re doing it and then he ll write me an australian script for it , so that s how we re coordinating it at the moment . the implications of this finding need further exploration , but at a minimum suggest that access to local medical care and advice for reproductive travellers is often filtered through general practitioners rather than ivf clinicians , and is more effectively obtained for australian women who are seeking egg donation compared to those seeking surrogacy . in response to the increasing numbers of people travelling for reproductive purposes , enabling more people to access local fertility services but while we would support measures to improve access to services , not least because these might also meet the needs of those who can not afford to travel , we would like to suggest that we should also be interested in what local fertility providers and regulators can learn from the experiences of reproductive travellers . as discussed in this article , our preliminary fieldwork has thrown up four themes : that the legal distinction between altruistic and commercial gamete donation and surrogacy is increasingly unsustainable ; that role of the law in cbr is profoundly equivocal ; that facebook is now the principal source of information and peer support for reproductive travellers and lastly , that domestic reproductive service providers are often sidestepped . it is clear that patients paying for treatment overseas feel as though they are more in control of their treatment , and that , in contrast to their experience of domestic fertility services , they do not have to be grateful for what they receive . this is a revealing illustration of the gap between the normal markers for success , as judged objectively , and the marker of high - quality care for patients , in which patient - centred care is not just a desirable extra , but is central to every aspect of what matters to them . the most striking example of this from our cbr study was an intended mother who explained that she and her partner had fallen in love with an egg donor , and that , for them , this trumped the discovery that she was a carrier of the tay sachs gene . what mattered instead was the lived experience of the practicalities of new parenthood : being able to get back into australia , obtain a medicare card to access medical treatment for the child , and later to enrol him or her in school . like the unenforced or ambiguous laws of the treating country , and the blind - eye approach of domestic regulators , the birth certificate both is , and is not , legal protection. we suggest that any attempt to further reform or refine laws relating to cbr must attend to the subjective and lived experience of law , which like the personal experience of treatment , may stand in contradiction to policy maker s understandings of participants motivations or of law s effect upon behaviour . our interviews explore both cross - border compensated surrogacy and egg donation and domestic uncompensated arrangements . though we are still at the beginning of our work , it is already clear that the picture is far more complex than might have been expected , both morally and legally . the law , as it currently stands , works in obstructive and confounding ways that push people outside its reach or encourage them to ignore its limits and this is ultimately counter - productive and likely to cause harm to the participants and their future children . in order to ensure that the law is capable of minimising the risk of harm to all participants , we argue that it is necessary to learn from the experience of those undertaking cross - border reproductive treatment , at and outside the limits of the law .

based on who report , 1 out of 10 infants born in 2010 were immature , resulting in 15 million immature births in a year . consequently , intravenous access is so crucial among them to provide them with fluids and intravenous therapy . in order to have intravenous access , the first option in infants is short peripheral catheters which are appropriate for short - term intravenous therapy . there are restrictions for infusion of fluids . in infants with prolonged hospitalization , usage of these catheters may lead to frequent catheterization . to solve this problem , various types of central conventional catheters are available that are inserted by physicians who have surgical skills , putting the infants in danger of pneumothorax and hemothorax . peripherally inserted central venous catheters ( picc ) are the other type of catheters which are inserted through peripheral veins and can diminish the complications of conventional central catheters . insertion of these catheters in central veins decreases some complications such as thrombosis , and catheter occlusion and leakage , compared to short peripheral catheters . on the other hand , as this procedure is conducted in the ward and bedside , patients monitoring is preserved and patients transfer out of the ward for catheter insertion is cancelled . another advantage in using these catheters is safe infusion of irritating drugs or those with high osmolality and non - physiologic acidity . in cases of intravenous therapies longer than 6 days , their insertion is recommended . in spite of having the above - mentioned advantages , these catheters are not risk free . thrombosis , leakage , phlebitis , catheter embolism , and extravascular fluid aggregation are their other complications . since these catheters are often inserted by professionally trained nurses , the rate of complications can be significantly diminished through promotion of nursing care quality and professional education . despite the available evidences from the studies conducted in developed countries concerning the advantage of picc , short peripheral catheters are vastly used in neonatal intensive care units ( nicus ) for long - term intravenous therapies , total parenteral nutrition , and injection of irritating drugs in iran , leaving the infants subjected to frequent catheterization and the resulting complications . meanwhile , most of these complications can be prevented by appropriate application of picc . to the best knowledge of the researcher , the only iranian related study is of jalalian - taghadomi et al . ( 2004 - 2005 ) on the patients who had undergone coronary bypass surgery , which was conducted to measure central venous pressure ( cvp ) with emphasis on the position of the catheter tip . with regard to aforementioned issues , the question is , what are the barriers for using piccs even after 40 years of their first use in infants in iran , despite their advantages ? the researcher , based on the study her observations in nicu , believes that perhaps the high cost of these catheters has restricted their use , although studies have reported its cost efficacy . since its insertion needs staffs training , less number of educated staffs can be a prohibiting factor in this regard . with regard to these barriers in other countries , no article was found on searching the available databases . it was necessary to investigate the experiences of the physicians and nurses who were working in these wards and were familiar with its advantages , with regard to the existing barriers in its application . as the obtained data are the product of the participants experiences and to investigate the phenomena deeply , qualitative research seems to be an appropriate method to find the answer for the study question . therefore , this study was designed and conducted to discover the barriers in spreading the usage of picc in the nicus . this was a descriptive explorative qualitative study conducted to discover the barriers of application of picc among the nurses and secondary year residents of neonatology and neonatal specialists working in three hospitals of alzahra , shadid beheshti , and amin , affiliated to isfahan university of medical sciences . gholami motlagh et al . , quoting from kazemi et al . , state that descriptive approach in qualitative research is used when there is little information about a specific phenomenon . after the research project was approved by the ethical committee of the university and research permission was issued in nursing and midwifery faculty , purposive and snowball sampling was conducted in 4 months ( december 2011april 2012 ) up to data saturation . data were collected using semi - structured deep interviews and informal observations and field notes . the interviews were conducted inside the ward classroom in the morning shifts ( except for one case ) . after taking a written informed consent , the anonymous interviews were recorded and coded to the relevant participants . in some cases , it was needed to refer to the participant to clarify some obscure points after transcription of the interviews . after the analysis and conducting interviews with 14 participants , the data were almost saturated and the sampling ended . to increase the rigor of the data , we used peer deliberating and peer review , and data collected in three different environments and from the individuals with different educational qualifications and occupations ( triangulation of place and individuals ) through interviews , informal observations , and field note recording ( triangulation methods ) . also , the narrations and extracted codes were given to some experts well - acquainted with qualitative research , and the subjects viewpoints were considered and followed . the data analysis was conducted by thematic analysis suggested by brown and clark ( 2006 ) through deductive method . most of the thematic analysis steps are similar to other qualitative researches . in the present study , after each interview , the interviews were listened to and transcribed verbatim . this process went on for the other interviews sequentially . in the first step of analysis , after reading the obtained data frequently and extracting primary ideas , primary codes were formed . next , the related sub - themes were categorized forming main themes . in the last step , the extracted themes were reviewed to assure the relevancy with the codes . the data analysis was conducted by thematic analysis suggested by brown and clark ( 2006 ) through deductive method . most of the thematic analysis steps are similar to other qualitative researches . in the present study , after each interview , the interviews were listened to and transcribed verbatim . to assure precise transcription , the interviews were listened to several times . this process went on for the other interviews sequentially . in the first step of analysis , after reading the obtained data frequently and extracting primary ideas , primary codes were formed . next , the related sub - themes were categorized forming main themes . in the last step , the extracted themes were reviewed to assure the relevancy with the codes . the data were obtained from interviews with 14 participants . the participants comprised eight female nurses , five male physicians , and a female physician . the findings included 175 primary codes , 12 sub - themes , and 3 main themes . the main themes were barriers related to procedure and maintenance , barriers related to persons providing care , and barriers related to management and planning [ table 1 ] . main themes and sub - themes participants experiences showed that the concern about complications is one of the barriers in usage of these catheters . perhaps one of the reasons the physicians do not agree ( to use them ) is their fear due to its high risk . in fact , all we do , if not appropriately done , have some complications . some of the participants talked about usage of these catheters as a time - consuming procedure and described that application of picc may take a long time . nurses ability to take care of the catheter played an important role in their idea . concerning this skill , we have no skill at the beginning of the work , but the guys ( nurses ) get gradually experienced and can insert ( the catheters ) by themselves . a nurse as a participant talks about nursing care : the nurse should be able to protect this ( the catheter ) . it means that the patients , if cared well from the arrival to discharge , i mean good ( appropriate ) nursing care , never face positive crp or any other problems . participants talked about lack of teamwork and professional reliance , lack of self - perseverance to make changes , resistance to change , and parents objections it is due to the fact that our teamwork is not good . another participant talked about the necessity of self - perseverance to make changes as a personal factor : now i , in hospital , am working on picc ; yet nobody has accepted my word , i should not give up , but some ( personnel ) support me well , our matron supports me well . regarding the association between lack of skill , high cost , and parents objection , one of the participants said thus : specially at the beginning , we had a lot of waste , it goes without saying that it makes problems as patients accompanying persons ask , where is it ( the catheter ) that we bought for us $ 10.00? some nurses talked about their fear and concern about the legal issues related to insertion of these catheters . they stated that insertion is not yet in their job description , and this worries them . some other participants were worried about the less number of nurses working in nicu and mentioned that it can act as an obstacle in taking appropriate care of the catheter . they believed that due to the high price , usage of these catheters may not be economical . a participating nurse stated : something we faced while working here is the high price these catheter impose to the patients accompanying persons . most of the times , the high price has been a problem - making factor . due to its high cost , not all hospitals accept that , you see , a small hospital usually does not tolerate expensive equipments . some of the participants talked about inadequate access to these catheters and claimed that provision of the needed catheters and their accessory equipments can increase their usage . educating the personnel about these catheters was also important in their opinion . one of the participants explained about the effect of more education about catheters , based on her own experience , as follows : in a seminar in tehran , there was a female doctor coming from a foreign country . i think one of the hospitals had educated her about the catheters , yes in fact she had gone to a hospital to be educated she had precisely been trained for this ( catheter ) and worked with that(catheter ) . another participant indicated thus : a nurse can do this ( insert the catheter ) , but just a well - trained nurse untrained nurses should not try it , as it is an access way to a central vein , if not careful , the vein may be ruptured . i mean he / she should know about venous anatomy and its possible complications , a known person , not everybody . participants experiences showed that the concern about complications is one of the barriers in usage of these catheters . a participant said : perhaps one of the reasons the physicians do not agree ( to use them ) is their fear due to its high risk . in fact , all we do , if not appropriately done , have some complications . some of the participants talked about usage of these catheters as a time - consuming procedure and described that application of picc may take a long time . nurses ability to take care of the catheter played an important role in their idea . concerning this skill , we have no skill at the beginning of the work , but the guys ( nurses ) get gradually experienced and can insert ( the catheters ) by themselves . a nurse as a participant talks about nursing care : the nurse should be able to protect this ( the catheter ) . it means that the patients , if cared well from the arrival to discharge , i mean good ( appropriate ) nursing care , never face positive crp or any other problems . participants talked about lack of teamwork and professional reliance , lack of self - perseverance to make changes , resistance to change , and parents objections it is due to the fact that our teamwork is not good . another participant talked about the necessity of self - perseverance to make changes as a personal factor : now i , in hospital , am working on picc ; yet nobody has accepted my word , i should not give up , but some ( personnel ) support me well , our matron supports me well . regarding the association between lack of skill , high cost , and parents objection , one of the participants said thus : specially at the beginning , we had a lot of waste , it goes without saying that it makes problems as patients accompanying persons ask , where is it ( the catheter ) that we bought for us $ 10.00? some nurses talked about their fear and concern about the legal issues related to insertion of these catheters . they stated that insertion is not yet in their job description , and this worries them . some other participants were worried about the less number of nurses working in nicu and mentioned that it can act as an obstacle in taking appropriate care of the catheter . they believed that due to the high price , usage of these catheters may not be economical . a participating nurse stated : something we faced while working here is the high price these catheter impose to the patients accompanying persons . most of the times , the high price has been a problem - making factor . due to its high cost , not all hospitals accept that , you see , a small hospital usually does not tolerate expensive equipments . some of the participants talked about inadequate access to these catheters and claimed that provision of the needed catheters and their accessory equipments can increase their usage . educating the personnel about these catheters was also important in their opinion . one of the participants explained about the effect of more education about catheters , based on her own experience , as follows : in a seminar in tehran , there was a female doctor coming from a foreign country . i think one of the hospitals had educated her about the catheters , yes in fact she had gone to a hospital to be educated she had precisely been trained for this ( catheter ) and worked with that(catheter ) . another participant indicated thus : a nurse can do this ( insert the catheter ) , but just a well - trained nurse untrained nurses should not try it , as it is an access way to a central vein , if not careful , the vein may be ruptured . i mean he / she should know about venous anatomy and its possible complications , a known person , not everybody . with regard to the findings , the results were collected in three themes : barriers related to the procedure and maintenance , barriers related to persons providing care , and barriers related to management and planning . with regard to the barriers related to procedure and maintenance , one of the participants main concern was the incidence of complications . on the other hand , the participants believed that as their usage is limited , their complications have remained unknown leading to prohibition of their usage . meanwhile , all their complications are almost known , and numerous studies have been conducted on their complications , comparing them with other peripheral and central catheters . in a retrospective study conducted in china compared one of their most important complications , infection , in the two groups of picc and peripheral intravenous catheter ( piv ) , and showed that there was no significant difference concerning incidence of septicemia in these two groups . this study showed a possibility of infection in the use of peripheral intravenous catheters too ; therefore , if there is a concern for complications in the use of picc , it must also be there for piv . when there is no chance of intravenous access through peripheral catheters , the only way is to send the infant to op for central catheter that is inserted under general anesthesia and results in , according to various studies , more complications compared to picc . meanwhile , there are studies reporting more complications for picc compared to central catheters inserted through surgery . turcotte et al . in a systematic review study compared two types of catheters and showed that there was no significant difference between them concerning infection , but thrombotic complications were notably more in picc . they concluded that there was no clear evidence showing that picc is better than central catheters in critical care environments and they also found it essential to conduct comparative studies to investigate the level of complications . however , possible complications should not be a reason for ignoring insertion , as the complications can be diminished by high - quality nursing care and nurses education concerning these complications . although apparently picc insertion takes more time compared to peripheral intravenous catheters , it should be noted that on one hand , venous access is not convenient in infants due to prolonged hospitalization , and on the other hand , picc , if cared and maintained well , can remain working for a long time with no need for peripheral intravenous catheters . 2004 ) reported the average time of insertion for picc and peripheral intravenous catheters as 19 ( 15 - 25 ) min and 5 min ( 3 - 12 ) , respectively , in patients ranging from infants to those of 14 years of age . it should be mentioned that in the above study , all the patients were not infants , and the subjects age range possibly led to shorter average of time for insertion . on the other hand , the subjects were fresh patients recently hospitalized and prepared for surgery , whose peripheral veins were intact . participants experiences showed that skill and experience in insertion is an essential element to insert these catheters . lourenco and ohara ( 2010 ) believed insertion of picc needs technical and clinical judgment skills and informed confident and efficient decision making , and the professionals administering them should have acquired theoretical and practical related knowledge through special training courses . nurses capability concerning taking care of these catheters is of great importance from the viewpoint of the participants . nursing care can obviously affect the incidence of complications and the outcomes for the patients . nurses responsibility concerning maintenance of intravenous access tools is increased and their key role in prevention of complications related to these tools is highlighted by the increase in need for intravenous therapies . usage of long - term tools give the nurses the chance of emphasizing on important caring aspects and patients monitoring , instead of spending a lot of effort on temporary ( short - term ) intravenous tools . however , caring is the main duty of nurses and they should obtain necessary skills concerning taking care of intravenous tools to preserve them in order to provide the patients with a better care . with regard to the barriers related to persons providing care , lack of teamwork and inter - professional trust was among the factors affecting treatment team 's function from the viewpoint of the participants . it is obvious that teamwork increases nurses self - confidence and self - efficacy . on the contrary , playing the role of a mere operator and lack of active function in patients treatment diminish nurses efficiency and self - confidence and delay their professional progress leading to their lower attention to patients needs . the participants mentioned personal factors such as lack of self - perseverance , resisting against changes , and outdated information as the reasons for not using these catheters . lourenco and ohara ( 2006 ) investigated the scientific knowledge level of nurses concerning picc and showed that nurses constantly need to update their knowledge and information concerning this procedure to promote their quality of care toward infants . however , many individuals may gradually lose their knowledge through time due to forgetfulness and this fact reveals the necessity for updating their knowledge . most of the participants considered the role of the parents so important and added that giving adequate explanation to the parents is crucial . with regard to direction of caring progress toward the family - centered cares , parents , in addition to physicians and nurses , play a key role in taking care of the infants , and their satisfaction with the treatment is also important . obtaining a written or an oral consent is essential to insert picc . they concluded that after medical and nursing team found the picc insertion to be necessary , the patients and their families should be guided . family members have the right to know about the options of treatment , benefits , risks , and expected costs , as well as the experience and competency of those staffs administering the procedure . therefore , it is an absolute right of the parents to be informed about the advantages and risks of these catheters so that they could decide freely . with regard to barriers related to management and planning , participants experiences revealed their concern in relation with legal issues of the catheters insertion . they stated that this procedure has not been listed in job description of the nurses , and authorities support plays an important role in spreading their usage . in nurses job description issued by ministry of health meanwhile , in countries where picc is used , it is inserted by nurses . in paragraph 1 of resolution 258 of 2001 in federal nursing association , nurses have been licensed to insert picc . pettit and wyckoff ( 2007 ) in the second clinical guide printed for neonate care nurses argue that the nurses authorized to insert picc should consult with their state nursing board to determine whether this procedure is in their field of nursing interventions or not . insertion of picc may be considered as an advanced nursing intervention , and therefore needs to be approved by an interdisciplinary committee as a standard procedure . the imbalance between the number of nurses and the number of beds in nicus was also an important issue , based on some participants views . with regard to the observed nurse bed ratio , which was in some cases 1:3 , this concern seems logical as high workload decreases the quality of care , which should be considered by the authorities . participants experiences showed that high price and lack of medicare coverage was an obstacle in the usage of picc . at the first glance , high price of picc compared to piv seems to impose high costs to the patients , but in the long term , picc is more economical . 2004 ) reported that although the cost of picc is 1.54 folds more than that of piv , they reasoned that better satisfaction with picc has made it cost - effective . xu et al . ( 2008 ) reported that in a time interval of more than 10 days , picc is more cost - effective compared to piv . based on participants experiences , lack of proper training in insertion and taking care of picc was among the major barriers , as the catheter should be inserted by experienced individuals . some of them indicated lack of information about these catheters and inadequate knowledge as the factors for not using them , which need a precise programming to be solved . now , insertion of these catheters is not in the bs and ms curricula of the nursing students , and no specific organization is responsible for its education and insertion . web search with persian key words concerning its education in iran yielded no scheduled education on it . education about these catheters promotes nurses skill and quality of care , reduces complications , and increases their cost efficacy . anh and susan ( 2005 ) showed that with designing an educational intervention based on albert bandora 's social learning theory , an increase is seen in the nurses knowledge and self - efficacy concerning picc , leading to a notable reduction in the level of catheter obstruction ( 29%-8.5% ) in a 6-month period . with regard to the current need which is felt for picc insertion , an increase in its use is predicted . based on the findings of the present study , there are still problems in picc application , which can delay the trend of its progress . it is suggested to have a precise program for organized education of the staffs about insertion and its care , with an emphasis on prevention of the complications , based on international standards in order to use them as a safe treatment option for the infants hospitalized for a prolonged time . as the problem in picc usage prevails all over iran and this qualitative study was conducted in just one city , the findings can not be generalized .

background : by increasing the survival of immature newborns , intravenous access methods , used to provide intravenous therapy , became more important . more attention has been recently paid on peripherally inserted central venous catheters in newborns , although it is yet unknown in iran . in this study , we tried to discover the barriers to spread the usage of peripherally inserted central venous catheters ( picc ) in the neonatal intensive care units of hospitals affiliated to isfahan university of medical sciences.materials and methods : in this descriptive explorative qualitative research , conducted from december 2011 to april 2012 with purposeful sampling and snowball method , participants were selected from nurses and residents of neonatology and neonatal specialists working in alzahra , shahid beheshty , and amin hospitals , until data saturation occurred . data were analyzed with thematic analysis proposed by broun and clarke in 2006.results:data analysis yielded 175 initial codes , 12 sub - themes , and 3 main themes . the main themes included barriers related to procedure and maintenance , barriers related to persons providing care , and barriers related to management and planning.conclusions:one of the major problems in premature newborns during hospitalization is long - term and safe intravascular access ; therefore , more use of picc is needed . a complete planning is also needed to eliminate barriers and to provide required catheters . educating the personnel is also necessary .

I M Data analysis R Barriers related to procedure and maintenance Barriers related to persons providing care Barriers related to management and planning D

based on who report , 1 out of 10 infants born in 2010 were immature , resulting in 15 million immature births in a year . consequently , intravenous access is so crucial among them to provide them with fluids and intravenous therapy . in order to have intravenous access , the first option in infants is short peripheral catheters which are appropriate for short - term intravenous therapy . in infants with prolonged hospitalization , usage of these catheters may lead to frequent catheterization . peripherally inserted central venous catheters ( picc ) are the other type of catheters which are inserted through peripheral veins and can diminish the complications of conventional central catheters . insertion of these catheters in central veins decreases some complications such as thrombosis , and catheter occlusion and leakage , compared to short peripheral catheters . on the other hand , as this procedure is conducted in the ward and bedside , patients monitoring is preserved and patients transfer out of the ward for catheter insertion is cancelled . in cases of intravenous therapies longer than 6 days , their insertion is recommended . thrombosis , leakage , phlebitis , catheter embolism , and extravascular fluid aggregation are their other complications . despite the available evidences from the studies conducted in developed countries concerning the advantage of picc , short peripheral catheters are vastly used in neonatal intensive care units ( nicus ) for long - term intravenous therapies , total parenteral nutrition , and injection of irritating drugs in iran , leaving the infants subjected to frequent catheterization and the resulting complications . meanwhile , most of these complications can be prevented by appropriate application of picc . to the best knowledge of the researcher , the only iranian related study is of jalalian - taghadomi et al . ( 2004 - 2005 ) on the patients who had undergone coronary bypass surgery , which was conducted to measure central venous pressure ( cvp ) with emphasis on the position of the catheter tip . with regard to aforementioned issues , the question is , what are the barriers for using piccs even after 40 years of their first use in infants in iran , despite their advantages ? the researcher , based on the study her observations in nicu , believes that perhaps the high cost of these catheters has restricted their use , although studies have reported its cost efficacy . since its insertion needs staffs training , less number of educated staffs can be a prohibiting factor in this regard . with regard to these barriers in other countries , no article was found on searching the available databases . it was necessary to investigate the experiences of the physicians and nurses who were working in these wards and were familiar with its advantages , with regard to the existing barriers in its application . as the obtained data are the product of the participants experiences and to investigate the phenomena deeply , qualitative research seems to be an appropriate method to find the answer for the study question . therefore , this study was designed and conducted to discover the barriers in spreading the usage of picc in the nicus . this was a descriptive explorative qualitative study conducted to discover the barriers of application of picc among the nurses and secondary year residents of neonatology and neonatal specialists working in three hospitals of alzahra , shadid beheshti , and amin , affiliated to isfahan university of medical sciences . gholami motlagh et al . , state that descriptive approach in qualitative research is used when there is little information about a specific phenomenon . after the research project was approved by the ethical committee of the university and research permission was issued in nursing and midwifery faculty , purposive and snowball sampling was conducted in 4 months ( december 2011april 2012 ) up to data saturation . data were collected using semi - structured deep interviews and informal observations and field notes . the interviews were conducted inside the ward classroom in the morning shifts ( except for one case ) . after taking a written informed consent , the anonymous interviews were recorded and coded to the relevant participants . in some cases , it was needed to refer to the participant to clarify some obscure points after transcription of the interviews . after the analysis and conducting interviews with 14 participants , the data were almost saturated and the sampling ended . to increase the rigor of the data , we used peer deliberating and peer review , and data collected in three different environments and from the individuals with different educational qualifications and occupations ( triangulation of place and individuals ) through interviews , informal observations , and field note recording ( triangulation methods ) . also , the narrations and extracted codes were given to some experts well - acquainted with qualitative research , and the subjects viewpoints were considered and followed . the data analysis was conducted by thematic analysis suggested by brown and clark ( 2006 ) through deductive method . most of the thematic analysis steps are similar to other qualitative researches . in the present study , after each interview , the interviews were listened to and transcribed verbatim . in the first step of analysis , after reading the obtained data frequently and extracting primary ideas , primary codes were formed . next , the related sub - themes were categorized forming main themes . in the last step , the extracted themes were reviewed to assure the relevancy with the codes . the data analysis was conducted by thematic analysis suggested by brown and clark ( 2006 ) through deductive method . most of the thematic analysis steps are similar to other qualitative researches . in the present study , after each interview , the interviews were listened to and transcribed verbatim . in the first step of analysis , after reading the obtained data frequently and extracting primary ideas , primary codes were formed . next , the related sub - themes were categorized forming main themes . in the last step , the extracted themes were reviewed to assure the relevancy with the codes . the data were obtained from interviews with 14 participants . the participants comprised eight female nurses , five male physicians , and a female physician . the findings included 175 primary codes , 12 sub - themes , and 3 main themes . the main themes were barriers related to procedure and maintenance , barriers related to persons providing care , and barriers related to management and planning [ table 1 ] . main themes and sub - themes participants experiences showed that the concern about complications is one of the barriers in usage of these catheters . perhaps one of the reasons the physicians do not agree ( to use them ) is their fear due to its high risk . in fact , all we do , if not appropriately done , have some complications . some of the participants talked about usage of these catheters as a time - consuming procedure and described that application of picc may take a long time . nurses ability to take care of the catheter played an important role in their idea . concerning this skill , we have no skill at the beginning of the work , but the guys ( nurses ) get gradually experienced and can insert ( the catheters ) by themselves . it means that the patients , if cared well from the arrival to discharge , i mean good ( appropriate ) nursing care , never face positive crp or any other problems . participants talked about lack of teamwork and professional reliance , lack of self - perseverance to make changes , resistance to change , and parents objections it is due to the fact that our teamwork is not good . regarding the association between lack of skill , high cost , and parents objection , one of the participants said thus : specially at the beginning , we had a lot of waste , it goes without saying that it makes problems as patients accompanying persons ask , where is it ( the catheter ) that we bought for us $ 10.00? some nurses talked about their fear and concern about the legal issues related to insertion of these catheters . they stated that insertion is not yet in their job description , and this worries them . some other participants were worried about the less number of nurses working in nicu and mentioned that it can act as an obstacle in taking appropriate care of the catheter . they believed that due to the high price , usage of these catheters may not be economical . most of the times , the high price has been a problem - making factor . some of the participants talked about inadequate access to these catheters and claimed that provision of the needed catheters and their accessory equipments can increase their usage . educating the personnel about these catheters was also important in their opinion . one of the participants explained about the effect of more education about catheters , based on her own experience , as follows : in a seminar in tehran , there was a female doctor coming from a foreign country . i think one of the hospitals had educated her about the catheters , yes in fact she had gone to a hospital to be educated she had precisely been trained for this ( catheter ) and worked with that(catheter ) . another participant indicated thus : a nurse can do this ( insert the catheter ) , but just a well - trained nurse untrained nurses should not try it , as it is an access way to a central vein , if not careful , the vein may be ruptured . participants experiences showed that the concern about complications is one of the barriers in usage of these catheters . a participant said : perhaps one of the reasons the physicians do not agree ( to use them ) is their fear due to its high risk . some of the participants talked about usage of these catheters as a time - consuming procedure and described that application of picc may take a long time . nurses ability to take care of the catheter played an important role in their idea . concerning this skill , we have no skill at the beginning of the work , but the guys ( nurses ) get gradually experienced and can insert ( the catheters ) by themselves . it means that the patients , if cared well from the arrival to discharge , i mean good ( appropriate ) nursing care , never face positive crp or any other problems . participants talked about lack of teamwork and professional reliance , lack of self - perseverance to make changes , resistance to change , and parents objections it is due to the fact that our teamwork is not good . regarding the association between lack of skill , high cost , and parents objection , one of the participants said thus : specially at the beginning , we had a lot of waste , it goes without saying that it makes problems as patients accompanying persons ask , where is it ( the catheter ) that we bought for us $ 10.00? some nurses talked about their fear and concern about the legal issues related to insertion of these catheters . they stated that insertion is not yet in their job description , and this worries them . some other participants were worried about the less number of nurses working in nicu and mentioned that it can act as an obstacle in taking appropriate care of the catheter . they believed that due to the high price , usage of these catheters may not be economical . a participating nurse stated : something we faced while working here is the high price these catheter impose to the patients accompanying persons . most of the times , the high price has been a problem - making factor . due to its high cost , not all hospitals accept that , you see , a small hospital usually does not tolerate expensive equipments . some of the participants talked about inadequate access to these catheters and claimed that provision of the needed catheters and their accessory equipments can increase their usage . educating the personnel about these catheters was also important in their opinion . one of the participants explained about the effect of more education about catheters , based on her own experience , as follows : in a seminar in tehran , there was a female doctor coming from a foreign country . i think one of the hospitals had educated her about the catheters , yes in fact she had gone to a hospital to be educated she had precisely been trained for this ( catheter ) and worked with that(catheter ) . another participant indicated thus : a nurse can do this ( insert the catheter ) , but just a well - trained nurse untrained nurses should not try it , as it is an access way to a central vein , if not careful , the vein may be ruptured . with regard to the findings , the results were collected in three themes : barriers related to the procedure and maintenance , barriers related to persons providing care , and barriers related to management and planning . with regard to the barriers related to procedure and maintenance , one of the participants main concern was the incidence of complications . meanwhile , all their complications are almost known , and numerous studies have been conducted on their complications , comparing them with other peripheral and central catheters . in a retrospective study conducted in china compared one of their most important complications , infection , in the two groups of picc and peripheral intravenous catheter ( piv ) , and showed that there was no significant difference concerning incidence of septicemia in these two groups . this study showed a possibility of infection in the use of peripheral intravenous catheters too ; therefore , if there is a concern for complications in the use of picc , it must also be there for piv . when there is no chance of intravenous access through peripheral catheters , the only way is to send the infant to op for central catheter that is inserted under general anesthesia and results in , according to various studies , more complications compared to picc . turcotte et al . they concluded that there was no clear evidence showing that picc is better than central catheters in critical care environments and they also found it essential to conduct comparative studies to investigate the level of complications . although apparently picc insertion takes more time compared to peripheral intravenous catheters , it should be noted that on one hand , venous access is not convenient in infants due to prolonged hospitalization , and on the other hand , picc , if cared and maintained well , can remain working for a long time with no need for peripheral intravenous catheters . 2004 ) reported the average time of insertion for picc and peripheral intravenous catheters as 19 ( 15 - 25 ) min and 5 min ( 3 - 12 ) , respectively , in patients ranging from infants to those of 14 years of age . it should be mentioned that in the above study , all the patients were not infants , and the subjects age range possibly led to shorter average of time for insertion . on the other hand , the subjects were fresh patients recently hospitalized and prepared for surgery , whose peripheral veins were intact . participants experiences showed that skill and experience in insertion is an essential element to insert these catheters . lourenco and ohara ( 2010 ) believed insertion of picc needs technical and clinical judgment skills and informed confident and efficient decision making , and the professionals administering them should have acquired theoretical and practical related knowledge through special training courses . nurses capability concerning taking care of these catheters is of great importance from the viewpoint of the participants . nurses responsibility concerning maintenance of intravenous access tools is increased and their key role in prevention of complications related to these tools is highlighted by the increase in need for intravenous therapies . usage of long - term tools give the nurses the chance of emphasizing on important caring aspects and patients monitoring , instead of spending a lot of effort on temporary ( short - term ) intravenous tools . however , caring is the main duty of nurses and they should obtain necessary skills concerning taking care of intravenous tools to preserve them in order to provide the patients with a better care . with regard to the barriers related to persons providing care , lack of teamwork and inter - professional trust was among the factors affecting treatment team 's function from the viewpoint of the participants . it is obvious that teamwork increases nurses self - confidence and self - efficacy . on the contrary , playing the role of a mere operator and lack of active function in patients treatment diminish nurses efficiency and self - confidence and delay their professional progress leading to their lower attention to patients needs . the participants mentioned personal factors such as lack of self - perseverance , resisting against changes , and outdated information as the reasons for not using these catheters . most of the participants considered the role of the parents so important and added that giving adequate explanation to the parents is crucial . with regard to direction of caring progress toward the family - centered cares , parents , in addition to physicians and nurses , play a key role in taking care of the infants , and their satisfaction with the treatment is also important . family members have the right to know about the options of treatment , benefits , risks , and expected costs , as well as the experience and competency of those staffs administering the procedure . therefore , it is an absolute right of the parents to be informed about the advantages and risks of these catheters so that they could decide freely . with regard to barriers related to management and planning , participants experiences revealed their concern in relation with legal issues of the catheters insertion . they stated that this procedure has not been listed in job description of the nurses , and authorities support plays an important role in spreading their usage . in nurses job description issued by ministry of health meanwhile , in countries where picc is used , it is inserted by nurses . pettit and wyckoff ( 2007 ) in the second clinical guide printed for neonate care nurses argue that the nurses authorized to insert picc should consult with their state nursing board to determine whether this procedure is in their field of nursing interventions or not . insertion of picc may be considered as an advanced nursing intervention , and therefore needs to be approved by an interdisciplinary committee as a standard procedure . the imbalance between the number of nurses and the number of beds in nicus was also an important issue , based on some participants views . with regard to the observed nurse bed ratio , which was in some cases 1:3 , this concern seems logical as high workload decreases the quality of care , which should be considered by the authorities . participants experiences showed that high price and lack of medicare coverage was an obstacle in the usage of picc . at the first glance , high price of picc compared to piv seems to impose high costs to the patients , but in the long term , picc is more economical . 2004 ) reported that although the cost of picc is 1.54 folds more than that of piv , they reasoned that better satisfaction with picc has made it cost - effective . ( 2008 ) reported that in a time interval of more than 10 days , picc is more cost - effective compared to piv . based on participants experiences , lack of proper training in insertion and taking care of picc was among the major barriers , as the catheter should be inserted by experienced individuals . some of them indicated lack of information about these catheters and inadequate knowledge as the factors for not using them , which need a precise programming to be solved . now , insertion of these catheters is not in the bs and ms curricula of the nursing students , and no specific organization is responsible for its education and insertion . web search with persian key words concerning its education in iran yielded no scheduled education on it . education about these catheters promotes nurses skill and quality of care , reduces complications , and increases their cost efficacy . anh and susan ( 2005 ) showed that with designing an educational intervention based on albert bandora 's social learning theory , an increase is seen in the nurses knowledge and self - efficacy concerning picc , leading to a notable reduction in the level of catheter obstruction ( 29%-8.5% ) in a 6-month period . based on the findings of the present study , there are still problems in picc application , which can delay the trend of its progress . it is suggested to have a precise program for organized education of the staffs about insertion and its care , with an emphasis on prevention of the complications , based on international standards in order to use them as a safe treatment option for the infants hospitalized for a prolonged time . as the problem in picc usage prevails all over iran and this qualitative study was conducted in just one city , the findings can not be generalized .

using multigenerational and extended pedigrees from a genetic isolate our group identified several loci significantly linked to attention deficit hyperactivity disorder ( adhd ) , namely on 4q , 5q , 8q , 11q and 17p . regions on 11q and 17p replicated linkage described by others . minimal critical regions for each one of these regions , defined by recombination events in affected individuals as well as the 1-lod - support criterion , have been reported elsewhere . given the significant linkage to different genomic regions , the existence of unique large size families linked to multiple regions , and the fact that these families came from an isolated population , we hypothesized that two - locus interaction contributions to adhd were plausible . to screen for possible interacting regions , we initially performed a correlation subset analysis between the linked regions . we used 134 nuclear families from the paisa genetic isolate and single - nucleotide polymorphisms ( snps ) spanning each minimal critical region with a resolution of 200 kb ( mapping resolution as well as snps selection criteria have been published in detail elsewhere ) . we calculated the non - parametric linkage ( npl ) score using genehunter with the weight function weight10 , where families that demonstrated nominal npl ( npl>1.00 ) to a region were included in the analysis ( that is , were coded as 1 for weight10 ) and those families not demonstrating linkage were excluded ( that is , were coded as 0 for weight10 ) . to measure heterogeneity the weight function weight01 ( defined analogously ) was used , where only those families that did not demonstrate linkage to a region were included in the analysis . of the entire possible set of pair - wise comparisons , two gave increases in the npl statistic greater than two units when compared with linkage analyses using all families . selecting families linked to 17p increased the nonparametric linkage statistic from 0.55 to 3.88 on 11q ( 17p11q interaction ) and selecting families linked to 4q increased the npl statistic from 0.55 to 3.24 in an overlapping region on 11q ( 4q11q interaction ; figure 1a , supplementary information ) . using the method implemented in genehunter - twolocus , we found a maximal non - parametric score of 6.08 ( p<1 10 ) produced by snps rs1038426 ( 4q ) and rs1293344 ( 11q ; 4q11q interaction ; figure 1b , supplementary information ) and a maximal non - parametric score of 5.51 ( p<1 10 ) , produced by snps rs9227 ( 17p ) and rs1293344 ( 11q ; 17p11q interaction ; figure 1c , supplementary information ) . furthermore , power analyses to detect two interacting loci while considering adhd as a binary trait and also to detect interacting loci underlying quantitative traits , shows that , in general , our discovery sample exhibits exceptional power to detect two - locus interactions ( see procedures attached to the supplementary information ) . using a very stringent linkage disequilibrium map with a resolution of 68 kb ( two times stronger that the resolution recommended for covering this genomic area ) we also demonstrated that throughout the scanned region there was a remarkable total absence of linkage disequilibrium gaps , meaning that our genotyped markers covered any variation inside of the 4q - linked region . with this map , we identified a unique region of association and linkage of 240 kb . additional analysis showed that lphn3 common variant confers susceptibility to adhd affects brain metabolism and predicts effectiveness of stimulant medication . we showed that three markers harbored in lphn3 passed the test of heterogeneity and were significant after adjusting for multiple tests : rs6551665 ( odds ratio ( or)=1.23 , 95% confidence interval ( ci ) 1.091.37 , p=3.46 10 ) , rs1947274 ( or=1.23 , 95% ci=1.091.38 , p=5.41 10 ) and rs2345039 ( or=1.21 , 95% ci=1.081.35 , p=8.97 10 ) . the marker rs6551665 was genotyped in all the samples and showed a linkage disequilibrium r with these other two markers > 90% . given the demonstrated association of adhd susceptibility to rs6551665 ( g , allele ) , a snp harbored inside the genomic region coding for lphn3 , we next performed an association analysis using snps on 11q and 17p and conditioning on the fact of being a carrier of the g variant of susceptibility at rs6551665 . either one case or control with at least one copy of the rs6551665 lphn3 susceptibility g variant was selected per family . because of the rareness of individuals homozygous for the rs6551665 g allele we pooled them with g heterozygotes , when we conditioned on the fact of being a carrier of the g variant of susceptibility at rs6551665 , we were able to narrow down signals at 11q and 17p . however , the signal at 11q pointed to a region containing only three genes , whereas the signal at 17p spanned a genomic region containing dozens of genes . furthermore , given that testing three - locus interactions ( lphn311q17p ) will demand evaluation of thousands of models ( 22 for a dichotomic trait ) , we focused in this manuscript to describe results of the lphn311q interaction . g ; or=4.47 , ci 2.308.69 , p<0.000005 , pcorrected<0.005 ; figure 1a , table 1a ) . this haplotype spans 166 kbps from intron 7 of the gene coding neural cell adhesion molecule 1 ( ncam1 ) , encompasses the tetratricopeptide repeat domain 12 ( ttc12 ) and the ankyrin repeat and kinase domain containing 1 ( ankk1 ) , and is adjacent to the 5 untranslated region of the dopamine receptor d2 ( drd2 ; figure 1a ) . to avoid the potential effects of genetic stratification , a known cause of type i error , we performed a transmission disequilibrium test analysis in the entire set of multigenerational , nuclear and trio paisa families . the or for the transmission of the susceptibility variants on 4q and 11q is 3.14 ( 95% ci=1.496.62 ; p<0.0027 ) compared with transmission of neither variant ( table 1b ) . both the association analysis and the transmission disequilibrium test demonstrate that the significance of the association on chromosome 11q is lost when not conditioning on the presence of the susceptibility variant within lphn3 . looking for replication , we performed a transmission disequilibrium test analyses for three additional samples : one from germany and two primarily european - american samples consisting of 95 trios collected at the national human genome research institute , bethesda , md , usa ( us1 ) and 240 trios from a sample collected at children 's hospital of philadelphia , philadelphia , pa , usa ( us2 ; table 1b ) . all these three samples were used for the replication of the lphn3 association to adhd . the us2 sample was not genotyped at identical snps on 11q , so we tested two tag - snps that fully describe the variation ( r=1 , rs754672 , rs965560 ) for the ceu sample ( the international hapmap consortium , 2005 ; http://hapmap.ncbi.nlm.nih.gov ) . overall , all of these results show a similar pattern of interaction and suggest that the haplotype on 11q interacts cooperatively with the lphn3 susceptibility variant to increase the risk to adhd . a meta - analysis of the transmission disequilibrium test results from the four samples , using a random effects model , demonstrated a significant association to the transmission of both susceptibility variants on chromosome 4q and 11q ( or=2.46 , 95% ci=1.633.70 , p<0.00001 ; figure 1b and table 1c ) . to define how the above described lphn311q interaction modulates the original effects of the lphn3 susceptibility variant on brain metabolism , we next examined proton magnetic resonance spectroscopy ( h - mrs ) data of 18 individuals from the paisa genetic isolate to four metabolites , namely , n - acetylaspartate , myoinositol , choline and glutamine ( for all taken as the ratio to creatine ) , in several brain regions making up part of the frontal striatal the full two - locus interaction model was fit to the data by using linear regression , where y is the quantitative mrs metabolite phenotype , is the mean effect , a is the age at diagnosis , s is a code for gender ( males=0 , females=1 ) , d describes disease status ( unaffected=0 , affected=1 ) , xi , i=1 , 2 , is a variable modeling an additive effect ( 1 for homozygote for allele 1 , 0 for a heterozygote and 1 for a homozygote for allele 2 ) , zi is a dummy variable for a dominant effect ( 0.5 for homozygote for allele 1 , 0.5 for a heterozygote and 0.5 for a homozygote for allele 2 ) , ai and di refer to additive and dominant coefficients estimated for the single locus effect , and iaa , iad , ida and idd represent epistatic coefficients in the following model : this model was compared with a nested model lacking interaction coefficients by a likelihood ratio test ( lrt ) that follows a distribution with four degrees of freedom . they are myoinositol in the right posterior cingulate gyrus , myoinositol in the left posterior cingulate gyrus and choline in the right medial cingulate gyrus . examining the coefficients for each disclosed that for myoinositol in the right and left posterior cingulate , the iad coefficient for an interaction between an additive effect from the haplotype on 11q and a dominant effect from rs6551665 was contributing to the better fitting model ( iad left cingulate gyrus p=0.00342 ; iad right cingulate gyrus p=0.00298 ) . thus , the means were plotted for an additive effect from the 11q haplotype and a dominant effect from rs6551665 ( where values from individuals that were aa or ag at rs6551665 were grouped ) . the results demonstrate that having two copies of the susceptibility haplotype on chromosome 11 and gg at rs6551665 correlates with a significant decrease in myoinositol in the two regions ( figure 2a and b ) . the results for choline in the right medial cingulate demonstrated that the iad coefficient for an interaction between an additive effect from rs6551665 and a dominant effect from the 11q haplotype was contributing to the better fitting model ( p=0.00968 ) . the results were plotted for an additive effect from rs6551665 and a dominant effect from the haplotype on 11q ( where having one or two copies of the susceptibility haplotype were grouped ) . the results demonstrate that having ag at rs6551665 and at least one copy of the susceptibility haplotype on 11q is related to an increase in choline in the right medial cingulate and that having ag at rs6551665 and no copies of the susceptibility haplotype on 11q is related to a decreased level of choline in the right medial cingulate region ( figure 2c ) . we next attempted to study the effect of the lphn311q interaction with regards to the pharmacogenetic consequences that the lphn3 adhd - susceptibility variant had on the treatment response to stimulant medication . in all , 82 individuals with complete genotype and phenotype information from the us1 sample were included in the analysis . these individuals were sampled from the original 240 individual sample described by our group with complete demographic information of this sample presented in the supplementary information . then we examined the relationship of swan scale questions individually as well as inattentive and hyperactive combined dimensions ( questions 19 indicate inattentive symptoms and questions 1018 indicate hyperactive symptoms before and after starting methylphenidate medication ) . we observed a significantly better fitting model for question 18 ( hyperactive impulsive dimension ) when the iad coefficient for interaction between additive effects at lphn3 and dominant effects at 11q are included ( pcorrected=0.0036 ) . overall results demonstrate that having gg at rs6551665 and two copies of the susceptibility haplotype on 11q is correlated with a significant improvement of symptoms after the treatment with stimulant medication , and that having aa at rs6551665 and fewer than two copies of the susceptibility haplotype on 11q is correlated with a poor response to stimulant medication treatment ( figure 2d ) . although statistically significant , the importance of the genetic lphn311q interaction regarding the treatment response to stimulant medication requires larger studies to both replicate and assess the contribution of this interaction to adhd symptoms and response to stimulant medication . single marker and haplotype case control association analysis revealed a single strongly associated haplotype ( rs677642rs877137 , g g ; or=4.47 , ci 2.308.69 , p<0.000005 , pcorrected<0.005 ; figure 1a , table 1a ) . this haplotype spans 166 kbps from intron 7 of the gene coding neural cell adhesion molecule 1 ( ncam1 ) , encompasses the tetratricopeptide repeat domain 12 ( ttc12 ) and the ankyrin repeat and kinase domain containing 1 ( ankk1 ) , and is adjacent to the 5 untranslated region of the dopamine receptor d2 ( drd2 ; figure 1a ) . to avoid the potential effects of genetic stratification , a known cause of type i error , we performed a transmission disequilibrium test analysis in the entire set of multigenerational , nuclear and trio paisa families . the or for the transmission of the susceptibility variants on 4q and 11q is 3.14 ( 95% ci=1.496.62 ; p<0.0027 ) compared with transmission of neither variant ( table 1b ) . both the association analysis and the transmission disequilibrium test demonstrate that the significance of the association on chromosome 11q is lost when not conditioning on the presence of the susceptibility variant within lphn3 . looking for replication , we performed a transmission disequilibrium test analyses for three additional samples : one from germany and two primarily european - american samples consisting of 95 trios collected at the national human genome research institute , bethesda , md , usa ( us1 ) and 240 trios from a sample collected at children 's hospital of philadelphia , philadelphia , pa , usa ( us2 ; table 1b ) . all these three samples were used for the replication of the lphn3 association to adhd . the us2 sample was not genotyped at identical snps on 11q , so we tested two tag - snps that fully describe the variation ( r=1 , rs754672 , rs965560 ) for the ceu sample ( the international hapmap consortium , 2005 ; http://hapmap.ncbi.nlm.nih.gov ) . overall , all of these results show a similar pattern of interaction and suggest that the haplotype on 11q interacts cooperatively with the lphn3 susceptibility variant to increase the risk to adhd . a meta - analysis of the transmission disequilibrium test results from the four samples , using a random effects model , demonstrated a significant association to the transmission of both susceptibility variants on chromosome 4q and 11q ( or=2.46 , 95% ci=1.633.70 , p<0.00001 ; figure 1b and table 1c ) . to define how the above described lphn311q interaction modulates the original effects of the lphn3 susceptibility variant on brain metabolism , we next examined proton magnetic resonance spectroscopy ( h - mrs ) data of 18 individuals from the paisa genetic isolate to four metabolites , namely , n - acetylaspartate , myoinositol , choline and glutamine ( for all taken as the ratio to creatine ) , in several brain regions making up part of the frontal striatal the full two - locus interaction model was fit to the data by using linear regression , where y is the quantitative mrs metabolite phenotype , is the mean effect , a is the age at diagnosis , s is a code for gender ( males=0 , females=1 ) , d describes disease status ( unaffected=0 , affected=1 ) , xi , i=1 , 2 , is a variable modeling an additive effect ( 1 for homozygote for allele 1 , 0 for a heterozygote and 1 for a homozygote for allele 2 ) , zi is a dummy variable for a dominant effect ( 0.5 for homozygote for allele 1 , 0.5 for a heterozygote and 0.5 for a homozygote for allele 2 ) , ai and di refer to additive and dominant coefficients estimated for the single locus effect , and iaa , iad , ida and idd represent epistatic coefficients in the following model : this model was compared with a nested model lacking interaction coefficients by a likelihood ratio test ( lrt ) that follows a distribution with four degrees of freedom . they are myoinositol in the right posterior cingulate gyrus , myoinositol in the left posterior cingulate gyrus and choline in the right medial cingulate gyrus . examining the coefficients for each disclosed that for myoinositol in the right and left posterior cingulate , the iad coefficient for an interaction between an additive effect from the haplotype on 11q and a dominant effect from rs6551665 was contributing to the better fitting model ( iad left cingulate gyrus p=0.00342 ; iad right cingulate gyrus p=0.00298 ) . thus , the means were plotted for an additive effect from the 11q haplotype and a dominant effect from rs6551665 ( where values from individuals that were aa or ag at rs6551665 were grouped ) . the results demonstrate that having two copies of the susceptibility haplotype on chromosome 11 and gg at rs6551665 correlates with a significant decrease in myoinositol in the two regions ( figure 2a and b ) . the results for choline in the right medial cingulate demonstrated that the iad coefficient for an interaction between an additive effect from rs6551665 and a dominant effect from the 11q haplotype was contributing to the better fitting model ( p=0.00968 ) . the results were plotted for an additive effect from rs6551665 and a dominant effect from the haplotype on 11q ( where having one or two copies of the susceptibility haplotype were grouped ) . the results demonstrate that having ag at rs6551665 and at least one copy of the susceptibility haplotype on 11q is related to an increase in choline in the right medial cingulate and that having ag at rs6551665 and no copies of the susceptibility haplotype on 11q is related to a decreased level of choline in the right medial cingulate region ( figure 2c ) . we next attempted to study the effect of the lphn311q interaction with regards to the pharmacogenetic consequences that the lphn3 adhd - susceptibility variant had on the treatment response to stimulant medication . in all , 82 individuals with complete genotype and phenotype information from the us1 sample were included in the analysis . these individuals were sampled from the original 240 individual sample described by our group with complete demographic information of this sample presented in the supplementary information . then we examined the relationship of swan scale questions individually as well as inattentive and hyperactive combined dimensions ( questions 19 indicate inattentive symptoms and questions 1018 indicate hyperactive symptoms before and after starting methylphenidate medication ) . we observed a significantly better fitting model for question 18 ( hyperactive impulsive dimension ) when the iad coefficient for interaction between additive effects at lphn3 and dominant effects at 11q are included ( pcorrected=0.0036 ) . overall results demonstrate that having gg at rs6551665 and two copies of the susceptibility haplotype on 11q is correlated with a significant improvement of symptoms after the treatment with stimulant medication , and that having aa at rs6551665 and fewer than two copies of the susceptibility haplotype on 11q is correlated with a poor response to stimulant medication treatment ( figure 2d ) . although statistically significant , the importance of the genetic lphn311q interaction regarding the treatment response to stimulant medication requires larger studies to both replicate and assess the contribution of this interaction to adhd symptoms and response to stimulant medication . variants within lphn3 were recently demonstrated to be associated with adhd in samples derived from various populations . the demonstrated or was 1.2 ( 95% ci=1.091.38 , p<5.42 10 ) for having adhd in individuals carrying susceptibility variants within lphn3 . in this analysis , when rs6551665 within lphn3 is examined concomitantly with the haplotype on 11q the or is 2.46 ( 95% ci=1.683.70 , p<0.00001 ) . however , the association with adhd is demonstrated when conditioning on the susceptibility variant within lphn3 . thus , the haplotype on 11q seems to function as a modifier of lphn3 susceptibility . the haplotype on chromosome 11q spans 166 kbps , from intron 7 of ncam1 , encompasses ttc12 and ankk1 and is adjacent to drd2 . snps and haplotypes across this identical region were previously demonstrated to be associated with nicotine , alcohol and drug dependence . the function of ttc12 is not well described , although expression has been reported in testis , prostate , lung , lymphocytes and numerous tumors . although there are conflicting results for replication , drd2 is a putative candidate gene for adhd given its involvement in dopamine metabolism . of note , the commonly tested polymorphism drd2 taq1a was found to be within exon 8 of ankk1 , where it causes a glutamine to lysine amino acid change . ankk1 was found to be expressed in placenta and spinal cord but to date has not been observed in the developing or adult brain . ncam1 is a member of the immunoglobin superfamily and is thought to have a fundamental role in normal neural development , function and plasticity . mouse knockout models of ncam1 have been described to mimic schizophrenia based on brain morphological changes as well as reduced prepulse inhibition of the startle response . furthermore , ncam1 mouse models have also been demonstrated to have hyperactivity , aggression , anxiety and abnormal social behaviors . in humans , genetic variation has been suggested to be associated with neural tube defects and bipolar disorder . isoform variation in postmortem brain and cerebrospinal fluid has been described to be associated with schizophrenia , bipolar disorder and autism , suggesting ncam1 has many pleiotropic effects . although we have genetic evidence for locus interaction in the development of adhd , the specific biological mechanism and gene products are not clear . characterization of homeostatic and pathophysiological mechanisms of gene products on 11q and lphn3 are necessary in order to determine the precise mechanistic interaction and any discussion of the mechanism until then is purely speculative . the fact that the lphn311q interaction better describes the proton mrs metabolite differences in the cingulate gyrus , with the strongest findings demonstrated for myoinositol in the posterior cingulate gyrus , might be compatible with recent findings of gray matter volume reduction in adhd patients in the posterior cingulate and with reduced activation of the posterior cingulate gyrus detected by functional magnetic resonance imaging in adhd patients during sustained tasks and inhibition failure . overall this region is hypothesized to be important for the interplay of attention and motivation and in particular visuospatial attention . it is worth mentioning that the main difference regarding h - mrs data between this report and the original report of arcos - burgos et al . is that additional brain metabolites were known at the time of the making of this report . analyses of association of one locus marker data to h - mrs data are part of a different manuscript that is currently prepared for submission . evaluation of h - mrs data in the context of two - locus interaction had the main goal to show that the two - locus interaction was able of predict genetic effects on brain metabolism much better than a model of one - locus . as exemplified by the few reproducible genes discovered to date for adhd , there are numerous difficulties that must be overcome in mapping disorders that are thought to have complex origins , including low penetrance , heterogeneity , pleiotropy and epistasis . assaying and accounting for these potential complexities are of extreme importance when searching for the specific genetic variations that underlie disease susceptibility . in this case , accounting for epistasis disclosed a variant that would have been missed by traditional association analysis . in summary , we demonstrate that an interaction between rs6551665 within lphn3 and a region on chromosome 11q is involved in adhd susceptibility and substantially increases the or for having adhd compared to examining the lphn3 common variant associated with adhd on its own .

in previous studies of a genetic isolate , we identified significant linkage of attention deficit hyperactivity disorder ( adhd ) to 4q , 5q , 8q , 11q and 17p . the existence of unique large size families linked to multiple regions , and the fact that these families came from an isolated population , we hypothesized that two - locus interaction contributions to adhd were plausible . several analytical models converged to show significant interaction between 4q and 11q ( p<1 108 ) and 11q and 17p ( p<1 106 ) . as we have identified that common variants of the lphn3 gene were responsible for the 4q linkage signal , we focused on 4q11q interaction to determine that single - nucleotide polymorphisms ( snps ) harbored in the lphn3 gene interact with snps spanning the 11q region that contains drd2 and ncam1 genes , to double the risk of developing adhd . this interaction not only explains genetic effects much better than taking each of these loci effects by separated but also differences in brain metabolism as depicted by proton magnetic resonance spectroscopy data and pharmacogenetic response to stimulant medication . these findings not only add information about how high order genetic interactions might be implicated in conferring susceptibility to develop adhd but also show that future studies of the effects of genetic interactions on adhd clinical information will help to shape predictive models of individual outcome .

Introduction Methods and Results LPHN311q interaction Effects of the LPHN311q interaction on brain metabolism and ADHD Effects of the LPHN311q interaction on ADHD pharmacogenetics Discussion Supplementary Material

using multigenerational and extended pedigrees from a genetic isolate our group identified several loci significantly linked to attention deficit hyperactivity disorder ( adhd ) , namely on 4q , 5q , 8q , 11q and 17p . regions on 11q and 17p replicated linkage described by others . minimal critical regions for each one of these regions , defined by recombination events in affected individuals as well as the 1-lod - support criterion , have been reported elsewhere . given the significant linkage to different genomic regions , the existence of unique large size families linked to multiple regions , and the fact that these families came from an isolated population , we hypothesized that two - locus interaction contributions to adhd were plausible . to screen for possible interacting regions , we initially performed a correlation subset analysis between the linked regions . we used 134 nuclear families from the paisa genetic isolate and single - nucleotide polymorphisms ( snps ) spanning each minimal critical region with a resolution of 200 kb ( mapping resolution as well as snps selection criteria have been published in detail elsewhere ) . we calculated the non - parametric linkage ( npl ) score using genehunter with the weight function weight10 , where families that demonstrated nominal npl ( npl>1.00 ) to a region were included in the analysis ( that is , were coded as 1 for weight10 ) and those families not demonstrating linkage were excluded ( that is , were coded as 0 for weight10 ) . of the entire possible set of pair - wise comparisons , two gave increases in the npl statistic greater than two units when compared with linkage analyses using all families . selecting families linked to 17p increased the nonparametric linkage statistic from 0.55 to 3.88 on 11q ( 17p11q interaction ) and selecting families linked to 4q increased the npl statistic from 0.55 to 3.24 in an overlapping region on 11q ( 4q11q interaction ; figure 1a , supplementary information ) . using the method implemented in genehunter - twolocus , we found a maximal non - parametric score of 6.08 ( p<1 10 ) produced by snps rs1038426 ( 4q ) and rs1293344 ( 11q ; 4q11q interaction ; figure 1b , supplementary information ) and a maximal non - parametric score of 5.51 ( p<1 10 ) , produced by snps rs9227 ( 17p ) and rs1293344 ( 11q ; 17p11q interaction ; figure 1c , supplementary information ) . furthermore , power analyses to detect two interacting loci while considering adhd as a binary trait and also to detect interacting loci underlying quantitative traits , shows that , in general , our discovery sample exhibits exceptional power to detect two - locus interactions ( see procedures attached to the supplementary information ) . using a very stringent linkage disequilibrium map with a resolution of 68 kb ( two times stronger that the resolution recommended for covering this genomic area ) we also demonstrated that throughout the scanned region there was a remarkable total absence of linkage disequilibrium gaps , meaning that our genotyped markers covered any variation inside of the 4q - linked region . with this map , we identified a unique region of association and linkage of 240 kb . additional analysis showed that lphn3 common variant confers susceptibility to adhd affects brain metabolism and predicts effectiveness of stimulant medication . we showed that three markers harbored in lphn3 passed the test of heterogeneity and were significant after adjusting for multiple tests : rs6551665 ( odds ratio ( or)=1.23 , 95% confidence interval ( ci ) 1.091.37 , p=3.46 10 ) , rs1947274 ( or=1.23 , 95% ci=1.091.38 , p=5.41 10 ) and rs2345039 ( or=1.21 , 95% ci=1.081.35 , p=8.97 10 ) . given the demonstrated association of adhd susceptibility to rs6551665 ( g , allele ) , a snp harbored inside the genomic region coding for lphn3 , we next performed an association analysis using snps on 11q and 17p and conditioning on the fact of being a carrier of the g variant of susceptibility at rs6551665 . because of the rareness of individuals homozygous for the rs6551665 g allele we pooled them with g heterozygotes , when we conditioned on the fact of being a carrier of the g variant of susceptibility at rs6551665 , we were able to narrow down signals at 11q and 17p . however , the signal at 11q pointed to a region containing only three genes , whereas the signal at 17p spanned a genomic region containing dozens of genes . furthermore , given that testing three - locus interactions ( lphn311q17p ) will demand evaluation of thousands of models ( 22 for a dichotomic trait ) , we focused in this manuscript to describe results of the lphn311q interaction . this haplotype spans 166 kbps from intron 7 of the gene coding neural cell adhesion molecule 1 ( ncam1 ) , encompasses the tetratricopeptide repeat domain 12 ( ttc12 ) and the ankyrin repeat and kinase domain containing 1 ( ankk1 ) , and is adjacent to the 5 untranslated region of the dopamine receptor d2 ( drd2 ; figure 1a ) . to avoid the potential effects of genetic stratification , a known cause of type i error , we performed a transmission disequilibrium test analysis in the entire set of multigenerational , nuclear and trio paisa families . the or for the transmission of the susceptibility variants on 4q and 11q is 3.14 ( 95% ci=1.496.62 ; p<0.0027 ) compared with transmission of neither variant ( table 1b ) . both the association analysis and the transmission disequilibrium test demonstrate that the significance of the association on chromosome 11q is lost when not conditioning on the presence of the susceptibility variant within lphn3 . looking for replication , we performed a transmission disequilibrium test analyses for three additional samples : one from germany and two primarily european - american samples consisting of 95 trios collected at the national human genome research institute , bethesda , md , usa ( us1 ) and 240 trios from a sample collected at children 's hospital of philadelphia , philadelphia , pa , usa ( us2 ; table 1b ) . all these three samples were used for the replication of the lphn3 association to adhd . overall , all of these results show a similar pattern of interaction and suggest that the haplotype on 11q interacts cooperatively with the lphn3 susceptibility variant to increase the risk to adhd . a meta - analysis of the transmission disequilibrium test results from the four samples , using a random effects model , demonstrated a significant association to the transmission of both susceptibility variants on chromosome 4q and 11q ( or=2.46 , 95% ci=1.633.70 , p<0.00001 ; figure 1b and table 1c ) . to define how the above described lphn311q interaction modulates the original effects of the lphn3 susceptibility variant on brain metabolism , we next examined proton magnetic resonance spectroscopy ( h - mrs ) data of 18 individuals from the paisa genetic isolate to four metabolites , namely , n - acetylaspartate , myoinositol , choline and glutamine ( for all taken as the ratio to creatine ) , in several brain regions making up part of the frontal striatal the full two - locus interaction model was fit to the data by using linear regression , where y is the quantitative mrs metabolite phenotype , is the mean effect , a is the age at diagnosis , s is a code for gender ( males=0 , females=1 ) , d describes disease status ( unaffected=0 , affected=1 ) , xi , i=1 , 2 , is a variable modeling an additive effect ( 1 for homozygote for allele 1 , 0 for a heterozygote and 1 for a homozygote for allele 2 ) , zi is a dummy variable for a dominant effect ( 0.5 for homozygote for allele 1 , 0.5 for a heterozygote and 0.5 for a homozygote for allele 2 ) , ai and di refer to additive and dominant coefficients estimated for the single locus effect , and iaa , iad , ida and idd represent epistatic coefficients in the following model : this model was compared with a nested model lacking interaction coefficients by a likelihood ratio test ( lrt ) that follows a distribution with four degrees of freedom . examining the coefficients for each disclosed that for myoinositol in the right and left posterior cingulate , the iad coefficient for an interaction between an additive effect from the haplotype on 11q and a dominant effect from rs6551665 was contributing to the better fitting model ( iad left cingulate gyrus p=0.00342 ; iad right cingulate gyrus p=0.00298 ) . the results demonstrate that having two copies of the susceptibility haplotype on chromosome 11 and gg at rs6551665 correlates with a significant decrease in myoinositol in the two regions ( figure 2a and b ) . the results for choline in the right medial cingulate demonstrated that the iad coefficient for an interaction between an additive effect from rs6551665 and a dominant effect from the 11q haplotype was contributing to the better fitting model ( p=0.00968 ) . the results were plotted for an additive effect from rs6551665 and a dominant effect from the haplotype on 11q ( where having one or two copies of the susceptibility haplotype were grouped ) . the results demonstrate that having ag at rs6551665 and at least one copy of the susceptibility haplotype on 11q is related to an increase in choline in the right medial cingulate and that having ag at rs6551665 and no copies of the susceptibility haplotype on 11q is related to a decreased level of choline in the right medial cingulate region ( figure 2c ) . we next attempted to study the effect of the lphn311q interaction with regards to the pharmacogenetic consequences that the lphn3 adhd - susceptibility variant had on the treatment response to stimulant medication . we observed a significantly better fitting model for question 18 ( hyperactive impulsive dimension ) when the iad coefficient for interaction between additive effects at lphn3 and dominant effects at 11q are included ( pcorrected=0.0036 ) . overall results demonstrate that having gg at rs6551665 and two copies of the susceptibility haplotype on 11q is correlated with a significant improvement of symptoms after the treatment with stimulant medication , and that having aa at rs6551665 and fewer than two copies of the susceptibility haplotype on 11q is correlated with a poor response to stimulant medication treatment ( figure 2d ) . although statistically significant , the importance of the genetic lphn311q interaction regarding the treatment response to stimulant medication requires larger studies to both replicate and assess the contribution of this interaction to adhd symptoms and response to stimulant medication . this haplotype spans 166 kbps from intron 7 of the gene coding neural cell adhesion molecule 1 ( ncam1 ) , encompasses the tetratricopeptide repeat domain 12 ( ttc12 ) and the ankyrin repeat and kinase domain containing 1 ( ankk1 ) , and is adjacent to the 5 untranslated region of the dopamine receptor d2 ( drd2 ; figure 1a ) . to avoid the potential effects of genetic stratification , a known cause of type i error , we performed a transmission disequilibrium test analysis in the entire set of multigenerational , nuclear and trio paisa families . the or for the transmission of the susceptibility variants on 4q and 11q is 3.14 ( 95% ci=1.496.62 ; p<0.0027 ) compared with transmission of neither variant ( table 1b ) . both the association analysis and the transmission disequilibrium test demonstrate that the significance of the association on chromosome 11q is lost when not conditioning on the presence of the susceptibility variant within lphn3 . looking for replication , we performed a transmission disequilibrium test analyses for three additional samples : one from germany and two primarily european - american samples consisting of 95 trios collected at the national human genome research institute , bethesda , md , usa ( us1 ) and 240 trios from a sample collected at children 's hospital of philadelphia , philadelphia , pa , usa ( us2 ; table 1b ) . all these three samples were used for the replication of the lphn3 association to adhd . overall , all of these results show a similar pattern of interaction and suggest that the haplotype on 11q interacts cooperatively with the lphn3 susceptibility variant to increase the risk to adhd . a meta - analysis of the transmission disequilibrium test results from the four samples , using a random effects model , demonstrated a significant association to the transmission of both susceptibility variants on chromosome 4q and 11q ( or=2.46 , 95% ci=1.633.70 , p<0.00001 ; figure 1b and table 1c ) . to define how the above described lphn311q interaction modulates the original effects of the lphn3 susceptibility variant on brain metabolism , we next examined proton magnetic resonance spectroscopy ( h - mrs ) data of 18 individuals from the paisa genetic isolate to four metabolites , namely , n - acetylaspartate , myoinositol , choline and glutamine ( for all taken as the ratio to creatine ) , in several brain regions making up part of the frontal striatal the full two - locus interaction model was fit to the data by using linear regression , where y is the quantitative mrs metabolite phenotype , is the mean effect , a is the age at diagnosis , s is a code for gender ( males=0 , females=1 ) , d describes disease status ( unaffected=0 , affected=1 ) , xi , i=1 , 2 , is a variable modeling an additive effect ( 1 for homozygote for allele 1 , 0 for a heterozygote and 1 for a homozygote for allele 2 ) , zi is a dummy variable for a dominant effect ( 0.5 for homozygote for allele 1 , 0.5 for a heterozygote and 0.5 for a homozygote for allele 2 ) , ai and di refer to additive and dominant coefficients estimated for the single locus effect , and iaa , iad , ida and idd represent epistatic coefficients in the following model : this model was compared with a nested model lacking interaction coefficients by a likelihood ratio test ( lrt ) that follows a distribution with four degrees of freedom . they are myoinositol in the right posterior cingulate gyrus , myoinositol in the left posterior cingulate gyrus and choline in the right medial cingulate gyrus . examining the coefficients for each disclosed that for myoinositol in the right and left posterior cingulate , the iad coefficient for an interaction between an additive effect from the haplotype on 11q and a dominant effect from rs6551665 was contributing to the better fitting model ( iad left cingulate gyrus p=0.00342 ; iad right cingulate gyrus p=0.00298 ) . the results demonstrate that having two copies of the susceptibility haplotype on chromosome 11 and gg at rs6551665 correlates with a significant decrease in myoinositol in the two regions ( figure 2a and b ) . the results for choline in the right medial cingulate demonstrated that the iad coefficient for an interaction between an additive effect from rs6551665 and a dominant effect from the 11q haplotype was contributing to the better fitting model ( p=0.00968 ) . the results were plotted for an additive effect from rs6551665 and a dominant effect from the haplotype on 11q ( where having one or two copies of the susceptibility haplotype were grouped ) . the results demonstrate that having ag at rs6551665 and at least one copy of the susceptibility haplotype on 11q is related to an increase in choline in the right medial cingulate and that having ag at rs6551665 and no copies of the susceptibility haplotype on 11q is related to a decreased level of choline in the right medial cingulate region ( figure 2c ) . we next attempted to study the effect of the lphn311q interaction with regards to the pharmacogenetic consequences that the lphn3 adhd - susceptibility variant had on the treatment response to stimulant medication . these individuals were sampled from the original 240 individual sample described by our group with complete demographic information of this sample presented in the supplementary information . overall results demonstrate that having gg at rs6551665 and two copies of the susceptibility haplotype on 11q is correlated with a significant improvement of symptoms after the treatment with stimulant medication , and that having aa at rs6551665 and fewer than two copies of the susceptibility haplotype on 11q is correlated with a poor response to stimulant medication treatment ( figure 2d ) . although statistically significant , the importance of the genetic lphn311q interaction regarding the treatment response to stimulant medication requires larger studies to both replicate and assess the contribution of this interaction to adhd symptoms and response to stimulant medication . ankk1 was found to be expressed in placenta and spinal cord but to date has not been observed in the developing or adult brain . mouse knockout models of ncam1 have been described to mimic schizophrenia based on brain morphological changes as well as reduced prepulse inhibition of the startle response . although we have genetic evidence for locus interaction in the development of adhd , the specific biological mechanism and gene products are not clear . characterization of homeostatic and pathophysiological mechanisms of gene products on 11q and lphn3 are necessary in order to determine the precise mechanistic interaction and any discussion of the mechanism until then is purely speculative . the fact that the lphn311q interaction better describes the proton mrs metabolite differences in the cingulate gyrus , with the strongest findings demonstrated for myoinositol in the posterior cingulate gyrus , might be compatible with recent findings of gray matter volume reduction in adhd patients in the posterior cingulate and with reduced activation of the posterior cingulate gyrus detected by functional magnetic resonance imaging in adhd patients during sustained tasks and inhibition failure . overall this region is hypothesized to be important for the interplay of attention and motivation and in particular visuospatial attention . it is worth mentioning that the main difference regarding h - mrs data between this report and the original report of arcos - burgos et al . analyses of association of one locus marker data to h - mrs data are part of a different manuscript that is currently prepared for submission . evaluation of h - mrs data in the context of two - locus interaction had the main goal to show that the two - locus interaction was able of predict genetic effects on brain metabolism much better than a model of one - locus . in summary , we demonstrate that an interaction between rs6551665 within lphn3 and a region on chromosome 11q is involved in adhd susceptibility and substantially increases the or for having adhd compared to examining the lphn3 common variant associated with adhd on its own .

macular edema is a frequent cause of decreased visual acuity in patients with central retinal vein occlusion [ 1 , 2 ] . the mechanism of macular edema ( me ) in central retinal vein occlusion ( crvo ) is not completely understood . one of the main factors in the development of macular edema apart from increased venous pressure is the elevated level of vascular endothelial factor ( vegf ) with subsequent break down of the blood - retinal barrier and leakage [ 35 ] . a minor role exists for upregulation of other inflammatory mediators ( interleukin- ( il- ) 1 , il-2 , il-5 , il-8 , il-9 , il-10 , il-12 , il-13 , eotaxin , granulocyte colony stimulating factor , interferon - inducible 10 kda protein , monocyte chemotactic protein-1 , and interferon- ) as well as dysregulation of endothelial tight junctions [ 68 ] . the current standard of treatment based on several large randomized control trials ( rcts ) is anti - vegf injections [ 9 , 10 ] . corticosteroids decrease expression of vegf and several other inflammatory mediators and therefore may indirectly reduce vegf levels in the vitreous cavity [ 6 , 11 , 12 ] . corticosteroids also have an anti - inflammatory role reducing vascular permeability , inhibiting leukocyte movement , suppressing homing and migration of inflammatory cells , stabilizing endothelial tight junctions , and inhibiting prostaglandins and other cytokines [ 1315 ] . it is important to note that whilst anti - vegfs like ranibizumab and bevacizumab target vegf already present in the vitreous , corticosteroids affect the expression and production of vegf as well as other mediators . in addition it would seem that their mechanisms of action would seem synergistic and not competitive to one another . there are two major steroids that are currently being used in the treatment of central retinal vein occlusion : triamcinolone acetonide and the dexamethasone implant . both these drugs were studied in two large randomized controlled trials score for triamcinolone and geneva for the dexamethasone implant . this was one of the first large scale rcts that looked into the effectiveness of steroids as a treatment regimen . before the use of steroids the standard of care was observation , particularly after grid laser photocoagulation proved its ineffectiveness in the cvos study . the score study was a multicenter randomized clinical trial that included 271 participants randomized into 3 groups : observation , 1 mg triamcinolone , and 4 mg triamcinolone groups . the study used a preservative - free , nondispersive formulation to avoid a postinjection inflammatory reaction ( allergan inc . , irvine , california ; 4 mg brand name , trivaris ) . this drug is not available in common clinical practice and the more commonly used kenalog ( bristol - myers , squibb , princeton , new jersey , alcon inc . patients were injected on their initial visit and retreated at 4-month intervals according to their originally assigned treatments . patients in the observation group were not purely nave in that they could receive intravitreal triamcinolone when there was loss of 15 letters or more present on 2 consecutive 4-month intervals . the data from these patients was analyzed in the observation group which means that it might have altered some of the final results and indirectly the comparison between the two groups . to help put the results of this study in their proper context it is important to understand the major differences between score and another major study , the cruise study . the cruise study helped establish ranibizumab and anti - vegfs as the preferred treatment modality for crvo . with regard to baseline criteria the average duration of edema was 4 months , with 29% of participants having edema for less than 3 months , 81% of patients having edema for 6 months or less , and 19% of patients having an edema for more than 7 months . for cruise , the mean duration was 3.3 months with duration of <3 months in 69% of patients . the mean baseline va was 51 letters ( snellen equivalent , approximately 20/100 ) in score , compared to 48.3 letters in cruise . furthermore , only 2% of patients in score had ischemic crvo by the previous cvos definition of > 10 da of capillary nonperfusion . an average of 42% of patients had poor baseline va ranging from 20/125 to 20/400 and this is in contrast to cruise , where the numbers of patients with va ranging from 20/200 to 20/500 in the sham , 0.3 mg , and 0.5 mg ranibizumab groups were 27% , 30.3% , and 30% , respectively . patients with rapd were excluded from cruise , but it was not an exclusion criteria in score . in a recent post hoc analysis for score , higher baseline visual acuities , and less severe anatomical abnormalities of the retina ( center point thickness and areas of retinal hemorrhage , thickening , and fluorescein leakage ) were predictive of better visual acuity outcomes . in addition with regard to center point thickness outcomes , shorter duration of macular edema was associated with better outcomes . this meant that differences in baseline va and duration of edema between both studies might have favored better outcomes for cruise . the primary outcome and major end point results for the score - crvo are summarized in table 2 . the study showed that 6.8% , 26.5% , and 25.6% gained 15 letters or more at 12 months for the observation , 1 mg , and 4 mg groups , respectively . there was a change in mean visual acuity letter score of loss of 1 - 2 letters compared with a mean loss of 12 in the observation group . when subgroup analysis was done for patients with pseudophakia at baseline , a mean gain of 2 letters was seen in the 1 mg group compared to a loss of 1 letter in the 4 mg group and 14 letters loss in the observation group . this is in contrast to the results of cruise that showed that at 6 months 46.2% and 47.7% of patients showed 15 letters or more improvement in the 0.3 mg and 0.5 mg ranibizumab groups , compared to 16.9% in the sham group . in addition , the mean letter gains were 12.7 , 14.9 , and 0.8 in the 0.3 mg , 0.5 mg , and the sham groups , respectively . however , it is to be noted that at 12 months the group in score with baseline va ( 20/8020/100 ) showed the best gains with 7.8 letters gained in the 1 mg group and 2.8 letters in the 4 mg groups compared to 13 letters loss in observation . it is also to be noted that the percentage of patients with 15 letter gains was 47% , 38% , and 6% in the 1 mg , 4 mg , and observation groups , respectively . when put in context , perhaps had the differences in the baseline criteria of both studies been similar , the differences in visual outcomes could have been smaller ( table 2 ) . the score study showed that although at month 4 the median decrease in central macular thickness was greater in the 4 mg group ( 196 m decrease ) than the 1 mg ( 77 m decrease ) and the observation group ( 125 m decrease ) , the percentage of patients with cmt < 250 m was similar for the 3 study groups at the end of the first year of followup . this meant that the natural history of rvo ( also previously documented in the cvos study ) shows a gradual decrease in macular thickness despite differences in visual outcomes . however , perhaps the chronic nature of the edema leads to structural damage to the inner retina and even after edema resolved high va can not be achieved . this was also demonstrated in the cruise study which showed that the sham / observation group that once shifted to prn ranibizumab after 6 months showed improvement in va but did not catch up to the 0.3 or the 0.5 mg dose in terms of bcva at the end of the first year . the major complication during this trial was glaucoma ; 35% of patients in the 4 mg triamcinolone group initiated iop lowering medications compared with 20% in the 1 mg group and 8% in the observation group . four patients required tube surgery in the triamcinolone group during the 24 months of the study but it was deemed by the investigator that the cause of elevated iop was neovascular glaucoma . cataract was the second most common complication , 18% in the observation group compared with 26% and 33% in the 1 mg and 4 mg triamcinolone groups . no eyes in the observation or 1 mg group had cataract surgery during the first year and only 4 eyes in the 4 mg group . the geneva study was in fact two randomized , prospective , sham controlled clinical trials that looked at the effect of using two different doses of the dexamethasone implant ( 0.35 mg and 0.7 mg ) compared with a sham procedure group in patients with branch and central retinal vein occlusion . dexamethasone is a potent , water soluble steroid that is delivered to the vitreous cavity using an intravitreal implant ( ozurdex , allergan , inc . , irvine , ca ) . the drug - copolymer complex gradually releases the drug over several months after insertion . although it was initially estimated that the duration of action of the drug would be 6 months , a recent large scale rct in diabetic macular edema patients ( mead study ) showed that the actual efficacy is closer to 4 months . the primary end point for geneva study was 6 months and patients received only a single injection during this period leaving a chance for undertreatment . the extension open label phase of geneva followed up patients to 12 months and allowed patients to receive a 0.7 mg dex implant at 6 months if they met criteria for reinjection . 99% of patients in all groups were reinjected meaning that a single dex implant is ineffective at treating crvo . one measured the proportion of eyes achieving at least a 15-letter improvement from baseline bcva at day 180 and the other looked at the time needed to reach a 15-letter improvement from baseline . these outcomes are different from other trials such as score and cruise , which looked only at proportion achieving 15 letters or more improvement , making it difficult for cross study comparisons [ 9 , 16 ] . again we will cautiously try to make comparisons with cruise which was the major anti - vegf trial . attempts to compare patients based on baseline criteria and demographics are difficult because geneva used a combined pool of both brvo and crvo patients . in addition , there were twice as many patients with brvo ( 66% ) than crvo ( 34% ) . nonetheless , the mean baseline va was approximately 54 letters in the 3 groups compared with 48 letters in cruise . the average duration of edema was 5.2 months in geneva compared with 3.3 months in cruise . this is important because a recent post hoc analysis of data from geneva showed that each one - month increase in me duration was associated with a significantly lower likelihood of achieving better visual outcomes at 6 and 12 months . this association was stronger in brvo patients but was found to be weaker in crvo patients and was not statistically significant . this is in line with the data from score that found that chronicity in crvo was not a predictive factor for va outcomes . in addition , the percentage of patients having edema less than 3 months was 15.3%18% , compared to 69% in cruise indicating that patients in the geneva study had a more chronic edema [ 9 , 20 ] . at 6 months , the cumulative response rate ( time to achieve 15 letters of improvement from baseline ) in patients with both brvo and crvo was 41% in the 0.7 mg group , 50% in the 0.35 mg group , and 23% in the sham group . at day 180 , the proportion of eyes achieving 15-letter improvements in the dex group ( 22% ) was not statistically different than the sham group ( 18% ) . however there was a significant difference between them from day 30 to day 90 that disappeared as they reached the 180-day followup . the mean increase from baseline va was significantly greater in both dex implant groups than sham with the greatest difference between them at day 60 ( 7 letters ) . subgroup analysis for crvo patients showed there was a significant difference between mean change in bcva in the dex implant groups , compared to the sham group on days 30 , 60 , and 90 of followup . however , at 6 months there was no significant difference between the different groups with mean gain of 2 letters in the 0.35 mg group , 0 letters in the 0.7 mg group , and 2 letters in the sham group . the peak letter gain was approximately 10 letters in the 0.35 mg group and 9 letters in the 0.7 mg group at day 60 . in addition , the percentage of patients showing 15-letter improvements was significantly higher in the 0.35 mg and 0.7 mg doses compared to sham on days 30 , 60 , and 90 reaching a peak at day 60 with 33% of patients in the 0.35 mg group , 29% in the 0.7 mg group , and 9% in the sham group . however , by day 180 , the differences were not significant ( 18% in the 0.7 mg group , 17% in the 0.35 mg group , and 12% in sham group ) . it would appear that the maximum va gains are achieved at 60 days and that these gains are gradually lost as the drug loses efficacy over time . these fluctuations were also seen during the extension phase after the second injection that showed a peak in bcva around the 240-day mark that gradually decreased to preinjection levels at 360 days . the dex implant seems to have a short duration of action and by 6 months was no longer effective . had patients been allowed a second injection at 4 months or injections were guided based on an oct fluid based strategy final visual outcomes might have been higher . a post hoc analysis showed that the duration of me at baseline affected final visual outcomes ; eyes with a shorter duration of me ( < 90 days ) showed greater response than patients with longer duration of me . at day 60 , the number of patients achieving more than 15-letter improvement was 38% in the 0.7 mg group and 35% in the 0.35 mg group in eyes with edema < 90 days , compared to 27% in both dex groups in eyes with edema > 90 days . however in the subgroup analysis , patients with crvo did not show any difference in treatment response with regard to the duration of edema . these changes were also mirrored in the post hoc analysis of score . perhaps these differences indicate that with regard to steroids the duration of crvo is not a predictive factor to final visual outcomes . another possible explanation is that steroids might have a potent effect on chronic edema as demonstrated by the fame study for dme . therefore , with equal potency for acute and chronic edema , outcomes would be similar regardless of duration . there have been no large rcts comparing the efficacy of anti - vegf in patients with short and long durations of edema in patients with crvo . this means we still have unanswered questions as to whether steroids are the better treatment option in patients with chronic crvo . the only adverse events that occurred significantly more frequently in the dex implant treatment groups than the sham group were ocular pain , ocular hypertension , and anterior chamber cells . there was no significant difference between the incidence of cataract in the 0.7 mg dex implant group ( 7.3% ) , 0.35 mg dex implant group ( 4.1% ) , and sham group ( 4.5% ) . patients who received more than 2 injections had no differences in adverse events compared to the group that received only one implant except for cataract . the incidence of cataract adverse events ( aes ) was 29.8% ( 90/302 dex 0.7/0.7 mg group ) and 19.8% ( 56/283 dex 0.35/0.7 mg group ) in patients receiving 2 dex implants . this was higher compared to the group that received only a single injection during the first year , with 7.6% ( 5/66 ) and 7.7% ( 6/78 ) developing cataract in phakic patients receiving the 0.7 mg dex implant and 0.35 mg dex implant groups , respectively . in the group that did not receive any injections at all the rate of cataract adverse events was 5.7% ( 5/88 ) but the patient numbers were much lower . in the mead study for dme , patients were divided into 3 groups : 0.35 mg dex implant , 0.7 mg dex implant , and sham . patients were then followed up for 36 months and that gave an opportunity for long term complications to be examined noting of course that the mean number of treatments in the mead study for the 0.7 mg and 0.35 mg group was 4 - 5 versus 2 injections only in the geneva extension study . in the mead study , unlike geneva , there was a higher percentage of cataract formation with 67.9% , 54.1% , and 20.4% in the 0.7 mg implant , 0.35 mg implant , and sham groups , respectively . in addition cataract surgery rates were 59.2% , 52.3% , and 7.2% in the 0.7 mg , 0.35 mg , and the sham groups , respectively . this higher percentage can be explained by the longer duration of followup and the repeated treatments . with regard to ocular hypertension , there was a significant difference between the dex implant groups and the sham group . the intraocular pressure peaked at day 60 which also correlated with the peak visual gains achieved by patients in the dex group . this trend continued during the extension study with patients receiving the second injection again peaking at day 240 before dropping at day 360 . at the end of the study , the percentage of eyes receiving iop lowering medications in the dex implant group was 24% at 6 months , with an additional 10.3% at the end of 12 months [ 17 , 20 ] . six eyes in the group that received 2 dex implants and 6 eyes in the single dex group required laser or iop reducing surgeries of which 4 had neovascular glaucoma . it is interesting that the peak iop correlated with the peak improvements in va , which probably meant the iop increase was drug related and with the diminishing effect of the implant iop improved . the mead study for dme showed similar results with one - third of patients in each dex implant group showing an increase in iop requiring treatment during the study . however , as in geneva very few patients required incisional surgery for iop reduction ( 0.3% ) . mean iop also showed similar fluctuations as in geneva where iop peaked close to 3 months before returning to baseline levels at 6 months . because of the long term followup , it was found that in mead the incidence of iop related adverse events remained the same from year 1 to year 3 of the study with no apparent increase . interestingly the same number of patients using iop lowering medications remained the same from year to year . secondly , it would seem that only a certain percentage of patients seem to be affected and those who are not susceptible do not witness iop increases later with chronic use . in addition , iop increase is dependent on the effects of dexamethasone and once its levels decrease in the vitreous , intraocular pressure normalizes . no large randomized studies have compared between steroids and anti - vegfs . a small study by gado as and macky ta was conducted on 60 patients with nonischemic crvo in which patients were randomized into two groups , one receiving bevacizumab and the other receiving ozurdex implants . the study showed that there was no significant difference in bcva or macular thickness between the two groups at 6 months . , conducted on 32 patients , showed similar results at 9 months . a third study by chiquet et al . , conducted on 102 patients randomized to receive anti - vegfs and dexamethasone implants showed that at 3 months there was significantly better visual outcomes in the dex group with no difference in the cmt . however , these differences were not maintained and by the first year there was no difference in anatomical or visual outcomes . in addition elevated iop > 21 mmhg was more frequent in the dex group ( 21% ) compared to the anti - vegf group ( 3% , p = 0.008 ) . it is possible that the different outcomes of the three major rcts ( cruise , score , and geneva ) can be partly explained by differences in the initial baseline criteria . a recent study by thom et al . attempted to compare between trials using a combination of multinomial and indirect bayesian comparison models . it showed that there was a trend for greater ranibizumab associated visual gains compared with dexamethasone at months 1 and 6 in a common clinical context , although results were not classically significant . the difference in visual outcomes ( or lack thereof in certain cases ) , as well as the higher incidence of complications , means that anti - vegf would be the more preferable first line drug . nonetheless , steroids can be used as a second - line drug in resistant cases or as an adjunct from the start . a retrospective study by sharareh et al . looked at 18 patients categorized as complete or partial responders to bevacizumab that were given dexamethasone implants . the study showed that both subgroups responded with an improvement in both central macular thickness ( average 147 micrometers ) and visual acuity ( mean improvement of 0.25 logmar ) . the omar study compared between the effects of ozurdex and triamcinolone acetonide in cases of refractory cystoid macular edema despite repeated bevacizumab therapy due to retinal vein occlusion . it showed that adding steroids improved central macular thickness significantly ( p < 0.0001 ) . however , final bcva did not change significantly after steroid introduction ( p = 0.06 ) . as an adjunct , a case series by singer et al . showed that dexamethasone implant with bevacizumab showed a synergistic effect in crvo and brvo patients , increasing va and prolonging the time between injections , compared to either of these medications alone . at 6 months , 64% of patients had a maximum visual acuity gain of 3 lines compared to baseline and had a mean increase of 16.8 letters . compared between patients who received bevacizumab alone and patients who received combination therapy with dexamethasone implants . at 6 months , there was a greater reduction in mean cmt in the combined group compared to the monotherapy group , despite no significant differences in va . however several studies comparing between the effects of anti - vegfs and combined anti - vegfs and triamcinolone found no significant difference in bcva or crt between the two subgroups [ 25 , 3234 ] . these data show that dexamethasone implants and , to a lesser extent , ta may be a suitable option in resistant cases as monotherapy or as adjunct therapy to ranibizumab or bevacizumab . however more large scale rcts are needed to show true benefit of this treatment modality . as monotherapy , steroids alone have not been proven to be superior to anti - vegfs . with the higher incidence of side effects , especially after repeated dosing , anti - vegfs still remain the first - line drug for treating crvo . as an initial therapy , combined steroids and anti - vegfs in crvo have not proven any advantage over anti - vegfs alone and as such can not be recommended based on current data . nonetheless , the role of steroids as an alternative therapy in resistant cases ( alone or in combination with anti - vegfs ) remains a viable option . dexamethasone and triamcinolone both appear as viable options in resistant cases . in conclusion , there remains a limited role for steroids in current practice .

with the current widespread use of anti - vegfs in the treatment of central retinal vein occlusion ( crvo ) , the role for steroids has become greatly diminished . recent large scale randomized control trials ( rcts ) have established the efficacy and safety of anti - vegfs in the treatment of crvo . steroids are known to cause elevations in intraocular pressure as well as increase the risk of cataract formation . with that in mind many ophthalmologists are injecting steroids less frequently . this paper aims to review some of the data pertaining to the use of steroids either as a first line monotherapy , adjunct therapy , or an alternative therapy to help answer the question : is there currently any role for steroids in the management of crvo ?

1. Introduction 2. Steroids as Monotherapy 3. Steroids versus Anti-VEGF 4. Steroids as Adjunct or Alternative Therapy to Anti-VEGF 5. Summary

macular edema is a frequent cause of decreased visual acuity in patients with central retinal vein occlusion [ 1 , 2 ] . the mechanism of macular edema ( me ) in central retinal vein occlusion ( crvo ) is not completely understood . one of the main factors in the development of macular edema apart from increased venous pressure is the elevated level of vascular endothelial factor ( vegf ) with subsequent break down of the blood - retinal barrier and leakage [ 35 ] . a minor role exists for upregulation of other inflammatory mediators ( interleukin- ( il- ) 1 , il-2 , il-5 , il-8 , il-9 , il-10 , il-12 , il-13 , eotaxin , granulocyte colony stimulating factor , interferon - inducible 10 kda protein , monocyte chemotactic protein-1 , and interferon- ) as well as dysregulation of endothelial tight junctions [ 68 ] . the current standard of treatment based on several large randomized control trials ( rcts ) is anti - vegf injections [ 9 , 10 ] . corticosteroids decrease expression of vegf and several other inflammatory mediators and therefore may indirectly reduce vegf levels in the vitreous cavity [ 6 , 11 , 12 ] . corticosteroids also have an anti - inflammatory role reducing vascular permeability , inhibiting leukocyte movement , suppressing homing and migration of inflammatory cells , stabilizing endothelial tight junctions , and inhibiting prostaglandins and other cytokines [ 1315 ] . it is important to note that whilst anti - vegfs like ranibizumab and bevacizumab target vegf already present in the vitreous , corticosteroids affect the expression and production of vegf as well as other mediators . there are two major steroids that are currently being used in the treatment of central retinal vein occlusion : triamcinolone acetonide and the dexamethasone implant . this was one of the first large scale rcts that looked into the effectiveness of steroids as a treatment regimen . before the use of steroids the standard of care was observation , particularly after grid laser photocoagulation proved its ineffectiveness in the cvos study . patients in the observation group were not purely nave in that they could receive intravitreal triamcinolone when there was loss of 15 letters or more present on 2 consecutive 4-month intervals . the data from these patients was analyzed in the observation group which means that it might have altered some of the final results and indirectly the comparison between the two groups . to help put the results of this study in their proper context it is important to understand the major differences between score and another major study , the cruise study . the cruise study helped establish ranibizumab and anti - vegfs as the preferred treatment modality for crvo . for cruise , the mean duration was 3.3 months with duration of <3 months in 69% of patients . furthermore , only 2% of patients in score had ischemic crvo by the previous cvos definition of > 10 da of capillary nonperfusion . an average of 42% of patients had poor baseline va ranging from 20/125 to 20/400 and this is in contrast to cruise , where the numbers of patients with va ranging from 20/200 to 20/500 in the sham , 0.3 mg , and 0.5 mg ranibizumab groups were 27% , 30.3% , and 30% , respectively . patients with rapd were excluded from cruise , but it was not an exclusion criteria in score . in a recent post hoc analysis for score , higher baseline visual acuities , and less severe anatomical abnormalities of the retina ( center point thickness and areas of retinal hemorrhage , thickening , and fluorescein leakage ) were predictive of better visual acuity outcomes . there was a change in mean visual acuity letter score of loss of 1 - 2 letters compared with a mean loss of 12 in the observation group . when subgroup analysis was done for patients with pseudophakia at baseline , a mean gain of 2 letters was seen in the 1 mg group compared to a loss of 1 letter in the 4 mg group and 14 letters loss in the observation group . this is in contrast to the results of cruise that showed that at 6 months 46.2% and 47.7% of patients showed 15 letters or more improvement in the 0.3 mg and 0.5 mg ranibizumab groups , compared to 16.9% in the sham group . in addition , the mean letter gains were 12.7 , 14.9 , and 0.8 in the 0.3 mg , 0.5 mg , and the sham groups , respectively . however , it is to be noted that at 12 months the group in score with baseline va ( 20/8020/100 ) showed the best gains with 7.8 letters gained in the 1 mg group and 2.8 letters in the 4 mg groups compared to 13 letters loss in observation . it is also to be noted that the percentage of patients with 15 letter gains was 47% , 38% , and 6% in the 1 mg , 4 mg , and observation groups , respectively . when put in context , perhaps had the differences in the baseline criteria of both studies been similar , the differences in visual outcomes could have been smaller ( table 2 ) . the score study showed that although at month 4 the median decrease in central macular thickness was greater in the 4 mg group ( 196 m decrease ) than the 1 mg ( 77 m decrease ) and the observation group ( 125 m decrease ) , the percentage of patients with cmt < 250 m was similar for the 3 study groups at the end of the first year of followup . this meant that the natural history of rvo ( also previously documented in the cvos study ) shows a gradual decrease in macular thickness despite differences in visual outcomes . however , perhaps the chronic nature of the edema leads to structural damage to the inner retina and even after edema resolved high va can not be achieved . this was also demonstrated in the cruise study which showed that the sham / observation group that once shifted to prn ranibizumab after 6 months showed improvement in va but did not catch up to the 0.3 or the 0.5 mg dose in terms of bcva at the end of the first year . the major complication during this trial was glaucoma ; 35% of patients in the 4 mg triamcinolone group initiated iop lowering medications compared with 20% in the 1 mg group and 8% in the observation group . four patients required tube surgery in the triamcinolone group during the 24 months of the study but it was deemed by the investigator that the cause of elevated iop was neovascular glaucoma . cataract was the second most common complication , 18% in the observation group compared with 26% and 33% in the 1 mg and 4 mg triamcinolone groups . no eyes in the observation or 1 mg group had cataract surgery during the first year and only 4 eyes in the 4 mg group . the geneva study was in fact two randomized , prospective , sham controlled clinical trials that looked at the effect of using two different doses of the dexamethasone implant ( 0.35 mg and 0.7 mg ) compared with a sham procedure group in patients with branch and central retinal vein occlusion . dexamethasone is a potent , water soluble steroid that is delivered to the vitreous cavity using an intravitreal implant ( ozurdex , allergan , inc . , irvine , ca ) . the drug - copolymer complex gradually releases the drug over several months after insertion . although it was initially estimated that the duration of action of the drug would be 6 months , a recent large scale rct in diabetic macular edema patients ( mead study ) showed that the actual efficacy is closer to 4 months . the extension open label phase of geneva followed up patients to 12 months and allowed patients to receive a 0.7 mg dex implant at 6 months if they met criteria for reinjection . 99% of patients in all groups were reinjected meaning that a single dex implant is ineffective at treating crvo . one measured the proportion of eyes achieving at least a 15-letter improvement from baseline bcva at day 180 and the other looked at the time needed to reach a 15-letter improvement from baseline . again we will cautiously try to make comparisons with cruise which was the major anti - vegf trial . nonetheless , the mean baseline va was approximately 54 letters in the 3 groups compared with 48 letters in cruise . the average duration of edema was 5.2 months in geneva compared with 3.3 months in cruise . this is in line with the data from score that found that chronicity in crvo was not a predictive factor for va outcomes . in addition , the percentage of patients having edema less than 3 months was 15.3%18% , compared to 69% in cruise indicating that patients in the geneva study had a more chronic edema [ 9 , 20 ] . at 6 months , the cumulative response rate ( time to achieve 15 letters of improvement from baseline ) in patients with both brvo and crvo was 41% in the 0.7 mg group , 50% in the 0.35 mg group , and 23% in the sham group . at day 180 , the proportion of eyes achieving 15-letter improvements in the dex group ( 22% ) was not statistically different than the sham group ( 18% ) . however there was a significant difference between them from day 30 to day 90 that disappeared as they reached the 180-day followup . the mean increase from baseline va was significantly greater in both dex implant groups than sham with the greatest difference between them at day 60 ( 7 letters ) . subgroup analysis for crvo patients showed there was a significant difference between mean change in bcva in the dex implant groups , compared to the sham group on days 30 , 60 , and 90 of followup . however , at 6 months there was no significant difference between the different groups with mean gain of 2 letters in the 0.35 mg group , 0 letters in the 0.7 mg group , and 2 letters in the sham group . the peak letter gain was approximately 10 letters in the 0.35 mg group and 9 letters in the 0.7 mg group at day 60 . in addition , the percentage of patients showing 15-letter improvements was significantly higher in the 0.35 mg and 0.7 mg doses compared to sham on days 30 , 60 , and 90 reaching a peak at day 60 with 33% of patients in the 0.35 mg group , 29% in the 0.7 mg group , and 9% in the sham group . however , by day 180 , the differences were not significant ( 18% in the 0.7 mg group , 17% in the 0.35 mg group , and 12% in sham group ) . these fluctuations were also seen during the extension phase after the second injection that showed a peak in bcva around the 240-day mark that gradually decreased to preinjection levels at 360 days . at day 60 , the number of patients achieving more than 15-letter improvement was 38% in the 0.7 mg group and 35% in the 0.35 mg group in eyes with edema < 90 days , compared to 27% in both dex groups in eyes with edema > 90 days . however in the subgroup analysis , patients with crvo did not show any difference in treatment response with regard to the duration of edema . these changes were also mirrored in the post hoc analysis of score . perhaps these differences indicate that with regard to steroids the duration of crvo is not a predictive factor to final visual outcomes . therefore , with equal potency for acute and chronic edema , outcomes would be similar regardless of duration . there have been no large rcts comparing the efficacy of anti - vegf in patients with short and long durations of edema in patients with crvo . this means we still have unanswered questions as to whether steroids are the better treatment option in patients with chronic crvo . the only adverse events that occurred significantly more frequently in the dex implant treatment groups than the sham group were ocular pain , ocular hypertension , and anterior chamber cells . there was no significant difference between the incidence of cataract in the 0.7 mg dex implant group ( 7.3% ) , 0.35 mg dex implant group ( 4.1% ) , and sham group ( 4.5% ) . patients who received more than 2 injections had no differences in adverse events compared to the group that received only one implant except for cataract . the incidence of cataract adverse events ( aes ) was 29.8% ( 90/302 dex 0.7/0.7 mg group ) and 19.8% ( 56/283 dex 0.35/0.7 mg group ) in patients receiving 2 dex implants . this was higher compared to the group that received only a single injection during the first year , with 7.6% ( 5/66 ) and 7.7% ( 6/78 ) developing cataract in phakic patients receiving the 0.7 mg dex implant and 0.35 mg dex implant groups , respectively . in the group that did not receive any injections at all the rate of cataract adverse events was 5.7% ( 5/88 ) but the patient numbers were much lower . in the mead study for dme , patients were divided into 3 groups : 0.35 mg dex implant , 0.7 mg dex implant , and sham . patients were then followed up for 36 months and that gave an opportunity for long term complications to be examined noting of course that the mean number of treatments in the mead study for the 0.7 mg and 0.35 mg group was 4 - 5 versus 2 injections only in the geneva extension study . in the mead study , unlike geneva , there was a higher percentage of cataract formation with 67.9% , 54.1% , and 20.4% in the 0.7 mg implant , 0.35 mg implant , and sham groups , respectively . in addition cataract surgery rates were 59.2% , 52.3% , and 7.2% in the 0.7 mg , 0.35 mg , and the sham groups , respectively . with regard to ocular hypertension , there was a significant difference between the dex implant groups and the sham group . the intraocular pressure peaked at day 60 which also correlated with the peak visual gains achieved by patients in the dex group . at the end of the study , the percentage of eyes receiving iop lowering medications in the dex implant group was 24% at 6 months , with an additional 10.3% at the end of 12 months [ 17 , 20 ] . six eyes in the group that received 2 dex implants and 6 eyes in the single dex group required laser or iop reducing surgeries of which 4 had neovascular glaucoma . it is interesting that the peak iop correlated with the peak improvements in va , which probably meant the iop increase was drug related and with the diminishing effect of the implant iop improved . the mead study for dme showed similar results with one - third of patients in each dex implant group showing an increase in iop requiring treatment during the study . mean iop also showed similar fluctuations as in geneva where iop peaked close to 3 months before returning to baseline levels at 6 months . because of the long term followup , it was found that in mead the incidence of iop related adverse events remained the same from year 1 to year 3 of the study with no apparent increase . secondly , it would seem that only a certain percentage of patients seem to be affected and those who are not susceptible do not witness iop increases later with chronic use . in addition , iop increase is dependent on the effects of dexamethasone and once its levels decrease in the vitreous , intraocular pressure normalizes . no large randomized studies have compared between steroids and anti - vegfs . , conducted on 32 patients , showed similar results at 9 months . , conducted on 102 patients randomized to receive anti - vegfs and dexamethasone implants showed that at 3 months there was significantly better visual outcomes in the dex group with no difference in the cmt . however , these differences were not maintained and by the first year there was no difference in anatomical or visual outcomes . in addition elevated iop > 21 mmhg was more frequent in the dex group ( 21% ) compared to the anti - vegf group ( 3% , p = 0.008 ) . it is possible that the different outcomes of the three major rcts ( cruise , score , and geneva ) can be partly explained by differences in the initial baseline criteria . a recent study by thom et al . the difference in visual outcomes ( or lack thereof in certain cases ) , as well as the higher incidence of complications , means that anti - vegf would be the more preferable first line drug . nonetheless , steroids can be used as a second - line drug in resistant cases or as an adjunct from the start . looked at 18 patients categorized as complete or partial responders to bevacizumab that were given dexamethasone implants . the study showed that both subgroups responded with an improvement in both central macular thickness ( average 147 micrometers ) and visual acuity ( mean improvement of 0.25 logmar ) . the omar study compared between the effects of ozurdex and triamcinolone acetonide in cases of refractory cystoid macular edema despite repeated bevacizumab therapy due to retinal vein occlusion . compared between patients who received bevacizumab alone and patients who received combination therapy with dexamethasone implants . at 6 months , there was a greater reduction in mean cmt in the combined group compared to the monotherapy group , despite no significant differences in va . however several studies comparing between the effects of anti - vegfs and combined anti - vegfs and triamcinolone found no significant difference in bcva or crt between the two subgroups [ 25 , 3234 ] . these data show that dexamethasone implants and , to a lesser extent , ta may be a suitable option in resistant cases as monotherapy or as adjunct therapy to ranibizumab or bevacizumab . however more large scale rcts are needed to show true benefit of this treatment modality . as monotherapy , steroids alone have not been proven to be superior to anti - vegfs . with the higher incidence of side effects , especially after repeated dosing , anti - vegfs still remain the first - line drug for treating crvo . as an initial therapy , combined steroids and anti - vegfs in crvo have not proven any advantage over anti - vegfs alone and as such can not be recommended based on current data . nonetheless , the role of steroids as an alternative therapy in resistant cases ( alone or in combination with anti - vegfs ) remains a viable option . dexamethasone and triamcinolone both appear as viable options in resistant cases . in conclusion , there remains a limited role for steroids in current practice .

strong epidemiological evidence indicates that a sedentary lifestyle leads to an increased risk of developing certain cancers , including colon , prostate , breast , and endometrial cancers [ 13 ] . by contrast , physical activity has been shown to have a protective effect against their development . igf - i is a potent mitogen that promotes cellular proliferation and prevents apoptosis in normal and cancer cells [ 46 ] . igf - i axis hormones are important factors implicated in the beneficial influence of exercise . epidemiological studies have shown that increased levels of circulating igf - i or increased igf - i relative to igfbp-3 are associated with a higher risk of developing several forms of cancer , including prostate and breast cancers [ 712 ] . the combination of a low - fat diet and exercise program is known to reduce igf levels . [ 13 , 14 ] showed a decrease in the serum concentration of igf - i and an increase in igfbp-1 with the implementation of a low - fat diet and exercise program . furthermore , they reported that serum from men undergoing this form of intervention showed reduced cancer cell growth and increased apoptosis in lncap prostate cancer cells in vitro [ 13 , 15 ] . in conjunction with the changes in serum igf - i and igfbp-1 , reductions have also been shown in insulin and free testosterone , along with increases in sex hormone - binding globulin [ 15 , 16 ] . when igfbp-1 was added to preintervention serum , lncap cancer cells underwent apoptosis ; whereas , when igf - i was added to postintervention serum , the reduction in growth was eliminated . thus , the down - regulation of igf - i and up - regulation of igfbp-1 , as a result of the diet modification and exercise intervention program , may have protective effects against the development of cancerous cells . however , the training intensity and/or duration of exercise required to induce such favorable alterations has not yet been fully elucidated . a number of intervention studies have shown increased [ 1823 ] , decreased [ 24 , 25 ] , or unaltered [ 26 , 27 ] levels of igf - i after endurance or resistance training . similarly , many cross - sectional observational studies have examined the association of physical activity with igf - i levels in the general population ; however , their findings have been inconsistent [ 2832 ] . additionally , some reports have shown that circulating levels of igfbp-1 and igfbp-3 are also modulated with exercise [ 23 , 33 , 34 ] , whereas other groups saw no effects in their levels with moderate - intensity aerobic exercise or strength training [ 35 , 36 ] . the different responses of igf axis hormones to the intensity and/or duration of various types of exercise could be the reason why exercise is not necessarily protective against all forms of cancer . furthermore , it also has been reported that very heavy exercisers demonstrate high mortality ratios for lung , colorectal , and pancreatic cancers . whether exercise induces desirable ( i.e. , downregulation of igf - i and upregulation of igfbp-1 ) or undesirable ( i.e. , up - regulation of igf - i and down - regulation of igfbp-1 ) effects on cancer prevention is highly dependent on training intensity . as high - intensity exercise produces an acute increase in circulating igf - i levels [ 38 , 39 ] , the exercise intervention that induces a reduction in igf - i levels may be in the low or moderate categories . to the best of our knowledge , only one cross - sectional study has shown a reduction in serum igf - i levels and increase in igfbp-1 levels in participants of an exercise program , without emphasis on diet modification , in comparison to a control group . despite the fact that physical exercise is considered a strong intervention for cancer prevention , there are few studies that specifically assess the effects of exercise on specific biomarkers of cancer . in addition , it has been described in a previous review that persons at varying risks of developing cancer should be examined in exercise intervention studies , as the magnitude of protection caused by exercise may differ in high - versus low - risk individuals . furthermore , no previous intervention study has been conducted to examine whether an exercise regimen alone can elicit favorable changes in both igf - i and igfbp-1 levels for cancer prevention in healthy men . additionally , insulin is another factor which has the potential to influence the levels of igf - i and igfbp-1 after exercise training . hyperinsulinemia stimulates liver production of igf - i and suppresses igfbp-1 production [ 4 , 41 ] . it is also known that insulin itself promotes cellular growth in normal as well as malignant tissues [ 42 , 43 ] . in addition , leptin may also regulate changes in igf - i following exercise intervention . the lactate threshold ( lt ) represents the oxygen uptake ( vo2 ) or work rate above which there is a systemic rise in blood lactate levels during incremental exercise . the lt level corresponds to approximately 50% vo2max , and exercise at lt can be performed easily and safely even in the elderly and individuals with metabolic syndrome [ 46 , 47 ] . in a previous study by our group , low - intensity aerobic training at lt level caused an increase in insulin sensitivity ( si ) , with a concomitant decrease in the basal level of insulin . in this study we used this exercise training program as it was designed to have an insulin - sensitizing effect . we hypothesized that lt - level aerobic training , which does not stimulate igf - i production and reduces basal insulin levels , would result in simultaneous alterations in both the down - regulation of igf - i and up - regulation of igfbp-1 . thus , we investigated the effect of mild aerobic exercise training at lt level on the circulating levels of igf - i , igfbp-1 , and igfbp-3 to determine an optimal exercise intervention that can induce favorable changes in igf - i and igfbp-1 levels for cancer prevention in healthy men . fourteen healthy men ( 22.6 0.5 years ) who had not undergone any regular exercise for at least 2 years were examined . all subjects were nonsmokers , had no evidence of chronic disease , such as diabetes , hypertension or cancer , and were not taking any medication . all subjects were also asked to maintain their normal dietary habits and not to engage in any strenuous physical activity . before beginning the study , the nature , purpose , and risks of the study each subject 's body fat percentage was measured by hydrostatic weighing before commencing training and 2 days after the last training session . this was estimated based on the hydrostatic density with a correction for the residual lung volume . to measure physical fitness , a graded exercise test was performed on a mechanically braked ergometer ( electric bicycle ergometer , lode 's instrumenten b. v. , groningen , holland ) before commencing the training program and 2 days after the final training session . the work rate was initially set at 10 watts and increased every 4 seconds by 1 watt , until physical exhaustion . the volume of the expired gas was quantified with a twin - drum - type respirometer ( fukuda irika cr-20 , tokyo , japan ) , and both the o2 and co2 fractions were analyzed by a mass spectrometer ( arco-1000 , arco system inc . , blood samples were obtained from the earlobe every 30 seconds to measure blood lactate levels . the blood lactate concentration was plotted against the exercise workload for each subject , and the workload at the first breaking of lactate was used to calculate the exercise training intensity for each subject . the lt was determined for each subject based on a visual inspection , according to the estimations of three experts , who were blinded to the purpose of our study , and the average was used to establish the exercise intensity for training . cycle ergometer aerobic training at the lt level was performed at our laboratory for 60 minutes per day , five times a week for 6 weeks . blood samples were obtained from an antecubital vein each morning between 0700 and 0900 h , following overnight fasting , prior to training , and 1672 hours after the final training session . plasma glucose levels were measured spectrophotometrically using glucose oxidase ( glucose b - test ; wako pure chemical , osaka , japan ) ; serum insulin ( phadeseph insulin radioimmunoassay kit , shionogi , osaka , japan ) and leptin ( human leptin kit , linco research , missouri , usa ) concentrations were measured by radioimmunoassay ; and serum igf - i ( somatomedin - cii [ chiron ] measurement kit , chiron inc . , tokyo , japan ) , igfbp-1 ( dsl-7800 , diagnostic systems laboratories , inc . , texas , usa ) , and igfbp-3 ( ab tube igf - bp-3 eiken , diagnostic systems laboratories , inc . , texas , usa ) the intra- and interassay coefficient variations were 3.9% and 2.8% for igf - i , 4.6% and 6.0% for igfbp-1 , and 7.2% and 10.5% for igfbp-3 , respectively . ivgtts were performed before commencing the training program and 16 hours after the last training session . following overnight fasting , subjects were allowed to rest while lying down for at least 30 minutes prior to blood sampling . baseline samples for glucose and insulin were obtained , followed by glucose administration via the contralateral antecubital vein ( 300 mg / kg body weight ) within 2 minutes . subsequent samples were obtained at frequent intervals until 180 min , as previously described . insulin ( humalin ; shionogi , osaka , japan ) was infused ( 20 mu / kg ) via an antecubital vein between the periods of 2025 minutes post glucose administration . on the day before undergoing ivgtt , all subjects were provided with an evening meal consisting of 140 g carbohydrate , 30 g fat , and 33 g protein . the si index represents the net increase in glucose disappearance rate , which also depends on the rise in insulin above basal levels . the minimal model program was written using pascal programming ( borland international , ca ) on a macintosh iicx ( apple computer , ca ) . pearson 's correlation coefficient was used for the analysis of the correlation between the changes of variables after the exercise training . weight and body fat percentage did not change after the training period ( table 1 ) . by contrast , mild training significantly increased indices of aerobic fitness . both basal glucose and insulin levels significantly reduced after training ( table 2 ) . although iri , which was calculated using matthews ' formula , increased only slightly , the increase was significant . lt exercise was found to significantly decrease the circulating levels of igf - i , whereas igfbp-1 was significantly increased . there was no significant relationship between the baseline level of igf - i and baseline measures of vo2max ( r = 0.15 ) or lt - vo2 ( r = 0.20 ) . there were also no significant correlations between the change in circulating igf - i levels and the changes in aerobic fitness ( vo2max , r = 0.10 ) , basal insulin ( r = 0.08 ) , iri ( r = 0.12 ) , and si ( r = 0.41 ) after exercise intervention . similarly , relationships between the change in igfbp-1 and changes in basal insulin ( r = 0.33 ) and si ( r = 0.27 ) were not statistically significant . furthermore , the change in leptin levels after exercise was not significantly correlated with the changes in igf - i ( r = 0.22 ) or igfbp-1 ( r = 0.45 ) . conversely , there was a negative correlation between pre - training igf - i levels and individual changes in igf - i after training ( r = 0.77 ; p < .01 ) ( figure 1 ) . changes in vo2max showed a positive correlation with changes in igfbp-1 ( r = 0.61 ; p < .05 ) ( figure 2 ) . the main findings of this study were as follows : ( 1 ) short - term cycle ergometer aerobic training at lt level decreases circulating igf - i concentrations and increases igfbp-1 levels , without changing body weight , in previously sedentary men ; ( 2 ) there is an inverse relationship between pre - training igf - i levels and individual changes in igf - i after training , suggesting that individuals with a higher pre - training igf - i level will have a more substantial decrease in igf - i following exercise intervention ; and ( 3 ) increases in aerobic fitness ( vo2max ) positively correlates with changes in igfbp-1 level after training . it is known that alterations in the igf axis , including a reduction in igf - i and an increase in igfbp-1 , by lifestyle modification are associated with an in vitro reduction in prostate cancer cell ( lncap ) growth and increased apoptosis [ 13 , 16 ] . thus , the current insulin - sensitizing exercise program is a safe and easily - performed exercise program that simultaneously induces a down - regulation of igf - i and up - regulation of igfbp-1 . the physiological effects of decreased igf - i and increased igfbp-1 levels after mild aerobic training have not been clarified by this study . however , since igf - i infusion causes hypoglycemia , primarly by stimulating peripheral glucose uptake , and igfbps buffer the acute hypoglycemic effect of igf - i , we thus speculate that the alterations in igf - i and igfbp-1 levels may be an adaptive response to prevent hypoglycemia following insulin - sensitizing training . in a previous study of exercise - induced energy deficit , leptin administration significantly increased circulating levels of igf - i in healthy men and women . in this study , leptin levels were unchanged after the training program , and the individual changes in the levels of igf - i were not significantly correlated with the changes in leptin concentration . thus , leptin may not play a role in the alteration of igf - i after mild exercise in healthy men . the principal factor enhancing the production of igf - i in the liver is growth hormone , which stimulates igf - i synthesis and is further enhanced by insulin [ 41 , 52 ] . as fasting insulin levels are slightly but significantly decreased after exercise intervention , it could potentially contribute to the reduction in igf - i levels . some [ 19 , 22 , 39 ] , but not all [ 5355 ] , previous studies have reported increases in igf - i after acute exercise ; however , these increases are transient and typically return to baseline levels within 1015 min after exercise . increases in igf - i after acute exercise are considered to be unrelated to exercise - induced increases in growth hormone . moreover , increases in igf - i after acute exercise were observed in growth hormone - deficient subjects . we previously showed that acute bout of exercise at lt transiently increases growth hormone level . however , it is likely that resting levels of growth hormone are not influenced by low - intensity training . it is worthwhile noting that this study examines changes in systemic igf - i levels and that changes in local production ( i.e. , paracrine / autocrine effects ) are not assessed . thus , this study could not capture the potential effect of exercise on igf changes at the tissue level . a single bout of acute resistance exercise upregulates local ( i.e. , skeletal muscle ) igf - i [ 5961 ] . to the best of our knowledge , whether local igf - i is upregulated by low - intensity aerobic exercise a recent review suggested that local changes in igf - i are independent of changes in circulating igf - i , indicating that serum igf - i is not necessarily a reflection of local concentrations . thus , in this study , the reduced levels of systemic igf - i post - exercise intervention may not have been influenced by local igf - i . it has been considered that reduced insulin levels after exercise may contribute to the up - regulation of igfbp-1 . furthermore , exercise - induced changes in basal insulin levels did not significantly correlate with changes in igfbp-1 ; however , this may be due to the low number of subjects . conversely , the present results show a significant relationship between changes in vo2max and circulating igfbp-1 levels . additionally , it seems noteworthy that correlations between the change in igfbp-1 and changes in basal insulin ( r = 0.33 ) and si ( r = 0.27 ) were moderate and in the expected direction , although not statistically significant . although we are unable to explain the underlying mechanisms , the enhancement of aerobic fitness might be important in the up - regulation of igfbp-1 with low - intensity training . that low - intensity aerobic exercise ( 2 to 3 times / week at an intensity of 6080% maximal heart rate for 3045 min ) for 6 months improved si and increased igfbp-1 in healthy middle - aged men . although that study did not assess the level of aerobic fitness ( vo2max ) , fasting insulin levels decreased in the exercise group by 14% , with a 15% increase in igfbp-1 after training for 6 months . in this study a similar decrease in fasting insulin ( 13% ) caused a similar increase in igfbp-1 ( 16% ) after exercise intervention . this indicates that increases in igfbp-1 are regulated by changes in insulin induced by exercise training . a previous study showed that the changes in igf - i and igfbp-1 levels , induced by a combined low - fat diet and exercise program , were accompanied by a reduction in body weight . furthermore , in this cross - sectional study , participants in the exercise program with a lower igf - i and higher igfbp-1 had a much lower bmi , compared with the control group . by contrast , another report on the effects of aerobic exercise combined with a low - fat , high - complex carbohydrate diet showed reduced serum insulin and positive control over aspects of metabolic syndrome , including hypertension and hypertriglyceridemia , over a period of only 3 weeks , even though the subjects remained overweight or obese . examined igf - i and body mass changes associated with 7-day strenuous exercise and found a decrease in igf - i levels , even in weight - stable subjects . similarly , this study showed that alterations of circulating igf - i and igfbp-1 , basal insulin , and si after lt - level training were observed without changes in body weight and body fat percentage . thus , in conjunction with previous results , this suggests the importance of exercise alone , rather than changes in body composition , on the regulation of igf - i and igfbp-1 . there were some limitations of this study that should also be described . the short period ( 6 weeks ) of training intervention was a limiting factor in this study . further studies are needed to examine whether longer periods of exercise intervention induce sustained and greater impacts on the changes in insulin sensitivity , fasting insulin , and igf - i and igfbp-1 levels . information on dietary intake before and during the intervention was not recorded , despite the fact that diet is an important modifier of igf levels . because of this limitation , we are unable to perform a cross - sectional analysis of the relationship between dietary components and igf levels . since body weight did not decrease in this study , dietary intake does not need to be reduced during the exercise intervention . thus , reduced igf levels after the current aerobic training might not be affected by decreased dietary intake . the potential problems with the absence of a control group are the unwitting incorporation of a sampling bias . for example , the possibility that an inverse relationship between the initial ( baseline ) igf - i levels and the change in igf - i levels after training could be due to a convergence towards the mean effect can not be fully excluded . however , previous reports have shown that a single igf - i measurement is generally representative of the levels over a period of time and igf - i levels appear to have no detectable diurnal or circadian variation [ 29 , 65 ] . it is also worth mentioning that , although a lower level of igf - i is associated with a lower cancer risk in prospective healthy population , several studies have shown that lower igf - i is associated with an increased risk of cardiovascular disease , type ii diabetes , obesity [ 56 , 66 ] , osteoporosis , and cognitive decline . there are also reports of conflicting data showing that subjects with obesity or type ii diabetes have normal levels of total igf - i [ 67 , 68 ] and no significant correlation between igf - i and bone mineral density in women [ 69 , 70 ] . nindl and pierce have described in their recent review that the fact that there are studies claiming that both increases and decreases in igf - i concentrations have beneficial effects on health presents a contradictory situation . furthermore , they also stated that , even though local igf - i is consistently up - regulated with both acute and chronic exercises , circulating igf - i may actually decrease . however , despite lowering igf - i , the current exercise program would be expected to have beneficial effects on all of these various conditions . in this study , subjects were healthy men with a relatively low risk of developing cancer . although the changes in igf - i were most dramatic in men with high baseline levels , it is not clear whether this will apply to populations with different baseline levels . serum levels of igf - i are inversely associated with age [ 29 , 31 ] , and igf - i levels are higher in women than in men in western population . conversely , in the japanese population , the circulating igf - i level was found to be higher in men than in women . further work is needed to extend the present outcome to a wider population by examining whether the same exercise regimen at the lt can induce such favorable changes in individuals who have elevated igf - i levels and are at increased risk of developing cancer . in conclusion , we found that short - term aerobic exercisetraining at lt levels decreased circulating igf - i and increased igfbp-1 levels , without changing body composition , in previously sedentary men . these results are consistent with those of a previous rodent study demonstrating the beneficial effects of low - level exercise on cardiovascular , as well as cancer risk factors . it has been described that physical exercise deserves particular attention in the prevention of neoplasia , especially as it also exerts consistent beneficial effects on other major chronic diseases prevalent in the western world , such as atherosclerosis and type 2 diabetes . the current low - intensity aerobic training regimen is thus considered to be an effective approach in healthy men for down - regulating igf - i and up - regulating igfbp-1 levels .

increased concentrations of circulating insulin - like growth factor - i ( igf - i ) or igf - i relative to igf - binding proteins ( igfbps ) are associated with increased risk of developing several forms of cancer . conversely , exercise is linked with reduced risk . this study aims to investigate the effect of a low - intensity exercise program on circulating levels of igf - i , igfbp-1 , and igfbp-3 , in previously sedentary males . fourteen healthy men participated in cycle ergometer training at lactate threshold intensity for 60 min / day , 5 days / week for 6 weeks . after aerobic training , insulin sensitivity improved by 20% , while fasting insulin levels decreased by 13% . simultaneously , low - intensity aerobic training decreased the circulating levels of igf - i by 9% , while igfbp-1 levels increased by 16% . an interesting finding was that higher pretraining level of igf - i was associated with greater decline in igf - i with training . insulin - sensitizing low - intensity aerobic exercise is thus considered to be an effective method for downregulating igf - i and upregulating igfbp-1 levels .

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

strong epidemiological evidence indicates that a sedentary lifestyle leads to an increased risk of developing certain cancers , including colon , prostate , breast , and endometrial cancers [ 13 ] . igf - i is a potent mitogen that promotes cellular proliferation and prevents apoptosis in normal and cancer cells [ 46 ] . igf - i axis hormones are important factors implicated in the beneficial influence of exercise . epidemiological studies have shown that increased levels of circulating igf - i or increased igf - i relative to igfbp-3 are associated with a higher risk of developing several forms of cancer , including prostate and breast cancers [ 712 ] . the combination of a low - fat diet and exercise program is known to reduce igf levels . [ 13 , 14 ] showed a decrease in the serum concentration of igf - i and an increase in igfbp-1 with the implementation of a low - fat diet and exercise program . in conjunction with the changes in serum igf - i and igfbp-1 , reductions have also been shown in insulin and free testosterone , along with increases in sex hormone - binding globulin [ 15 , 16 ] . when igfbp-1 was added to preintervention serum , lncap cancer cells underwent apoptosis ; whereas , when igf - i was added to postintervention serum , the reduction in growth was eliminated . thus , the down - regulation of igf - i and up - regulation of igfbp-1 , as a result of the diet modification and exercise intervention program , may have protective effects against the development of cancerous cells . a number of intervention studies have shown increased [ 1823 ] , decreased [ 24 , 25 ] , or unaltered [ 26 , 27 ] levels of igf - i after endurance or resistance training . similarly , many cross - sectional observational studies have examined the association of physical activity with igf - i levels in the general population ; however , their findings have been inconsistent [ 2832 ] . additionally , some reports have shown that circulating levels of igfbp-1 and igfbp-3 are also modulated with exercise [ 23 , 33 , 34 ] , whereas other groups saw no effects in their levels with moderate - intensity aerobic exercise or strength training [ 35 , 36 ] . the different responses of igf axis hormones to the intensity and/or duration of various types of exercise could be the reason why exercise is not necessarily protective against all forms of cancer . furthermore , it also has been reported that very heavy exercisers demonstrate high mortality ratios for lung , colorectal , and pancreatic cancers . , downregulation of igf - i and upregulation of igfbp-1 ) or undesirable ( i.e. , up - regulation of igf - i and down - regulation of igfbp-1 ) effects on cancer prevention is highly dependent on training intensity . as high - intensity exercise produces an acute increase in circulating igf - i levels [ 38 , 39 ] , the exercise intervention that induces a reduction in igf - i levels may be in the low or moderate categories . to the best of our knowledge , only one cross - sectional study has shown a reduction in serum igf - i levels and increase in igfbp-1 levels in participants of an exercise program , without emphasis on diet modification , in comparison to a control group . despite the fact that physical exercise is considered a strong intervention for cancer prevention , there are few studies that specifically assess the effects of exercise on specific biomarkers of cancer . in addition , it has been described in a previous review that persons at varying risks of developing cancer should be examined in exercise intervention studies , as the magnitude of protection caused by exercise may differ in high - versus low - risk individuals . furthermore , no previous intervention study has been conducted to examine whether an exercise regimen alone can elicit favorable changes in both igf - i and igfbp-1 levels for cancer prevention in healthy men . additionally , insulin is another factor which has the potential to influence the levels of igf - i and igfbp-1 after exercise training . hyperinsulinemia stimulates liver production of igf - i and suppresses igfbp-1 production [ 4 , 41 ] . in addition , leptin may also regulate changes in igf - i following exercise intervention . the lactate threshold ( lt ) represents the oxygen uptake ( vo2 ) or work rate above which there is a systemic rise in blood lactate levels during incremental exercise . the lt level corresponds to approximately 50% vo2max , and exercise at lt can be performed easily and safely even in the elderly and individuals with metabolic syndrome [ 46 , 47 ] . in a previous study by our group , low - intensity aerobic training at lt level caused an increase in insulin sensitivity ( si ) , with a concomitant decrease in the basal level of insulin . in this study we used this exercise training program as it was designed to have an insulin - sensitizing effect . we hypothesized that lt - level aerobic training , which does not stimulate igf - i production and reduces basal insulin levels , would result in simultaneous alterations in both the down - regulation of igf - i and up - regulation of igfbp-1 . thus , we investigated the effect of mild aerobic exercise training at lt level on the circulating levels of igf - i , igfbp-1 , and igfbp-3 to determine an optimal exercise intervention that can induce favorable changes in igf - i and igfbp-1 levels for cancer prevention in healthy men . fourteen healthy men ( 22.6 0.5 years ) who had not undergone any regular exercise for at least 2 years were examined . all subjects were nonsmokers , had no evidence of chronic disease , such as diabetes , hypertension or cancer , and were not taking any medication . before beginning the study , the nature , purpose , and risks of the study each subject 's body fat percentage was measured by hydrostatic weighing before commencing training and 2 days after the last training session . this was estimated based on the hydrostatic density with a correction for the residual lung volume . to measure physical fitness , a graded exercise test was performed on a mechanically braked ergometer ( electric bicycle ergometer , lode 's instrumenten b. v. , groningen , holland ) before commencing the training program and 2 days after the final training session . the volume of the expired gas was quantified with a twin - drum - type respirometer ( fukuda irika cr-20 , tokyo , japan ) , and both the o2 and co2 fractions were analyzed by a mass spectrometer ( arco-1000 , arco system inc . the blood lactate concentration was plotted against the exercise workload for each subject , and the workload at the first breaking of lactate was used to calculate the exercise training intensity for each subject . the lt was determined for each subject based on a visual inspection , according to the estimations of three experts , who were blinded to the purpose of our study , and the average was used to establish the exercise intensity for training . cycle ergometer aerobic training at the lt level was performed at our laboratory for 60 minutes per day , five times a week for 6 weeks . blood samples were obtained from an antecubital vein each morning between 0700 and 0900 h , following overnight fasting , prior to training , and 1672 hours after the final training session . plasma glucose levels were measured spectrophotometrically using glucose oxidase ( glucose b - test ; wako pure chemical , osaka , japan ) ; serum insulin ( phadeseph insulin radioimmunoassay kit , shionogi , osaka , japan ) and leptin ( human leptin kit , linco research , missouri , usa ) concentrations were measured by radioimmunoassay ; and serum igf - i ( somatomedin - cii [ chiron ] measurement kit , chiron inc . , tokyo , japan ) , igfbp-1 ( dsl-7800 , diagnostic systems laboratories , inc . , texas , usa ) , and igfbp-3 ( ab tube igf - bp-3 eiken , diagnostic systems laboratories , inc . , texas , usa ) the intra- and interassay coefficient variations were 3.9% and 2.8% for igf - i , 4.6% and 6.0% for igfbp-1 , and 7.2% and 10.5% for igfbp-3 , respectively . on the day before undergoing ivgtt , all subjects were provided with an evening meal consisting of 140 g carbohydrate , 30 g fat , and 33 g protein . both basal glucose and insulin levels significantly reduced after training ( table 2 ) . lt exercise was found to significantly decrease the circulating levels of igf - i , whereas igfbp-1 was significantly increased . there was no significant relationship between the baseline level of igf - i and baseline measures of vo2max ( r = 0.15 ) or lt - vo2 ( r = 0.20 ) . there were also no significant correlations between the change in circulating igf - i levels and the changes in aerobic fitness ( vo2max , r = 0.10 ) , basal insulin ( r = 0.08 ) , iri ( r = 0.12 ) , and si ( r = 0.41 ) after exercise intervention . furthermore , the change in leptin levels after exercise was not significantly correlated with the changes in igf - i ( r = 0.22 ) or igfbp-1 ( r = 0.45 ) . conversely , there was a negative correlation between pre - training igf - i levels and individual changes in igf - i after training ( r = 0.77 ; p < .01 ) ( figure 1 ) . the main findings of this study were as follows : ( 1 ) short - term cycle ergometer aerobic training at lt level decreases circulating igf - i concentrations and increases igfbp-1 levels , without changing body weight , in previously sedentary men ; ( 2 ) there is an inverse relationship between pre - training igf - i levels and individual changes in igf - i after training , suggesting that individuals with a higher pre - training igf - i level will have a more substantial decrease in igf - i following exercise intervention ; and ( 3 ) increases in aerobic fitness ( vo2max ) positively correlates with changes in igfbp-1 level after training . it is known that alterations in the igf axis , including a reduction in igf - i and an increase in igfbp-1 , by lifestyle modification are associated with an in vitro reduction in prostate cancer cell ( lncap ) growth and increased apoptosis [ 13 , 16 ] . thus , the current insulin - sensitizing exercise program is a safe and easily - performed exercise program that simultaneously induces a down - regulation of igf - i and up - regulation of igfbp-1 . the physiological effects of decreased igf - i and increased igfbp-1 levels after mild aerobic training have not been clarified by this study . however , since igf - i infusion causes hypoglycemia , primarly by stimulating peripheral glucose uptake , and igfbps buffer the acute hypoglycemic effect of igf - i , we thus speculate that the alterations in igf - i and igfbp-1 levels may be an adaptive response to prevent hypoglycemia following insulin - sensitizing training . in a previous study of exercise - induced energy deficit , leptin administration significantly increased circulating levels of igf - i in healthy men and women . in this study , leptin levels were unchanged after the training program , and the individual changes in the levels of igf - i were not significantly correlated with the changes in leptin concentration . thus , leptin may not play a role in the alteration of igf - i after mild exercise in healthy men . the principal factor enhancing the production of igf - i in the liver is growth hormone , which stimulates igf - i synthesis and is further enhanced by insulin [ 41 , 52 ] . as fasting insulin levels are slightly but significantly decreased after exercise intervention , it could potentially contribute to the reduction in igf - i levels . some [ 19 , 22 , 39 ] , but not all [ 5355 ] , previous studies have reported increases in igf - i after acute exercise ; however , these increases are transient and typically return to baseline levels within 1015 min after exercise . increases in igf - i after acute exercise are considered to be unrelated to exercise - induced increases in growth hormone . moreover , increases in igf - i after acute exercise were observed in growth hormone - deficient subjects . however , it is likely that resting levels of growth hormone are not influenced by low - intensity training . it is worthwhile noting that this study examines changes in systemic igf - i levels and that changes in local production ( i.e. , paracrine / autocrine effects ) are not assessed . thus , this study could not capture the potential effect of exercise on igf changes at the tissue level . , skeletal muscle ) igf - i [ 5961 ] . to the best of our knowledge , whether local igf - i is upregulated by low - intensity aerobic exercise a recent review suggested that local changes in igf - i are independent of changes in circulating igf - i , indicating that serum igf - i is not necessarily a reflection of local concentrations . thus , in this study , the reduced levels of systemic igf - i post - exercise intervention may not have been influenced by local igf - i . it has been considered that reduced insulin levels after exercise may contribute to the up - regulation of igfbp-1 . furthermore , exercise - induced changes in basal insulin levels did not significantly correlate with changes in igfbp-1 ; however , this may be due to the low number of subjects . conversely , the present results show a significant relationship between changes in vo2max and circulating igfbp-1 levels . although we are unable to explain the underlying mechanisms , the enhancement of aerobic fitness might be important in the up - regulation of igfbp-1 with low - intensity training . that low - intensity aerobic exercise ( 2 to 3 times / week at an intensity of 6080% maximal heart rate for 3045 min ) for 6 months improved si and increased igfbp-1 in healthy middle - aged men . although that study did not assess the level of aerobic fitness ( vo2max ) , fasting insulin levels decreased in the exercise group by 14% , with a 15% increase in igfbp-1 after training for 6 months . in this study a similar decrease in fasting insulin ( 13% ) caused a similar increase in igfbp-1 ( 16% ) after exercise intervention . a previous study showed that the changes in igf - i and igfbp-1 levels , induced by a combined low - fat diet and exercise program , were accompanied by a reduction in body weight . furthermore , in this cross - sectional study , participants in the exercise program with a lower igf - i and higher igfbp-1 had a much lower bmi , compared with the control group . by contrast , another report on the effects of aerobic exercise combined with a low - fat , high - complex carbohydrate diet showed reduced serum insulin and positive control over aspects of metabolic syndrome , including hypertension and hypertriglyceridemia , over a period of only 3 weeks , even though the subjects remained overweight or obese . examined igf - i and body mass changes associated with 7-day strenuous exercise and found a decrease in igf - i levels , even in weight - stable subjects . similarly , this study showed that alterations of circulating igf - i and igfbp-1 , basal insulin , and si after lt - level training were observed without changes in body weight and body fat percentage . thus , in conjunction with previous results , this suggests the importance of exercise alone , rather than changes in body composition , on the regulation of igf - i and igfbp-1 . there were some limitations of this study that should also be described . the short period ( 6 weeks ) of training intervention was a limiting factor in this study . further studies are needed to examine whether longer periods of exercise intervention induce sustained and greater impacts on the changes in insulin sensitivity , fasting insulin , and igf - i and igfbp-1 levels . information on dietary intake before and during the intervention was not recorded , despite the fact that diet is an important modifier of igf levels . since body weight did not decrease in this study , dietary intake does not need to be reduced during the exercise intervention . thus , reduced igf levels after the current aerobic training might not be affected by decreased dietary intake . the potential problems with the absence of a control group are the unwitting incorporation of a sampling bias . for example , the possibility that an inverse relationship between the initial ( baseline ) igf - i levels and the change in igf - i levels after training could be due to a convergence towards the mean effect can not be fully excluded . however , previous reports have shown that a single igf - i measurement is generally representative of the levels over a period of time and igf - i levels appear to have no detectable diurnal or circadian variation [ 29 , 65 ] . it is also worth mentioning that , although a lower level of igf - i is associated with a lower cancer risk in prospective healthy population , several studies have shown that lower igf - i is associated with an increased risk of cardiovascular disease , type ii diabetes , obesity [ 56 , 66 ] , osteoporosis , and cognitive decline . there are also reports of conflicting data showing that subjects with obesity or type ii diabetes have normal levels of total igf - i [ 67 , 68 ] and no significant correlation between igf - i and bone mineral density in women [ 69 , 70 ] . nindl and pierce have described in their recent review that the fact that there are studies claiming that both increases and decreases in igf - i concentrations have beneficial effects on health presents a contradictory situation . furthermore , they also stated that , even though local igf - i is consistently up - regulated with both acute and chronic exercises , circulating igf - i may actually decrease . however , despite lowering igf - i , the current exercise program would be expected to have beneficial effects on all of these various conditions . in this study , subjects were healthy men with a relatively low risk of developing cancer . although the changes in igf - i were most dramatic in men with high baseline levels , it is not clear whether this will apply to populations with different baseline levels . serum levels of igf - i are inversely associated with age [ 29 , 31 ] , and igf - i levels are higher in women than in men in western population . conversely , in the japanese population , the circulating igf - i level was found to be higher in men than in women . further work is needed to extend the present outcome to a wider population by examining whether the same exercise regimen at the lt can induce such favorable changes in individuals who have elevated igf - i levels and are at increased risk of developing cancer . in conclusion , we found that short - term aerobic exercisetraining at lt levels decreased circulating igf - i and increased igfbp-1 levels , without changing body composition , in previously sedentary men . these results are consistent with those of a previous rodent study demonstrating the beneficial effects of low - level exercise on cardiovascular , as well as cancer risk factors . the current low - intensity aerobic training regimen is thus considered to be an effective approach in healthy men for down - regulating igf - i and up - regulating igfbp-1 levels .

huntington 's disease ( hd ) and parkinson 's disease ( pd ) are distinct neurodegenerative diseases that present with unique motor and cognitive symptoms . hd is an autosomal dominant inherited disease caused by the expansion of a polyglutamine repeat sequence in the huntingtin gene , which results in a progressive loss of medium spiny neurons in the striatum . it is marked by the loss of dopaminergic neurons of the substantia nigra pars compacta , which leads to abnormal basal ganglia circuitry , resulting in motor symptoms [ 2 , 3 ] . treatments for these diseases are symptomatic and new therapeutic targets are of the essence . emphasis is now being placed on the changes that occur at the synapse and the processes that underlie cognitive dysfunction as it has been shown that synaptic and cognitive dysfunction occurs long before the onset of clinical symptoms in the human [ 47 ] . glutamate receptors are currently viewed as valuable therapeutic targets in both hd and pd . n - methyl - d - aspartate ( nmda ) and alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa)-type glutamate receptors and their bound postsynaptic density - membrane associated guanylate kinases ( psd - maguks ) are critical for synapse development and plasticity [ 812 ] . maguks act as scaffolding molecules and are responsible for maintaining the structure of synapses , trafficking of receptors , and activating signalling molecules . psd-95 targets ampa receptors to the synapse through its interaction with stargazin and also binds directly to nmda receptor subunits ( glun2a and glun2b ) for synaptic targeting [ 14 , 15 ] . sap97 binds directly to the glua1 subunit of ampa receptors to traffic them to the psd and the glun2 subunits of nmda receptors and together with cask traffic nmda receptors through a unique secretory pathway to the psd . it is evident that maguks could play a role in the pathogenesis of some neurodegenerative diseases [ 19 , 20 ] . with regard to huntington 's disease , normal huntingtin is associated with nmdars via psd-95 but mutant huntingtin impairs the interaction between psd-95 and huntingtin , leading to excitotoxicity through increased nmda receptor activity , which is a key feature of this neurodegenerative disease [ 2224 ] . in the striatum of yac128 hd model mice , increased levels of psd-95 as well as increased psd-95-glun2b interactions are observed in extrasynaptic regions [ 25 , 26 ] . a reorganisation of postsynaptic density proteins , including a switch of psd-93 by psd-95 in the striatum of the r6/1 hd mouse model , has also been described . in the n171 - 82q transgenic hd mouse model , a decrease in striatal psd-95-like proteins was observed . in parkinson 's disease , there is a reduced interaction between nmdars and maguks in the striatum of 6-ohda lesioned pd animal models , as well as a change in the subcellular distribution and levels of psd-95 and sap97 . to date , animal models of hd and pd have provided valuable information on how synaptic structure and function may be altered in these human diseases ; however the results with respect to changes in the glutamatergic synapse vary between studies and between models [ 20 , 30 ] . to determine the changes that occur with neurodegenerative diseases of the human brain , it is imperative to examine the postmortem brain tissue of patients who died from these disorders . here we have used postmortem human brain tissue to investigate whether changes in synaptic protein expression occur in response to human neurodegenerative disease and thereby may play an important role in the changes in synapse function that occur in disease . we focused on the maguks psd-95 and sap97 as they play a major role in regulating glutamate receptor trafficking to synapses and also glutamate receptor localisation at synapses [ 810 , 18 , 31 ] . our data show that changes occurring in psd-95 , sap97 and glutamate receptor subunit expression in the human hd hippocampus differ from changes in the yac128 hd animal model , suggesting that unique changes occur in the human brain in response to neurodegenerative disease that vary across different brain regions . human brain tissue was obtained from the neurological foundation of new zealand human brain bank ( centre for brain research , university of auckland ) . the consent and research protocols used in this study the post - mortem human brain tissue was processed and dissected as described elsewhere [ 32 , 33 ] . briefly , brains were perfused via the basilar and carotid arteries with phosphate buffered saline ( 1% sodium nitrite ) and then with 15% formalin in 0.1 m phosphate buffer ( ph 7.4 ) . brains were dissected into the different functional parts including blocks of the striatum and hippocampus . the blocks were then cryoprotected in 20% sucrose 0.1 m phosphate buffer with 0.1% sodium - azide and frozen at 80c . the hippocampal and striatal frozen tissue blocks were cut into 50 m coronal sections on a microtome and stored in pbs - azide at 4c until used for immunohistochemistry . for hippocampal studies , a total of 34 human brains were examined , 12 control cases , 11 hd cases , and 11 pd cases ( see table 1 ) . for the striatum , a total of 21 brains were examined , 7 control cases , 8 hd cases , and 6 pd cases ( see table 2 ) . free - floating tissue sections were first incubated in pbs - triton ( 0.2% ) overnight at 4c . tissue was washed in citric acid buffer ( ph 4.5 ) and sections then heated in the microwave for 30 seconds on high power for antigen retrieval if needed . the sections were cooled to room temperature and washed with pbs - t ( 3 10 min ) . the sections were then incubated in 50% methanol , 0.9% hydrogen peroxide solution for further antigen retrieval and for blocking endogenous peroxidise activity . sections were incubated for 72 hours ( 4c ) in primary antibodies against mouse glua2 ( neuromab , 75 - 002 ) 1 : 500 , rabbit psd-95 ( sigma , hpa010122 ) 1 : 300 , rabbit sap97 ( abr , pa1 - 741 ) 1 : 1000 , and mouse glun1 ( millipore , mab363 ) 1 : 300 . all antibodies were checked for specificity by western blot analysis ( see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/938530 ) . sections were then washed and incubated overnight at room temperature with the respective biotinylated secondary antibodies , goat anti - mouse ( sigma , b7264 ) 1 : 500 and goat anti - rabbit ( sigma , b7389 ) 1 : 1000 . sections were again washed and incubated in the tertiary antibody , extravadin peroxidase ( sigma , e2886 ) 1 : 1000 for 4 hours at room temperature . the chromogen was 0.05% 3,3-diaminobenzidine tetrahydrochloride ( dab ; sigma , d5637 ) and 0.01% h2o2 in 0.1 m phosphate buffer , ph 7.4 for 1020 min . sections were mounted , dehydrated , and cleared in xylene before being coverslipped and imaged . immunohistochemical staining was repeated a minimum of 3 times for each control , hd and pd cases . twelve - month - old male and female yac128 transgenic hd mice expressing the human huntingtin protein containing a 128 cag repeat were utilised for this study . at this age mice were housed and tissue harvested according to protocols approved by the university of auckland animal ethics committee . coronal whole brain ( 30 m ) free - floating tissue sections were first incubated in pbs - triton ( 0.2% ) overnight at 4c . the sections were then incubated in 50% methanol , 0.9% hydrogen peroxide solution for further antigen retrieval and for blocking endogenous peroxidise activity . hereafter the sections were incubated in 5% normal goat serum in pbst for 1 hour at room temperature . sections were then incubated for 72 hours ( 4c ) in primary antibodies against mouse glua2 ( alomone ) 1 : 200 ( wt n = 6 , yac n = 5 ) , rabbit psd-95 ( sigma ) 1 : 200 ( wt n = 6 , yac n = 5 ) , rabbit sap97 ( abr ) 1 : 1000 ( wt n = 6 , yac n = 5 ) , and mouse glun1 ( neuromab ) 1 : 300 ( wt n = 5 , yac n = 6 ) . procedures for secondary and tertiary antibody incubations were exactly as for the human brain sections , except that a 1 : 500 dilution was used . images were first collected on a nikon te2000 inverted microscope in bright - field mode . the imaging conditions were optimised for each antibody but kept constant for all control and diseased cases immunostained with each antibody . for each human case ( control , hd , or pd ) , 10x z - stack images ( 1280 960 pixels ) were taken in each hippocampal and striatal region at 2 m apart at 40x magnification , allowing the cell bodies and apical and basal dendrites to be clearly visible . for the hippocampus , images were collected in the dentate gyrus and ca3 and ca1 regions . in the striatum images a minimum of 2 sections were collected from each hippocampal and striatal region in every human brain analysed . z - stack images were converted to z - projections in image j and the coloured images converted to grey scale images ( 8-bit ) . background was measured on each z - projection image individually and automatically subtracted for each image . the image was then inverted so that density measurements were made in arbitrary units where a value of 255 is a complete transparency and a value of 0 is complete darkness . a similar method for analysis of dab immunohistochemistry in the human brain using image j has been used successfully . densitometric values for each immunohistochemistry set were normalised to the average of the normal cases immunostained in parallel to get the relative change in intensity compared to the control group ( independent of experimental set ) . intensity values for hd and pd tissues are presented as a ratio of the grey value divided by the control ( non - diseased tissue ) density value sem . quantitative immunohistochemical changes in hippocampal synaptic protein levels were also examined by quantitative western blot analysis to validate the consistency of the quantified changes ( figure s1 ) . data are presented as mean sem , and one way analysis of variance ( anova ) or two - tailed student 's t - test was used to compare the densitometric measurements between control and diseased groups . protein extracts from the human hippocampus of control and hd cases were denatured in laemmli loading buffer ( sigma , s3401 ) at 95c for 5 min . protein extracts ( 30 g per sample ) were separated by gel electrophoresis ( nupage 412% bis - tris gel ; invitrogen , np0335 ) and transferred to polyvinylidene difluoride ( pvdf ) membrane ( amersham rpn303f ) . after blocking with 5% skim milk , the membranes were probed with primary antibodies and detected with species - specific horseradish - peroxidase - conjugated secondary antibodies ( millipore ) and developed using ecl reagents ( amersham , rpn2132 ) . primary antibodies used were directed against glur2 ( neuromab , 75 - 002 ) 1 : 500 , psd-95 ( sigma , hpa010122 ) 1 : 500 , sap97 ( abr , pa1 - 741 ) 1:500 , and nr1 ( millipore , mab363 ) 1 : 300 . the bands were visualised with the fuji film las-4000 scanner , and quantification of western blot intensity was analysed using the gel analyser on image j software ( nih usa , public domain ) ( figure s1 ) . sap97 , psd-95 , glua2 , and glun1 are known to play major roles at synapses within the dentate gyrus and areas ca3 and ca1 of the hippocampus [ 9 , 10 , 18 , 31 ] . we were interested to determine ( i ) whether specific subregional changes occur in these proteins in hd or pd human hippocampus or striatum and ( ii ) whether the two neurodegenerative diseases pd and hd , which have different mechanisms of causation [ 13 ] , differentially affect glutamatergic synaptic proteins in these brain regions . the hippocampus was a major focus as many hd and pd patients have dementia , depression , cognitive decline , and other nonmotor symptoms as well as the classic well - characterised motor symptoms . quantitative western blot analysis of hippocampal tissue from control and hd post - mortem human brain tissue revealed that significant changes in synaptic protein levels were occurring with hd . specifically , we observed that significant increases in the protein levels of psd-95 , sap97 , and the glun1 subunit of the nmda receptor occurred in hd hippocampal post - mortem tissue compared to controls ( figure s1 ) , suggesting that differential changes are occurring in synaptic proteins in hd . to provide more specific information on potential changes in immunoreactivity patterns of synaptic proteins in the principal neurons in hd and pd brain tissues compared to controls the expression of psd-95 , sap97 , and glutamate receptor subunits glun1 and glua2 were examined in the principle neurons of the dentate gyrus and ca3 and ca1 regions of the hippocampus , and in the caudate nucleus and putamen of the striatum . immunohistochemistry and imaging criteria were kept constant for control and diseased cases to enable detection of changes in expression levels of the different synaptic proteins . we first investigated expression changes in psd-95 in the human hippocampus and striatum ( figures 1(a)1(e ) ) . strong psd-95 immunostaining was evident in the cell bodies and in the dendrites in both control and diseased hippocampal and striatal neurons ( figures 1(c)1(e ) ) . in hd postmortem brain , a significant 1.52 0.26 fold increase in psd-95 was observed in neurons in area ca3 of the hippocampus ( n = 6 , control n = 8 ; p < 0.05 ) and in the dentate gyrus ( hd dg : 1.45 0.22 , n = 6 , control n = 8 , p = 0.05 ) . no significant change was measured in neurons in area ca1 ( hd ca1 : 1.45 0.31 , n = 6 , control n = 8 , p = 0.13 ) . in pd post - mortem tissue , a significant 1.37 0.19 fold increase in the neurons in the dentate gyrus ( pd n = 6 , control n = 8 ; p < 0.05 ) and a significant 2.44 0.73 fold increase in area ca1 ( pd n = 6 , control n = 8 ; p < 0.05 ; figure 1(a ) ) regions were also observed . however , psd-95 levels in the neurons in the ca3 region in pd were not significantly different from control levels ( 0.99 0.12 ; pd n = 4 , control n = 8) . in the striatum we observed stark changes in psd-95 expression . in the hd striatum the expression of psd-95 was significantly decreased in both the caudate nucleus ( hd mean = 0.40 0.12 , n = 3 , control n = 4 ; p < 0.005 ) and putamen ( hd mean = 0.51 0.09 , n = 3 , control n = 3 ; p < 0.005 ; figure 1(b ) ) . however , there were no changes in pd caudate nucleus ( 1.02 0.25 ; pd n = 4 , control n = 4 ) or putamen ( 1.16 0.22 ; pd n = 4 , control n = 4 ) . we next examined whether similar changes also occur in sap97 expression and observed that the changes in sap97 were different to those observed for psd-95 . immunohistochemical analysis of hippocampal and striatal sections in dentate gyrus , ca3 , ca1 , and caudate nucleus , and putamen revealed that increases in sap97 expression were found to occur in both the pd and hd human hippocampus but not in the striatum ( figures 2(a)2(d ) ) . sap97 immunostaining was evident in the neuronal cell body layers and dendritic regions throughout the hippocampus and striatum ( figures 2(c)2(e ) ) . consistent with its role in trafficking receptor complexes through the secretory pathway and along dendrites [ 18 , 36 ] , sap97 appeared diffusely along dendrites ( figure 2(e ) ) . sap97 expression in each hippocampal region was found to increase to a similar degree in both hd and pd and occurred in both cell body and dendritic regions of the hippocampus . in the dentate gyrus , sap97 was increased 2.06 0.25 fold in hd ( n = 6 ; p < 0.005 ) and similarly increased 1.91 0.22 in pd ( n = 9 ; p < 0.05 ) above control levels ( n = 6 ) . in the ca3 region , sap97 was increased 2.22 0.51 fold in hd ( n = 5 ; p < 0.05 ) and 1.62 0.26 fold in pd ( n = 10 ; p < 0.05 ) above control ( n = 6 ) . in the ca1 region sap97 was significantly increased 2.15 0.31 fold in hd ( n = 8 ; p < 0.005 ) and 1.88 0.35 in pd ( n = 6 ; p < in contrast , in the striatum there were no significant changes in the expression of sap97 in the caudate nucleus in hd ( n = 6 ) or pd ( n = 5 ) , nor in the putamen in hd ( n = 6 ) or pd ( n = 5 ) as compared to control ( caudate nucleus n = 5 , putamen n = 4 ; figure 2(b ) ) . overall these results indicate that sap97 expression is significantly altered throughout the hippocampus in human hd and pd , but not in the striatum . moreover , these data show that the maguk proteins psd-95 and sap97 are differentially affected by different neurodegenerative diseases in the human brain and that these changes can differ in hippocampus versus striatum . in the hippocampus , we performed quantitative immunohistochemistry to examine whether ampa receptor subunit expression is altered in pd or hd in the human hippocampus and striatum by examining the expression of the glua2 subunit common to both these receptor subtypes that could therefore reflect changes in either subtype of receptor ( figure 3 ) . glua2 immunostaining was again evident in the hippocampal and striatal neuronal cell bodies and the dendritic regions in both control and diseased human tissue ( figures 3(c)3(e ) ) . no significant changes in glua2 subunit expression were observed in the hd or pd hippocampus dentate gyrus , area ca3 , or area ca1 , or in the striatal caudate nucleus ( hd n = 4 , pd n = 5 , control n however , significant decreases in glua2 expression were observed in the putamen relative to control levels ( hd mean = 0.52 0.07 of control levels , n = 4 , p < 0.005 ; pd mean = 0.67 0.08 of control , n = 5 , p < 0.05 ; figures 3(a)3(d ) ) . we also examined potential changes in the expression of glun1 , the obligatory subunit of the nmda receptor ( figure 4 ) . strong glun1 immunostaining was evident throughout the somatic and dendritic regions in both hippocampal and striatal neurons ( figures 4(c)4(e ) ) . we observed significant increases in glun1 expression above control levels in neurons in the hd hippocampus ( figure 4(a ) ) . specifically , a significant 2.49 0.62 fold increase occurred in the dentate gyrus ( hd n = 8 , control n = 8 , p < 0.05 ) and a significant 3.17 0.89 fold increase in area ca1 ( hd n = 8 , control n = 7 , p < 0.05 ) . no changes occurred in pd hippocampus in either the dentate gyrus , area ca3 , or area ca1 ( pd n = 6 , control n = 7 ) . in the striatum , no change in glun1 expression was observed in either disease in the caudate nucleus or putamen ( hd n = 4 , pd n = 4 , control n = 4 ; figure 4(b ) ; p > 0.05 in all cases ) . together these data show that different changes in nmda and ampa receptor subunit expression levels occur in the hippocampal and striatal human brain regions in response to hd versus pd . we were particularly intrigued by the significant changes in the expression levels of psd-95 , sap97 , and glun1 in the human hd hippocampus ( figures 14 , figure s1 ) . although hd is considered a motor disorder , there is significant evidence that cognitive effects appear before the motor symptoms suggesting a role of hippocampal changes in the disease [ 1 , 3840 ] . in animal models of hd , striatal changes in glutamatergic synapse structure and function have been widely addressed ( e.g. [ 22 , 2426 , 61 ] ) ; however the hippocampus in yac128 hd model mice has been shown to be spared of atrophy and degeneration [ 41 , 42 ] . we wanted to determine whether the changes in maguk and glutamate receptor subunit expression we observed in the human hippocampus in hd were also occurring in the hippocampus of the yac128 mouse model of hd . immunohistochemistry was performed on hippocampal sections from yac128 mice at 12 months of age , when the animals are highly symptomatic and in the late stage of hd and therefore are comparable to tissue from post - mortem human hd patients . as expected , sap97 , psd-95 , glua2 and glun1 immunostaining was observed strongly in the dendritic regions of neurons in area ca1 , however , densitometry quantification revealed that none of the changes in sap97 , psd-95 , and glun1 that we observed in human post - mortem hd hippocampus occurred in yac128 mice hippocampus . we observed no significant changes in expression levels for any of these synaptic proteins compared to control tissue in either dentate gyrus , area ca1 , or area ca3 of yac128 hippocampus ( figure 5 ) . to ensure that our immunohistochemical analysis could detect changes in protein expression levels in yac128 tissue , we examined the expression changes of darpp-32 to act as a positive control in both yac128 and wildtype striatum . darpp-32 is a marker of dopaminergic neurons in the striatum and has been reported to decrease in yac128 striatum [ 41 , 4345 ] . indeed we observed that darpp-32 levels were significantly decreased in yac128 striatum to 0.42 0.12 of wildtype control levels ( n = 5 ; p < 0.05 ; figure 5 ) . therefore our data suggest that different subcellular hippocampal changes are occurring in human hd compared to animal models of the same disease . here we report changes that occur in sap97 , psd-95 , glun1 and glua2 in the human brain in response to the neurodegenerative diseases hd and pd . our data reveal that the overall patterns of change in these synaptic proteins are not the same in the post - mortem human hd versus pd brains , with the majority of the changes observed occurring in hd brains . we also report that the changes observed differed between the hippocampus versus striatum , as evidenced by increases in glutamatergic synaptic proteins in the hippocampus but only decreases in the striatum . moreover , hippocampal increases tended to occur across all hippocampal regions examined ( dentate gyrus , ca3 , and ca1 ) , whereas some putamen - specific changes were observed in the striatum . we predict that the hippocampal - specific increases in sap97 , psd-95 and glun1 are likely related to the nonmotor symptoms of hd such as cognitive decline or dementia [ 38 , 46 , 47 ] , while the striatal - specific decreases in psd-95 and glua2 are related to striatal degeneration and behavioural motor symptoms . as the hippocampal changes did not occur in the yac128 hd mouse model one of the major increases that we observed occurred in sap97 in the hippocampus of hd and pd brains . this suggests a hippocampal - specific role of sap97 in hd and pd as no changes in sap97 expression levels occurred in the striatum despite other significant subcellular pathology [ 1 , 2 ] . therefore sap97 expression changes do not appear to be involved in the subcellular striatal changes that underlie the motor symptoms of pd and hd . it remains to be determined whether the specific sap97 hippocampal changes are a pathological hallmark of the cognitive changes seen in hd and pd patients [ 4 , 38 , 46 , 47 ] . at the cellular level , the known role of sap97 in regulating ampa and nmda receptor - mediated postsynaptic currents [ 9 , 10 , 31 ] and nmda receptor - dependent excitotoxicity , suggests that the increases in sap97 alter the trafficking , synaptic expression and/or localisation of nmda and ampa receptors in the diseased brain . changes in psd-95 also occurred in the hippocampus in hd and pd post - mortem tissue , and the lack of change in striatal pd tissue suggests it also does not play a role in the striatal pathology of pd . however , subcellular decreases in psd-95 levels do appear to play a major role in the subcellular striatal pathology of hd . as psd-95 is a major component of the postsynaptic density at excitatory synapses , these data suggest that the decrease in psd-95 observed could be secondary to a loss of synapses . this is consistent with previous work in human hd tissue in which golgi impregnation has shown alterations in the number and size of dendritic spines of striatal medium spiny neurons . similar spine pathology has also been observed in transgenic r6 hd mice [ 50 , 51 ] . however , no similar decrease was observed for sap97 which is also a major component of the psd , suggesting that changes beyond synapse number may also underlie the decrease in psd-95 . the consequence of this decrease is not known , but it may be an attempt to reduce nmda receptor - mediated excitotoxicity [ 9 , 48 ] . in addition , given the importance of psd-95 in the synaptic targeting of ampars , the reduced expression of psd-95 in the hd striatum may also relate to the observed hd striatal reduction in glua2 . with regards to changes in glutamate receptor subunit expression , our data show that glun1 and glua2 expression levels are differentially affected by hippocampal versus striatal subcellular pathology that occurs in pd or hd human brain . for example , glun1 was not altered in the human hd striatum , despite early work showing that nmda receptor binding was significantly decreased the putamen in human hd tissue . glun1 was significantly increased in the human hd hippocampus , which may again reflect a role in cognitive changes in hd . in contrast , no changes in glun1 were observed in pd post - mortem brain . however , the precise sub - synaptic location of the expression of the obligatory glun1 subunit will be important to determine in both the hd and pd human striatum and hippocampus , and this may explain the differential changes observed . in the yac128 animal model of hd , extrasynaptic nmda receptors in the striatum are upregulated and their blockade reverses the hd - induced signalling and motor learning deficits [ 5355 ] . whether the observed upregulation of glun1 in the human hippocampus represents an increase in extrasynaptic receptors is not yet known . our observed lack of change in glun1 in the yac128 hippocampus suggests the change in nmda receptor distribution in hd may be restricted to the striatum , or alternatively that total nmda receptors level remain the same but are simply redistributed to extrasynaptic regions . it will be of significant interest to determine whether the upregulation of sap97 observed in human hd and pd brain is specific to - or sap97 isoforms that regulate synaptic versus extrasynaptic nmda receptors . in contrast to glun1 , the decrease in glua2-containing ampars was restricted to the putamen in both hd and pd post - mortem brain tissue suggesting that changes in ampar subunits do not contribute to hippocampal changes in hd and pd . however , a decrease in glua2-containing receptors in the putamen in pd and hd could alter ampar - mediated synaptic transmission in the basal ganglia and consequently play a more dominant role in the motor symptoms of pd and hd . currently , it is unknown whether the observed increases in hippocampal maguk and glur subunit expression levels helps or hinders neuronal survival , synaptic transmission , synaptic plasticity , or cognition and , whether the increases represent the active decline of neuronal structure and function or represent proactive changes in an attempt to restore lost synapse function . moreover , these increases may differ depending on the hd grade , cag repeat length , or sex of the patient . it is also not yet known if the observed changes in synaptic proteins occur at synapses . unfortunately , the use of human tissue precludes quantification of proteins at synaptic sites via double immunolabelling due to the lipofuscin - induced autofluorescence that interferes with synaptic staining . reduced synaptic plasticity is routinely seen in huntington 's disease mouse models [ 5759 ] , which are proposed to underlie the observed cognitive changes . whether similar changes in synaptic plasticity occur in the human hd brain has so far not been able to be determined but remains of significant interest . the major changes in glutamatergic synaptic protein expression that we observed in the hd human hippocampus were not found to occur in the yac128 animal model of hd . in addition , we observed that the staining pattern of psd-95 , sap97 , glun1 and glua2 were not identical in human and animal tissue . our observed diffuse dendritic expression of these synaptic proteins in human tissue is consistent with previous work in the human brain revealing the mostly diffuse expression of psd-95 . whether this expression pattern reflects a higher proportion of synaptic protein trafficking along dendrites in the human tissue remains to be determined . the changes in synaptic protein expression that we observed in the human hd tissue may reflect changes that only occur at end - stage hd . our yac128 mice were highly symptomatic , late - stage hd at the time of our experiments . therefore future studies are required to examine whether any changes in synaptic proteins occur later when the yac128 mice are end stage ( at ~18 months in our colony ) . it will also be important to determine whether these human versus mice differences are consistent in other hd animal models . previous work on yac128 mice has largely focused on striatal synaptic changes [ 2226 , 55 , 61 ] , and the lack of hippocampal changes we observed likely reflects the previous observation that the hippocampus in yac128 ( but not r6/2 ) hd model mice are spared of atrophy and degeneration [ 41 , 42 , 62 ] . this suggests that the previously described cognitive dysfunction , depressive behaviour , increased mutant huntingtin nuclear localization , and decreased hippocampal neurogenesis observed in yac128 mice [ 41 , 6365 ] are independent of changes in the expression of the synaptic proteins examined in the present study . previous work has described increased psd-95 localisation and psd-95-glun2b interactions in striatal extrasynaptic fractions . together with previous work showing an increase in extrasynaptic nmda receptors , these data suggest that a redistribution of psd proteins occurs in yac128 animals . therefore despite a lack of change in synaptic protein levels detected in our study in yac128 animals , a change in their subsynaptic distribution or function could underlie yac128 subcellular pathology . however , our immunohistochemical analysis does reveal that synaptic proteins are differentially altered in the hd human versus mouse brain and that unique changes may occur in the human hippocampus with hd making further study of human tissue of significant importance . inconsistencies in reported changes in protein expression are not restricted to human versus animal data but also occur between different animal models of the same disease . we observed no change in psd-95 expression in the hippocampus of yac128 mice ; however in contrast in hd r6/1 mice a significant decrease in psd-95 levels has been observed . other studies examining changes in synaptic proteins in animal models of hd and pd have also shown differential changes in psd-95 as well as glun1 [ 19 , 24 , 2729 , 6769 ] . the reasons underlying these conflicting data likely result not only from the mouse model employed but also from the symptomatic stage and quantitative methodology used . we believe that it is imperative to assess which of the changes described in animal models are directly reflective of the changes occurring in the human brain . however , unlike human cellular studies , animal models enable examination of neuronal changes occurring in the pre - symptomatic and symptomatic phases of the disease . human tissue from presymptomatic and symptomatic phases the changes that occur at these stages will be important to determine , but currently cellular human brain studies are largely restricted to end stage studies . inherent variability in the measurements of changes in protein expression is also more evident in data from human brain tissue . this is not occurring in animal studies and is likely due to the use of genetically similar laboratory animals , enabling changes to be more easily deciphered . however , despite the inter - patient variation , our observed changes in sap97 , psd-95 , glua2 , and glun1 were routinely observed across all patients within a disease group , reflecting consistent changes across the spectrum of patients . the overall changes in sap97 , psd-95 , glua2 , and glun1 levels in hd versus pd postmortem human brains represent unique disease - related changes occurring differentially in discrete brain regions . these changes likely reflect the different origins , symptoms , and potentially different neurodegenerative mechanisms of these two diseases . we hypothesise that the hippocampal increases in sap97 , psd-95 , and glun1 may be a pathological hallmark of the cognitive changes seen in hd patients [ 4 , 38 , 46 ] . overall , a lack of human data has made it difficult to predict therapeutic outcomes in the human and to extrapolate animal model data to the human . however , here we have shown that unique changes in synaptic protein expression occur in the human hippocampus and striatum which establish a baseline comparison for animal models and should be considered in future studies .

nmda and ampa - type glutamate receptors and their bound membrane - associated guanylate kinases ( maguks ) are critical for synapse development and plasticity . we hypothesised that these proteins may play a role in the changes in synapse function that occur in huntington 's disease ( hd ) and parkinson 's disease ( pd ) . we performed immunohistochemical analysis of human postmortem brain tissue to examine changes in the expression of sap97 , psd-95 , glua2 and glun1 in human control , and hd- and pd - affected hippocampus and striatum . significant increases in sap97 and psd-95 were observed in the hd and pd hippocampus , and psd95 was downregulated in hd striatum . we observed a significant increase in glun1 in the hd hippocampus and a decrease in glua2 in hd and pd striatum . parallel immunohistochemistry experiments in the yac128 mouse model of hd showed no change in the expression levels of these synaptic proteins . our human data show that major but different changes occur in glutamatergic proteins in hd versus pd human brains . moreover , the changes in human hd brains differ from those occurring in the yac128 hd mouse model , suggesting that unique changes occur at a subcellular level in the hd human hippocampus .

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

huntington 's disease ( hd ) and parkinson 's disease ( pd ) are distinct neurodegenerative diseases that present with unique motor and cognitive symptoms . emphasis is now being placed on the changes that occur at the synapse and the processes that underlie cognitive dysfunction as it has been shown that synaptic and cognitive dysfunction occurs long before the onset of clinical symptoms in the human [ 47 ] . n - methyl - d - aspartate ( nmda ) and alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa)-type glutamate receptors and their bound postsynaptic density - membrane associated guanylate kinases ( psd - maguks ) are critical for synapse development and plasticity [ 812 ] . it is evident that maguks could play a role in the pathogenesis of some neurodegenerative diseases [ 19 , 20 ] . in the striatum of yac128 hd model mice , increased levels of psd-95 as well as increased psd-95-glun2b interactions are observed in extrasynaptic regions [ 25 , 26 ] . a reorganisation of postsynaptic density proteins , including a switch of psd-93 by psd-95 in the striatum of the r6/1 hd mouse model , has also been described . in the n171 - 82q transgenic hd mouse model , a decrease in striatal psd-95-like proteins was observed . in parkinson 's disease , there is a reduced interaction between nmdars and maguks in the striatum of 6-ohda lesioned pd animal models , as well as a change in the subcellular distribution and levels of psd-95 and sap97 . to date , animal models of hd and pd have provided valuable information on how synaptic structure and function may be altered in these human diseases ; however the results with respect to changes in the glutamatergic synapse vary between studies and between models [ 20 , 30 ] . to determine the changes that occur with neurodegenerative diseases of the human brain , it is imperative to examine the postmortem brain tissue of patients who died from these disorders . here we have used postmortem human brain tissue to investigate whether changes in synaptic protein expression occur in response to human neurodegenerative disease and thereby may play an important role in the changes in synapse function that occur in disease . our data show that changes occurring in psd-95 , sap97 and glutamate receptor subunit expression in the human hd hippocampus differ from changes in the yac128 hd animal model , suggesting that unique changes occur in the human brain in response to neurodegenerative disease that vary across different brain regions . immunohistochemical staining was repeated a minimum of 3 times for each control , hd and pd cases . sap97 , psd-95 , glua2 , and glun1 are known to play major roles at synapses within the dentate gyrus and areas ca3 and ca1 of the hippocampus [ 9 , 10 , 18 , 31 ] . we were interested to determine ( i ) whether specific subregional changes occur in these proteins in hd or pd human hippocampus or striatum and ( ii ) whether the two neurodegenerative diseases pd and hd , which have different mechanisms of causation [ 13 ] , differentially affect glutamatergic synaptic proteins in these brain regions . the hippocampus was a major focus as many hd and pd patients have dementia , depression , cognitive decline , and other nonmotor symptoms as well as the classic well - characterised motor symptoms . specifically , we observed that significant increases in the protein levels of psd-95 , sap97 , and the glun1 subunit of the nmda receptor occurred in hd hippocampal post - mortem tissue compared to controls ( figure s1 ) , suggesting that differential changes are occurring in synaptic proteins in hd . to provide more specific information on potential changes in immunoreactivity patterns of synaptic proteins in the principal neurons in hd and pd brain tissues compared to controls the expression of psd-95 , sap97 , and glutamate receptor subunits glun1 and glua2 were examined in the principle neurons of the dentate gyrus and ca3 and ca1 regions of the hippocampus , and in the caudate nucleus and putamen of the striatum . immunohistochemistry and imaging criteria were kept constant for control and diseased cases to enable detection of changes in expression levels of the different synaptic proteins . we first investigated expression changes in psd-95 in the human hippocampus and striatum ( figures 1(a)1(e ) ) . in hd postmortem brain , a significant 1.52 0.26 fold increase in psd-95 was observed in neurons in area ca3 of the hippocampus ( n = 6 , control n = 8 ; p < 0.05 ) and in the dentate gyrus ( hd dg : 1.45 0.22 , n = 6 , control n = 8 , p = 0.05 ) . in pd post - mortem tissue , a significant 1.37 0.19 fold increase in the neurons in the dentate gyrus ( pd n = 6 , control n = 8 ; p < 0.05 ) and a significant 2.44 0.73 fold increase in area ca1 ( pd n = 6 , control n = 8 ; p < 0.05 ; figure 1(a ) ) regions were also observed . in the striatum we observed stark changes in psd-95 expression . in the hd striatum the expression of psd-95 was significantly decreased in both the caudate nucleus ( hd mean = 0.40 0.12 , n = 3 , control n = 4 ; p < 0.005 ) and putamen ( hd mean = 0.51 0.09 , n = 3 , control n = 3 ; p < 0.005 ; figure 1(b ) ) . we next examined whether similar changes also occur in sap97 expression and observed that the changes in sap97 were different to those observed for psd-95 . immunohistochemical analysis of hippocampal and striatal sections in dentate gyrus , ca3 , ca1 , and caudate nucleus , and putamen revealed that increases in sap97 expression were found to occur in both the pd and hd human hippocampus but not in the striatum ( figures 2(a)2(d ) ) . in the ca1 region sap97 was significantly increased 2.15 0.31 fold in hd ( n = 8 ; p < 0.005 ) and 1.88 0.35 in pd ( n = 6 ; p < in contrast , in the striatum there were no significant changes in the expression of sap97 in the caudate nucleus in hd ( n = 6 ) or pd ( n = 5 ) , nor in the putamen in hd ( n = 6 ) or pd ( n = 5 ) as compared to control ( caudate nucleus n = 5 , putamen n = 4 ; figure 2(b ) ) . overall these results indicate that sap97 expression is significantly altered throughout the hippocampus in human hd and pd , but not in the striatum . moreover , these data show that the maguk proteins psd-95 and sap97 are differentially affected by different neurodegenerative diseases in the human brain and that these changes can differ in hippocampus versus striatum . in the hippocampus , we performed quantitative immunohistochemistry to examine whether ampa receptor subunit expression is altered in pd or hd in the human hippocampus and striatum by examining the expression of the glua2 subunit common to both these receptor subtypes that could therefore reflect changes in either subtype of receptor ( figure 3 ) . no significant changes in glua2 subunit expression were observed in the hd or pd hippocampus dentate gyrus , area ca3 , or area ca1 , or in the striatal caudate nucleus ( hd n = 4 , pd n = 5 , control n however , significant decreases in glua2 expression were observed in the putamen relative to control levels ( hd mean = 0.52 0.07 of control levels , n = 4 , p < 0.005 ; pd mean = 0.67 0.08 of control , n = 5 , p < 0.05 ; figures 3(a)3(d ) ) . we also examined potential changes in the expression of glun1 , the obligatory subunit of the nmda receptor ( figure 4 ) . we observed significant increases in glun1 expression above control levels in neurons in the hd hippocampus ( figure 4(a ) ) . specifically , a significant 2.49 0.62 fold increase occurred in the dentate gyrus ( hd n = 8 , control n = 8 , p < 0.05 ) and a significant 3.17 0.89 fold increase in area ca1 ( hd n = 8 , control n = 7 , p < 0.05 ) . in the striatum , no change in glun1 expression was observed in either disease in the caudate nucleus or putamen ( hd n = 4 , pd n = 4 , control n = 4 ; figure 4(b ) ; p > 0.05 in all cases ) . together these data show that different changes in nmda and ampa receptor subunit expression levels occur in the hippocampal and striatal human brain regions in response to hd versus pd . we were particularly intrigued by the significant changes in the expression levels of psd-95 , sap97 , and glun1 in the human hd hippocampus ( figures 14 , figure s1 ) . although hd is considered a motor disorder , there is significant evidence that cognitive effects appear before the motor symptoms suggesting a role of hippocampal changes in the disease [ 1 , 3840 ] . in animal models of hd , striatal changes in glutamatergic synapse structure and function have been widely addressed ( e.g. we wanted to determine whether the changes in maguk and glutamate receptor subunit expression we observed in the human hippocampus in hd were also occurring in the hippocampus of the yac128 mouse model of hd . immunohistochemistry was performed on hippocampal sections from yac128 mice at 12 months of age , when the animals are highly symptomatic and in the late stage of hd and therefore are comparable to tissue from post - mortem human hd patients . as expected , sap97 , psd-95 , glua2 and glun1 immunostaining was observed strongly in the dendritic regions of neurons in area ca1 , however , densitometry quantification revealed that none of the changes in sap97 , psd-95 , and glun1 that we observed in human post - mortem hd hippocampus occurred in yac128 mice hippocampus . we observed no significant changes in expression levels for any of these synaptic proteins compared to control tissue in either dentate gyrus , area ca1 , or area ca3 of yac128 hippocampus ( figure 5 ) . to ensure that our immunohistochemical analysis could detect changes in protein expression levels in yac128 tissue , we examined the expression changes of darpp-32 to act as a positive control in both yac128 and wildtype striatum . here we report changes that occur in sap97 , psd-95 , glun1 and glua2 in the human brain in response to the neurodegenerative diseases hd and pd . our data reveal that the overall patterns of change in these synaptic proteins are not the same in the post - mortem human hd versus pd brains , with the majority of the changes observed occurring in hd brains . we also report that the changes observed differed between the hippocampus versus striatum , as evidenced by increases in glutamatergic synaptic proteins in the hippocampus but only decreases in the striatum . moreover , hippocampal increases tended to occur across all hippocampal regions examined ( dentate gyrus , ca3 , and ca1 ) , whereas some putamen - specific changes were observed in the striatum . we predict that the hippocampal - specific increases in sap97 , psd-95 and glun1 are likely related to the nonmotor symptoms of hd such as cognitive decline or dementia [ 38 , 46 , 47 ] , while the striatal - specific decreases in psd-95 and glua2 are related to striatal degeneration and behavioural motor symptoms . as the hippocampal changes did not occur in the yac128 hd mouse model one of the major increases that we observed occurred in sap97 in the hippocampus of hd and pd brains . this suggests a hippocampal - specific role of sap97 in hd and pd as no changes in sap97 expression levels occurred in the striatum despite other significant subcellular pathology [ 1 , 2 ] . at the cellular level , the known role of sap97 in regulating ampa and nmda receptor - mediated postsynaptic currents [ 9 , 10 , 31 ] and nmda receptor - dependent excitotoxicity , suggests that the increases in sap97 alter the trafficking , synaptic expression and/or localisation of nmda and ampa receptors in the diseased brain . changes in psd-95 also occurred in the hippocampus in hd and pd post - mortem tissue , and the lack of change in striatal pd tissue suggests it also does not play a role in the striatal pathology of pd . however , subcellular decreases in psd-95 levels do appear to play a major role in the subcellular striatal pathology of hd . this is consistent with previous work in human hd tissue in which golgi impregnation has shown alterations in the number and size of dendritic spines of striatal medium spiny neurons . in addition , given the importance of psd-95 in the synaptic targeting of ampars , the reduced expression of psd-95 in the hd striatum may also relate to the observed hd striatal reduction in glua2 . with regards to changes in glutamate receptor subunit expression , our data show that glun1 and glua2 expression levels are differentially affected by hippocampal versus striatal subcellular pathology that occurs in pd or hd human brain . for example , glun1 was not altered in the human hd striatum , despite early work showing that nmda receptor binding was significantly decreased the putamen in human hd tissue . glun1 was significantly increased in the human hd hippocampus , which may again reflect a role in cognitive changes in hd . in contrast , no changes in glun1 were observed in pd post - mortem brain . however , the precise sub - synaptic location of the expression of the obligatory glun1 subunit will be important to determine in both the hd and pd human striatum and hippocampus , and this may explain the differential changes observed . in the yac128 animal model of hd , extrasynaptic nmda receptors in the striatum are upregulated and their blockade reverses the hd - induced signalling and motor learning deficits [ 5355 ] . whether the observed upregulation of glun1 in the human hippocampus represents an increase in extrasynaptic receptors is not yet known . our observed lack of change in glun1 in the yac128 hippocampus suggests the change in nmda receptor distribution in hd may be restricted to the striatum , or alternatively that total nmda receptors level remain the same but are simply redistributed to extrasynaptic regions . it will be of significant interest to determine whether the upregulation of sap97 observed in human hd and pd brain is specific to - or sap97 isoforms that regulate synaptic versus extrasynaptic nmda receptors . in contrast to glun1 , the decrease in glua2-containing ampars was restricted to the putamen in both hd and pd post - mortem brain tissue suggesting that changes in ampar subunits do not contribute to hippocampal changes in hd and pd . however , a decrease in glua2-containing receptors in the putamen in pd and hd could alter ampar - mediated synaptic transmission in the basal ganglia and consequently play a more dominant role in the motor symptoms of pd and hd . currently , it is unknown whether the observed increases in hippocampal maguk and glur subunit expression levels helps or hinders neuronal survival , synaptic transmission , synaptic plasticity , or cognition and , whether the increases represent the active decline of neuronal structure and function or represent proactive changes in an attempt to restore lost synapse function . whether similar changes in synaptic plasticity occur in the human hd brain has so far not been able to be determined but remains of significant interest . the major changes in glutamatergic synaptic protein expression that we observed in the hd human hippocampus were not found to occur in the yac128 animal model of hd . in addition , we observed that the staining pattern of psd-95 , sap97 , glun1 and glua2 were not identical in human and animal tissue . our observed diffuse dendritic expression of these synaptic proteins in human tissue is consistent with previous work in the human brain revealing the mostly diffuse expression of psd-95 . the changes in synaptic protein expression that we observed in the human hd tissue may reflect changes that only occur at end - stage hd . therefore future studies are required to examine whether any changes in synaptic proteins occur later when the yac128 mice are end stage ( at ~18 months in our colony ) . this suggests that the previously described cognitive dysfunction , depressive behaviour , increased mutant huntingtin nuclear localization , and decreased hippocampal neurogenesis observed in yac128 mice [ 41 , 6365 ] are independent of changes in the expression of the synaptic proteins examined in the present study . however , our immunohistochemical analysis does reveal that synaptic proteins are differentially altered in the hd human versus mouse brain and that unique changes may occur in the human hippocampus with hd making further study of human tissue of significant importance . we observed no change in psd-95 expression in the hippocampus of yac128 mice ; however in contrast in hd r6/1 mice a significant decrease in psd-95 levels has been observed . other studies examining changes in synaptic proteins in animal models of hd and pd have also shown differential changes in psd-95 as well as glun1 [ 19 , 24 , 2729 , 6769 ] . inherent variability in the measurements of changes in protein expression is also more evident in data from human brain tissue . however , despite the inter - patient variation , our observed changes in sap97 , psd-95 , glua2 , and glun1 were routinely observed across all patients within a disease group , reflecting consistent changes across the spectrum of patients . the overall changes in sap97 , psd-95 , glua2 , and glun1 levels in hd versus pd postmortem human brains represent unique disease - related changes occurring differentially in discrete brain regions . we hypothesise that the hippocampal increases in sap97 , psd-95 , and glun1 may be a pathological hallmark of the cognitive changes seen in hd patients [ 4 , 38 , 46 ] . overall , a lack of human data has made it difficult to predict therapeutic outcomes in the human and to extrapolate animal model data to the human . however , here we have shown that unique changes in synaptic protein expression occur in the human hippocampus and striatum which establish a baseline comparison for animal models and should be considered in future studies .

monoterpenes are major components of essential oils , and are wide - spread secondary metabolites in the plant kingdom . allelopathic interactions due to monoterpenes may be common in nature ( e.g. , mller , 1970 ) . abrahim et al . , 2000 ) , causing morphological and physiological changes in plant seedlings ( einhellig and leather , 1988 ; fischer , 1991 ; dudai et al . , 2000 ) . they inhibit respiration in isolated mitochondria and mitosis , deteriorate membrane integrity of treated cells , interfere with cuticular waxes , enhance transpiration , cause lipid oxidation , and disrupt microtubules ( duke and oliva , 2004 ; dayan et al . , 2000 ; romagni et al . , 2000 ; schulz et al . , 2007 ; zunino and zygadlo , 2004 ; chaimovitsh et al . , 2010 ) . nonetheless , the molecular mechanism ( e.g. , primary target point ) for the allelopathic effects of monoterpenes is still obscure . ( 2005 ) examined the effects of five monoterpenes produced by s. leucophylla ( camphor , 1,8-cineole , -pinene , -pinene , and camphene ) on germination and seedling growth of a test plant , brassica campestris . the monoterpenes inhibited growth of b. campestris seedlings in a dose - dependent manner , and their inhibitory effects appeared to affect root growth more severely than hypocotyl growth . the monoterpenes did not alter the sizes of matured cells in roots but effectively inhibited dna synthesis and cell proliferation in the root apical meristem . therefore , the observed preferential inhibition of root growth relative to hypocotyl growth might be explained by the preferential inhibition of cell proliferation , because root growth requires both cell proliferation and cell elongation whereas hypocotyl growth only requires elongation of existing cells ( obroucheva , 1999 ) . thus , the different sensitivities of hypocotyls and roots to monoterpenes could give us a clue towards understanding their mode of action . in the present study , we examined the effects of a monoterpene , 1,8-cineole , on proliferation and elongation of plant cells by using by-2 suspension - cultured tobacco ( nicotiana tabacum ) cells as receiver cells . 1,8-cineole was chosen because it is easy to handle ( it is liquid at room temperature ) and has strong inhibitory effects ( nishida et al . , 2005 ) . tobacco by-2 cells were chosen for the following reasons : ( i ) in liquid suspension culture , by-2 cells form small cell clusters composed of several ( 216 ) cylindrical cells connected in tandem ( sakai et al . , 2004 ) . since most of the surface area of individual cells is exposed directly to culture media , cells are expected to exhibit clear , synchronous , and homogeneous responses to any compound applied to the culture medium ; ( ii ) by-2 cells can exhibit either a high proliferation rate or drastic elongation , depending on culture conditions . under conventional culture conditions , by-2 cells rapidly proliferate and multiply up to 100-fold within a week ( nagata et al . , 1992 ) . in contrast , when cultured in an auxin - depleted , cytokinin - containing medium , they elongate drastically and accumulate starch without proliferation ( sakai et al . , 1996 ) . thus , by-2 cells should provide us with a suitable system for examining the effects of monoterpene on cell proliferation and cell elongation ; ( iii ) a protoplast culture system , in which proliferation and elongation are induced separately depending on culture conditions ( hasezawa and syono , 1983 ) , is established . because protoplasts are suspended individually without any protection from a cell wall or neighboring cells ( at least initially , i.e. , before regeneration of cell wall ) , a protoplast culture system may provide us with a more sensitive way to examine the effects of monoterpene . we first examined the effects of 1,8-cineole on growth of tobacco seedlings to check whether the phenomena observed for b. campestris were also true for tobacco . finally , the effects of 1,8-cineole on cell proliferation and cell elongation were assessed quantitatively with by-2 cell cultures . we also assessed the effects of 1,8-cineole on starch synthesis , as an example of a physiological activity other than cell proliferation and cell elongation . based on the results , possible mechanisms for the differential inhibition of root growth and hypocotyl growth by monoterpenes as well as the mode of action of monoterpenes at cellular level are discussed . plant materials , chemicals , and treatment of seedlings with 1,8-cineole seeds of n. tabacum ( cv . bright yellow-2 , from which a by-2 cell line was established ) were sown on two layers of filter paper ( whatman no . 3 , diam 55 mm ) soaked with 3 ml of water in transparent , airtight containers ( 5.5 cm diam 9.6 cm height , 230 ml volume , 50 seeds per container ) . 1,8-cineole , purchased from aldrich ( milwaukee , wi , usa ) , was spotted onto a piece of filter paper ( 3 6 cm ) hanging from the cap of the container and allowed to volatilize into the airspace within the container . the doses of 1,8-cineole were expressed as the theoretical concentrations in the airspace within the container , which were calculated assuming that the spotted compounds volatilize completely without adsorption or leakage . for example , spotting of 17 l of 1,8-cineole solution ( f.w . = 154 , d = 0.921 ) to filter paper hanging within the 230-ml container gives final concentration of 440 m ; zero m ( control ) indicates no application of 1,8-cineole liquid . ( note that actual concentrations in the airspace may be lower than those theoretical , calculated values as a result of absorption . the seeds / seedlings in the containers were incubated for several days at 23c under a 14 : 10 h , l : d photoperiod . measurement of seedling and cell sizes the lengths of hypocotyls and primary roots were measured with digital calipers . only individuals that successfully germinated were used for measurements . for determination of cell size , seedlings were fixed , washed , and cleared according to the methods of yadegari et al . the cortex cells at the root - hair - forming region ( i.e. , differentiation / elongation zone in the upper part of the root ) of the primary roots were observed with a microscope ( bx-60 , olympus , tokyo , japan ) equipped with nomarski optics and their sizes measured . determination of mitotic index in the root apical meristem root tips ( 5 mm long ) were excised , fixed with faa solution ( formalin : acetic acid : 50% ethanol = 1 : 1 : 18 , v / v / v ) for 12 h , rinsed with 0.1 m sodium phosphate buffer ( ph 7.0 ) , and dialyzed against 0.1 m sodium phosphate buffer containing increasing concentrations of sucrose ( 10 , 20 , and 30% , successively ) . compound ( sakura finetech , torrance , ca , usa ) , quickly frozen with liquid nitrogen , and the frozen , serial sections ( 7 m thick ) were prepared using a cryostat ( ote cryostat , bright , uk ) at -26c . sections were mounted on gelatin - coated slides and air - dried , as described previously ( tamotsu et al . , 1994 ) . they were stained with 1 g / ml 4,6-diamidino-2-phenylindole ( dapi ) without removal of the o.c.t . compound , observed with a fluorescence microscope ( bx-60 , olympus ) under uv irradiation , and the percentages of the cells in a mitotic phase were counted as described in nishida et al . protoplast culture and treatment with 1,8-cineole protoplasts were prepared and cultured according to the method of hasezawa and syono ( 1983 ) with slight modifications . by-2 cells usually were propagated by inoculating 95 ml of fresh medium with 1.52 ml of suspension of stationary - phase cells at weekly intervals and incubating the culture at 26c in the dark on a gyratory shaker ( 130 rpm ) , as described by yasuda et al . ( 1988 ) . four - day - old by-2 cells were converted to protoplasts by treating them with enzyme solution ( 0.4 m mannitol , 1% cellulase yc , 0.1% pectolyase y23 , ph 5.5 ) for 90 min at 30c . the protoplasts were collected by centrifugation ( 260 g , 2 min ) , suspended in 0.4 m mannitol solution , centrifuged again , and suspended in either 2,4-d medium ( ls medium containing 3% sucrose , 0.4 m mannitol , and 0.2 mg / l 2,4-d ) or ba - naa medium ( ls medium containing 3% sucrose , 0.4 m mannitol , 1.0 mg / l ba , and 0.1 mg / l naa ) at a density of 0.51 10 cells / ml . the protoplast suspension was dispensed into 50-ml erlenmeyer flasks ( 4.6 ml per flask ) , and cultured at 26c in the dark without shaking . for treatment with 1,8-cineole , the protoplast - containing flasks were settled , one - by - one , in transparent , airtight containers ( 5.5 cm diam 9.6 cm height , 230 ml volume ) . 1,8-cineole was applied either by spotting appropriate volumes of 1,8-cineole liquid onto a piece of filter paper hanging from the cap of the container ( volatilization method ) or by adding it directly into the culture medium in the flask ( direct - addition method ) . in both cases , the caps of the containers were closed immediately after the application of 1,8-cineole , and the compound was allowed to volatilize into the airspace within the container . the final concentrations of 1,8-cineole in the airspace were calculated assuming that the compound volatilized completely without adsorption or leakage , as described above . cell culture and treatment with 1,8-cineole seven - day - old ( stationary - phase ) by-2 cells were transferred to either fresh d - medium ( modified ls medium containing 0.2 mg / l 2,4-d ) or fresh b - medium ( modified ls medium containing 1 mg / l ba ) at a 1:20 dilution , and dispensed into 50-ml erlenmeyer flasks ( 9.2 ml per flask ) . for treatment with 1,8-cineole , the cell suspension - containing flasks were settled , one - by - one , in transparent , airtight containers described above , and were fixed to prepare for shaking - culture . 1,8-cineole was applied via the direct - addition method only . after the addition of 1,8-cineole , the caps of the containers were closed immediately , and the cells were cultured for 2 days at 26c in the dark on a gyratory shaker ( 130 rpm ) . measurements of mitotic index , cell length , and starch content of the cells / protoplasts for measurements of mitotic index , protoplasts and cells were fixed with 1% formaldehyde , stained with 1 g / ml dapi , and examined with a fluorescence microscope ( bx-60 ) under uv - irradiation . for measurements of the cell ( or protoplast ) length , samples ( with or without fixation with 1% formaldehyde ) were observed with a microscope , photographed , and the length measured . the starch content of by-2 cells was measured according to the method of sakai et al . in short , by-2 cells present in 1 ml of cell suspension were collected by centrifugation , converted to protoplasts by incubation for 1 h at 30c in enzyme solution ( 0.4 m mannitol , 1% cellulase yc , 0.1% pectolyase y23 , ph 5.5 ) , and solubilized by adding 1/10 vols of 10% sds and incubation for additional 30 min at 37c . insoluble materials , including starch granules , were collected by centrifugation , re - suspended in 1 ml of hot 80% ethanol , and collected again by centrifugation . the starch was solubilized in hot water , extracted with perchloric acid , and quantified by the phenol - sulfuric acid method ( dubois et al . , 1956 ) using a dilution series of glucose solution as a standard . estimation of half - maximal inhibitory concentrations ( ic50 ) we emploied ic50 value as the parameter to represent the sensitivities of the biological activities of interest to 1,8-cineole . the ic50 values were simply estimated from semi - log graphs of dose response curves , without any mathematical model - fitting ( belz et al . , 2005 ) . effects of 1 , 8-cineole on the growth of tobacco seedlings in the absence of 1,8-cineole , germination of tobacco seeds proceeded rapidly and synchronously under our experimental conditions . almost 100% germination was achieved 5 days after sowing , and the length of hypocotyls and primary roots reached their maximum values by day 10 ( data not shown ) . thus , the effects of various concentrations of 1,8-cineole were examined on day 10.germination of tobacco seeds ( fig . 1 , top ) the germination rate suddenly dropped to 60% at 1,000 m , and further dropped to 12% at 2,000 m . the concentration of 1,8-cineole required to lower the germination rate to 50% of the control value ( ic50 ) was about 1,200 m . 1,8-cineole stimulated hypocotyl growth slightly in a dose - dependent manner within this low - concentration range ( rs = 0.94 , p < 0.05 ) , indicating hormesis ( duke et al . , 2006 ; calabrese et al . at higher concentration ranges , 1,8-cineole inhibited hypocotyl growth in a dose - dependent manner . root growth ( fig . 1 , bottom ) was not stimulated by any concentrations of 1,8-cineole tested ; 1,8-cineole shortened root length even in the low concentration range ( 200 m ) . the ic50 value for root growth was 410 m , indicating that root growth was more sensitive to 1,8-cineole than was hypocotyl growth . . germination rate ( top ) , hypocotyl length ( middle ) , and root length ( bottom ) were measured on day 10 . each data point for germination rate was calculated from the results of all 50 individuals contained in a single container , while each for hypocotyl and root length represents the average value from 30 individuals that successfully germinated . ic50 values calculated from the data are shown in respective graphsnext , relative contribution of cell elongation and cell proliferation to the root - growth inhibition by 1,8-cineole was examined ( fig . 2 ) . the concentration of 1,8-cineole was set at 440 m , where hypocotyl length was almost the same as the control value , while root length was reduced by approximately 50% ( fig . treatment with 440-m 1,8-cineole did not reduce the size of cells in the elongation / differentiation zone of tobacco roots , but it lowered the mitotic index in the root apical meristem to less than half the control value ( fig . 2b ) , indicating that cell proliferation in the root apical meristem was selectively inhibited , while cell expansion in the upper region of the root was largely unaffected with this concentration . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m of 1,8-cineole for 10 days , and the hypocotyl length ( top ) and root length ( bottom ) were measured . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m 1,8-cineole for 10 days , and the sizes of matured cells ( top ) and mitotic index in the root apical meristem ( bottom ) were measured . for cell length ( top ) , each point represents average value of 60 cells derived from more than 4 individuals . each point represents the average value from 8 ( control ) or 7 ( cineole ) individuals . n.s . ; not significant ( p > 0.05 ) , * * * ; p < 0.001 , student s t - testthus , the results obtained for tobacco seedlings ( i.e. , preferential inhibition of root growth over hypocotyl growth , relative insensitivity of cell elongation in the upper region of the root as compared to cell proliferation in the root apical meristem ) were essentially the same as those obtained previously for b. campestris seedlings ( koitabashi et al . , 1997 ; nishida et al . , 2005 ) . germination rate ( top ) , hypocotyl length ( middle ) , and root length ( bottom ) were measured on day 10 . each data point for germination rate was calculated from the results of all 50 individuals contained in a single container , while each for hypocotyl and root length represents the average value from 30 individuals that successfully germinated . ic50 values calculated from the data are shown in respective graphs effects of 1,8-cineole on growth of nicotiana tabacum seedlings . a effects on hypocotyl length and root length . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m of 1,8-cineole for 10 days , and the hypocotyl length ( top ) and root length ( bottom ) were measured . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m 1,8-cineole for 10 days , and the sizes of matured cells ( top ) and mitotic index in the root apical meristem ( bottom ) were measured . for cell length ( top ) , each point represents average value of 60 cells derived from more than 4 individuals . for mitotic index , each point represents the average value from 8 ( control ) or 7 ( cineole ) individuals . not significant ( p > 0.05 ) , * * * ; p < 0.001 , student s t - test effects of 1,8-cineole on the proliferation and elongation of protoplasts prepared from by-2 cultured tobacco cells we examined the effects of 1,8-cineole on cell proliferation and cell elongation by using protoplasts prepared from by-2 cultured tobacco cells . when cultured in 2,4-d medium , by-2 protoplasts proliferated actively ( with mitotic indices of about 3% from day 2 to day 6 ) , but their sizes did not increase significantly during the culture period ( fig . in contrast , when cultured in ba - naa medium , they ceased proliferation and elongated drastically by day 7 ( fig . thus , by-2 protoplasts cultured in 2,4-d medium and those cultured in ba - naa medium were used to examine the effects of 1,8-cineole on cell proliferation and on cell elongation , respectively . in the experiments with protoplast cultures , 1,8-cineole was applied using the two different modes , i.e. , the volatilization method and the direct - addition method ( fig . . four - day - old by-2 cells were converted to protoplasts and cultured in either 2,4-d medium a or ba - naa medium b , and the changes in the mitotic index ( ) and cell length ( ) were followed during the culture . each data point for mitotic index was determined by counting more than 1,000 cells / protoplasts . each point for the length of cells / protoplasts represents the average value from 30 ( for 2,4-d medium ) or 60 ( for ba - naa medium ) cells / protoplasts . 4effects of 1,8-cineole and its application methods on the proliferation and elongation of by-2 protoplasts / cells . a schematic representation of the experimental system . 1,8-cineole was applied to either filter paper hanging from the cap of the air - tight container ( volatilization method ) or the culture medium in the flask ( direct - addition method ) . the calculated final concentrations of 1,8-cineole in the airspace ( gas - phase ) were 0 to 3,000 m . in direct - addition method , the initial concentrations of 1,8-cineole in the culture medium ( liquid - phase ) were 50 times higher than the calculated final concentrations in the airspace ( gas - phase ) . ( b ) effects of 1,8-cineole on the proliferation of by-2 protoplasts grown in 2,4-d medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and the mitotic index was examined on d 2 . c effects of 1,8-cineole on the elongation of by-2 protoplasts grown in ba - naa medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and cell length was examined on day 0 and day 7 . estimated from the data are shown in the respective graphsfigure 4b shows the mitotic indices of by-2 protoplasts cultured in the presence of various concentrations of 1,8-cineole in 2,4-d medium for 2 days . in either of the two application modes , relatively high concentrations of 1,8-cineole were necessary to lower the mitotic index . with the protoplast culture system , ic50 values of 1,8-cineole for proliferation were estimated to be about 2,000 m for the volatilization method and 100 m for the direct - addition method . figure 4c shows the length of by-2 cells cultured in the presence of various concentrations of 1,8-cineole in ba - naa medium for 7 days . normally , the cell wall should have regenerated by day 7 . in either of the two application modes , 1,8-cineole lowered cell length in a dose - dependent manner and completely inhibited cell elongation at the highest concentration tested ( 1,000 m ) . ic50 values for cell elongation were about 140 m for the volatilization method and 30 m for the direct - addition method . these results demonstrate that ( i ) the direct addition method was more effective than the volatilization method , and ( ii ) in a by-2 protoplast culture system , cell elongation was more sensitive to 1,8-cineole than cell proliferation . four - day - old by-2 cells were converted to protoplasts and cultured in either 2,4-d medium a or ba - naa medium b , and the changes in the mitotic index ( ) and cell length ( ) were followed during the culture . each data point for mitotic index each point for the length of cells / protoplasts represents the average value from 30 ( for 2,4-d medium ) or 60 ( for ba - naa medium ) cells / protoplasts . vertical bars represent standard errors effects of 1,8-cineole and its application methods on the proliferation and elongation of by-2 protoplasts / cells . a schematic representation of the experimental system . 1,8-cineole was applied to either filter paper hanging from the cap of the air - tight container ( volatilization method ) or the culture medium in the flask ( direct - addition method ) . the calculated final concentrations of 1,8-cineole in the airspace ( gas - phase ) were 0 to 3,000 m . in direct - addition method , the initial concentrations of 1,8-cineole in the culture medium ( liquid - phase ) were 50 times higher than the calculated final concentrations in the airspace ( gas - phase ) . ( b ) effects of 1,8-cineole on the proliferation of by-2 protoplasts grown in 2,4-d medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and the mitotic index was examined on d 2 . c effects of 1,8-cineole on the elongation of by-2 protoplasts grown in ba - naa medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and cell length was examined on day 0 and day 7 . ic50 values estimated from the data are shown in the respective graphs effects of 1,8-cineole on proliferation , elongation , and starch accumulation of by-2 cells we examined the effects of 1,8-cineole on by-2 cells that retained intact cell walls . by-2 cells proliferate actively without significant increase of cell volume when cultured in d - medium that contains 0.2 mg / l 2,4-d , whereas they increase in cell volume ( mostly due to cell elongation ) and accumulate a large amount of starch without proliferation when cultured in b - medium that contains 1 mg / l ba ( sakai et al . , 1996 ) . accordingly , by-2 cells cultured in d - medium were used to examine the effects of 1,8-cineole on cell proliferation , while those cultured in b - medium were used to examine the effects on cell elongation and starch accumulation . as the differentiation of cells in the two culture media becomes clear 2 days after transfer ( sakai et al . , 1996 ) , based on the results of the experiments with the protoplast culture system , only the direct - addition method was used to apply 1,8-cineole.1,8-cineole lowered the mitotic index in a dose - dependent manner , and the ic50 value for proliferation was about 140 m ( fig . 5b ) , and at high concentrations ( 120 or 240 m ) , the cell size did not increase as compared to the value at the start of the culture ( 105 m ) . the ic50 value for cell elongation was about 30 m , and was lower than the ic50 value for cell proliferation . ic50 value for starch accumulation was about 80 m and was also lower than the ic50 value for cell proliferation . these results demonstrate that the inhibition by 1,8-cineole is not specific to cell proliferation . 5effects of 1,8-cineole on the proliferation and elongation of by-2 cells . a the effects of a range of doses of 1,8-cineole on the proliferation of by-2 cells cultured in d - medium are shown . b the effects of a range of doses of 1,8-cineole on elongation ( top ) and starch accumulation ( bottom ) in by-2 cells cultured in b - medium . the estimated final concentrations of 1,8-cineole in the airspace ( gas phase ) were 0 to 240 m , while the initial concentrations in the culture medium ( liquid phase ) immediately after application were 25 times higher . ic50 values estimated from the data are shown in respective graphs effects of 1,8-cineole on the proliferation and elongation of by-2 cells . a the effects of a range of doses of 1,8-cineole on the proliferation of by-2 cells cultured in d - medium are shown . b the effects of a range of doses of 1,8-cineole on elongation ( top ) and starch accumulation ( bottom ) in by-2 cells cultured in b - medium . the estimated final concentrations of 1,8-cineole in the airspace ( gas phase ) were 0 to 240 m , while the initial concentrations in the culture medium ( liquid phase ) immediately after application were 25 times higher . we found here that the effects of 1,8-cineole on n. tabacum seedlings were essentially the same as those on b. campestris seedlings ; 1,8-cineole preferentially inhibited root growth over hypocotyl growth ( fig . 1 ) . in the affected roots , cell size in the elongation zone ( upper region of the root ) was largely unaffected , whereas cell proliferation in the root apical region was severely inhibited ( fig . 2 ) . these results indicate that the mode of action of growth inhibition by 1,8-cineole is common within the two plant species . thus , we consider that tobacco , especially cultured tobacco cells , can be used as experimental material to examine the mode of action of 1,8-cineole at the cellular level . we compared the effectiveness of two modes of application of 1,8-cineole , i.e. , the volatilization method and the direct - addition method , by using a protoplast culture system ( fig . 4 ) , and we found that the direct - addition method was more effective . this result is quite reasonable when the predicted behavior of the applied 1,8-cineole is considered . in the volatilization method , thus , the concentrations of 1,8-cineole that the cells experience should gradually increase and finally reach the highest values determined by equilibrium between the liquid and gas phase . in the direct - addition method , the cells should experience the highest concentrations of 1,8-cineole immediately after application of the compound . thus , in the latter method , the actual concentration that the cells experience should be higher and the time at which the cells experience the highest concentration should be earlier . because protoplasts lose their cell wall and are completely separated from neighboring cells , we expected that they might exhibit higher sensitivity to externally added compounds as compared to cultured cells that have intact cell walls and neighboring cells . for this reason , initially we examined the effects of 1,8-cineole on by-2 cells using protoplasts ( figs . 3 and 4 ) however , ic50 values obtained for protoplasts and intact cells were essentially the same ( compare figs . 4 and 5 ) , indicating that cell wall and neighboring cells did not function as a barrier to the permeation of 1,8-cineole into the cells . in contrast to our initial expectation , 1,8-cineole inhibited cell elongation more efficiently than cell proliferation ; the ic50 value for cell proliferation was always higher than that for cell elongation , irrespective of the application methods ( fig . 4 ) or the condition ( i.e. , presence or absence of cell wall ) of receiver cells ( figs . 4 and 5 ) . moreover , 1,8-cineole also inhibited starch accumulation with an ic50 value lower than that for cell proliferation ( fig . 5 ) . these results demonstrated clearly that the inhibitory effect of 1,8-cineole was not specific to cell proliferation ; rather , 1,8-cineole appeared to affect a wide spectrum of cellular activities in an almost non - specific manner when target cells were efficiently exposed to the compound . despite numerous reports on inhibitory effects of monoterpenes , their mechanisms of action remain obscure . however , they also reported that the effects of citral and microtubule inhibitor oryzalin on plant seedlings are different from each other , suggesting that there should be additional target(s ) of the monoterpene . 5 ) also suggests the presence of additional target(s ) , as starch accumulation itself does not seem so tightly associated with microtubule function . a reduction in respiratory oxygen consumption resulting from monoterpene treatment has been reported in a number of studies ( e.g. , mller et al . , 1968 ; peuelas et al . , 1996 ) . moreover , monoterpenes affect the respiratory activity of isolated mitochondria ( mller et al . , 1969 ; abrahim et al . , 2000 , 2003a ) via uncoupling of oxidative phosphorylation and inhibition of electron transfer ( abrahim et al . , 2003b ) . 1993 ) , as well as lipid oxidation and deterioration of membrane integrity in plant cells exposed to monoterpenes ( lorber and mller , 1976 ; fischer , 1986 ; zunino and zygadlo , 2004 ) suggests that biological membranes , including mitochondrial membranes , are the primary target of monoterpenes . among all the possible effects on biological membranes , the deleterious effects on mitochondrial membranes should cause inhibition of mitochondrial energy metabolism and result in disturbances in a wide range of physiological and biochemical processes within the cell . our results rule out the possibility that preferential inhibition by 1,8-cineole of root growth is explained by preferential inhibition of cell proliferation ; 1,8-cineole inhibited multiple distinct biological processes ( cell proliferation , cell elongation , and starch synthesis ) in cultured cells , and the sensitivity of cell proliferation to 1,8-cineole was , contrary to our initial expectation , relatively low ( figs . 4 and 5 ) . then , in plant seedlings , why is root growth more sensitive than hypocotyl growth to 1,8-cineole ? one possibility is that the actual concentration of 1,8-cineole may be higher around roots ( liquid / solid phase ) than around hypocotyls ( gas - phase ) . in nature , soil colloids adsorb volatile monoterpenes in the atomosphere and exhibit toxicity for several months ( mller and moral , 1966 ) . under our experimental conditions for the seedling assay , monoterpenes within an air - tight container might absorb to the wet filter paper wad used as supporting substance , resulting in higher concentrations of the compound around the roots as compared to the concentrations around the aerial tissues . ( this also suggests that the actual concentrations of 1,8-cineole in the gas - phase should be lower than the calculated values . ) however , root growth was more sensitive to 1,8-cineole than hypocotyl growth even when the seedlings were grown under conditions in which both hypocotyls and roots were in equal contact with the filter paper , and therefore , the concentration of 1,8-cineole surrounding the two organs should have been equal ( nishida et al . , 2005 ) . thus , differences in the actual concentrations around roots and hypocotyls do not seem to play a critical role under our experimental conditions . another possible explanation is that root surfaces may have higher permeability to 1,8-cineole as compared to hypocotyl surfaces , resulting in higher doses of the compound within root tissues as compared to those within hypocotyl tissues . the surface of aerial parts of the plant body is covered with a well - developed cuticle layer , while that of roots is not ( bessire et al . , 2007 ) . in addition , a permeability assay , based on staining with toluidine blue dye ( bessire et al . , 2007 ) , demonstrated differential permeability ( and thus differential development of a cuticle layer ) within a root ; in various plant species , the root tip region that includes the root apical meristem exhibited higher permeability than that of the upper region of the same root , which corresponded to elongation / differentiation zone ( unpublished data ) . thus , differential permeability due to differential development of a cuticle layer might be involved in not only preferential inhibition of root growth over hypocotyl growth but also preferential inhibition of cell proliferation in the root apical region over cell elongation in the upper region of the same root . the relationship between development of the surface barriers , such as cuticle layer , and sensitivity to monoterpenes is now under further investigation .

volatile monoterpenes such as 1,8-cineole inhibit the growth of brassica campestris seedlings in a dose - dependent manner , and the growth - inhibitory effects are more severe for roots than hypocotyls . the preferential inhibition of root growth may be explained if the compounds inhibit cell proliferation more severely than cell elongation because root growth requires both elongation and proliferation of the constituent cells , whereas hypocotyl growth depends exclusively on elongation of existing cells . in order to examine this possibility , by-2 suspension - cultured tobacco ( nicotiana tabacum ) cells were treated with 1,8-cineole , and the inhibitory effects on cell proliferation and on cell elongation were assessed quantitatively . treatment with 1,8-cineole lowered both the mitotic index and elongation of the cells in a dose - dependent manner , and the half - maximal inhibitory concentration ( ic50 ) for cell elongation was lower than that for cell proliferation . moreover , 1,8-cineole also inhibited starch synthesis , with ic50 lower than that for cell proliferation . thus , the inhibitory effects of 1,8-cineole were not specific to cell proliferation ; rather , 1,8-cineole seemed inhibitory to a variety of physiological activities when it was in direct contact with target cells . based on these results , possible mechanisms for the mode of action of 1,8-cineole and for its preferential inhibition on root growth are discussed .

Introduction Methods and Materials Results Discussion

, primary target point ) for the allelopathic effects of monoterpenes is still obscure . ( 2005 ) examined the effects of five monoterpenes produced by s. leucophylla ( camphor , 1,8-cineole , -pinene , -pinene , and camphene ) on germination and seedling growth of a test plant , brassica campestris . the monoterpenes inhibited growth of b. campestris seedlings in a dose - dependent manner , and their inhibitory effects appeared to affect root growth more severely than hypocotyl growth . therefore , the observed preferential inhibition of root growth relative to hypocotyl growth might be explained by the preferential inhibition of cell proliferation , because root growth requires both cell proliferation and cell elongation whereas hypocotyl growth only requires elongation of existing cells ( obroucheva , 1999 ) . thus , the different sensitivities of hypocotyls and roots to monoterpenes could give us a clue towards understanding their mode of action . in the present study , we examined the effects of a monoterpene , 1,8-cineole , on proliferation and elongation of plant cells by using by-2 suspension - cultured tobacco ( nicotiana tabacum ) cells as receiver cells . tobacco by-2 cells were chosen for the following reasons : ( i ) in liquid suspension culture , by-2 cells form small cell clusters composed of several ( 216 ) cylindrical cells connected in tandem ( sakai et al . thus , by-2 cells should provide us with a suitable system for examining the effects of monoterpene on cell proliferation and cell elongation ; ( iii ) a protoplast culture system , in which proliferation and elongation are induced separately depending on culture conditions ( hasezawa and syono , 1983 ) , is established . we first examined the effects of 1,8-cineole on growth of tobacco seedlings to check whether the phenomena observed for b. campestris were also true for tobacco . finally , the effects of 1,8-cineole on cell proliferation and cell elongation were assessed quantitatively with by-2 cell cultures . we also assessed the effects of 1,8-cineole on starch synthesis , as an example of a physiological activity other than cell proliferation and cell elongation . based on the results , possible mechanisms for the differential inhibition of root growth and hypocotyl growth by monoterpenes as well as the mode of action of monoterpenes at cellular level are discussed . determination of mitotic index in the root apical meristem root tips ( 5 mm long ) were excised , fixed with faa solution ( formalin : acetic acid : 50% ethanol = 1 : 1 : 18 , v / v / v ) for 12 h , rinsed with 0.1 m sodium phosphate buffer ( ph 7.0 ) , and dialyzed against 0.1 m sodium phosphate buffer containing increasing concentrations of sucrose ( 10 , 20 , and 30% , successively ) . compound , observed with a fluorescence microscope ( bx-60 , olympus ) under uv irradiation , and the percentages of the cells in a mitotic phase were counted as described in nishida et al . four - day - old by-2 cells were converted to protoplasts by treating them with enzyme solution ( 0.4 m mannitol , 1% cellulase yc , 0.1% pectolyase y23 , ph 5.5 ) for 90 min at 30c . for treatment with 1,8-cineole , the protoplast - containing flasks were settled , one - by - one , in transparent , airtight containers ( 5.5 cm diam 9.6 cm height , 230 ml volume ) . in both cases , the caps of the containers were closed immediately after the application of 1,8-cineole , and the compound was allowed to volatilize into the airspace within the container . cell culture and treatment with 1,8-cineole seven - day - old ( stationary - phase ) by-2 cells were transferred to either fresh d - medium ( modified ls medium containing 0.2 mg / l 2,4-d ) or fresh b - medium ( modified ls medium containing 1 mg / l ba ) at a 1:20 dilution , and dispensed into 50-ml erlenmeyer flasks ( 9.2 ml per flask ) . for treatment with 1,8-cineole , the cell suspension - containing flasks were settled , one - by - one , in transparent , airtight containers described above , and were fixed to prepare for shaking - culture . after the addition of 1,8-cineole , the caps of the containers were closed immediately , and the cells were cultured for 2 days at 26c in the dark on a gyratory shaker ( 130 rpm ) . measurements of mitotic index , cell length , and starch content of the cells / protoplasts for measurements of mitotic index , protoplasts and cells were fixed with 1% formaldehyde , stained with 1 g / ml dapi , and examined with a fluorescence microscope ( bx-60 ) under uv - irradiation . for measurements of the cell ( or protoplast ) length , samples ( with or without fixation with 1% formaldehyde ) were observed with a microscope , photographed , and the length measured . estimation of half - maximal inhibitory concentrations ( ic50 ) we emploied ic50 value as the parameter to represent the sensitivities of the biological activities of interest to 1,8-cineole . effects of 1 , 8-cineole on the growth of tobacco seedlings in the absence of 1,8-cineole , germination of tobacco seeds proceeded rapidly and synchronously under our experimental conditions . thus , the effects of various concentrations of 1,8-cineole were examined on day 10.germination of tobacco seeds ( fig . the concentration of 1,8-cineole required to lower the germination rate to 50% of the control value ( ic50 ) was about 1,200 m . 1,8-cineole stimulated hypocotyl growth slightly in a dose - dependent manner within this low - concentration range ( rs = 0.94 , p < 0.05 ) , indicating hormesis ( duke et al . at higher concentration ranges , 1,8-cineole inhibited hypocotyl growth in a dose - dependent manner . the ic50 value for root growth was 410 m , indicating that root growth was more sensitive to 1,8-cineole than was hypocotyl growth . ic50 values calculated from the data are shown in respective graphsnext , relative contribution of cell elongation and cell proliferation to the root - growth inhibition by 1,8-cineole was examined ( fig . treatment with 440-m 1,8-cineole did not reduce the size of cells in the elongation / differentiation zone of tobacco roots , but it lowered the mitotic index in the root apical meristem to less than half the control value ( fig . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m of 1,8-cineole for 10 days , and the hypocotyl length ( top ) and root length ( bottom ) were measured . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m 1,8-cineole for 10 days , and the sizes of matured cells ( top ) and mitotic index in the root apical meristem ( bottom ) were measured . , preferential inhibition of root growth over hypocotyl growth , relative insensitivity of cell elongation in the upper region of the root as compared to cell proliferation in the root apical meristem ) were essentially the same as those obtained previously for b. campestris seedlings ( koitabashi et al . ic50 values calculated from the data are shown in respective graphs effects of 1,8-cineole on growth of nicotiana tabacum seedlings . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m of 1,8-cineole for 10 days , and the hypocotyl length ( top ) and root length ( bottom ) were measured . n. tabacum seedlings were grown in the absence ( control ) or presence ( cineole ) of 440 m 1,8-cineole for 10 days , and the sizes of matured cells ( top ) and mitotic index in the root apical meristem ( bottom ) were measured . not significant ( p > 0.05 ) , * * * ; p < 0.001 , student s t - test effects of 1,8-cineole on the proliferation and elongation of protoplasts prepared from by-2 cultured tobacco cells we examined the effects of 1,8-cineole on cell proliferation and cell elongation by using protoplasts prepared from by-2 cultured tobacco cells . thus , by-2 protoplasts cultured in 2,4-d medium and those cultured in ba - naa medium were used to examine the effects of 1,8-cineole on cell proliferation and on cell elongation , respectively . four - day - old by-2 cells were converted to protoplasts and cultured in either 2,4-d medium a or ba - naa medium b , and the changes in the mitotic index ( ) and cell length ( ) were followed during the culture . 4effects of 1,8-cineole and its application methods on the proliferation and elongation of by-2 protoplasts / cells . in direct - addition method , the initial concentrations of 1,8-cineole in the culture medium ( liquid - phase ) were 50 times higher than the calculated final concentrations in the airspace ( gas - phase ) . ( b ) effects of 1,8-cineole on the proliferation of by-2 protoplasts grown in 2,4-d medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and the mitotic index was examined on d 2 . c effects of 1,8-cineole on the elongation of by-2 protoplasts grown in ba - naa medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and cell length was examined on day 0 and day 7 . in either of the two application modes , relatively high concentrations of 1,8-cineole were necessary to lower the mitotic index . in either of the two application modes , 1,8-cineole lowered cell length in a dose - dependent manner and completely inhibited cell elongation at the highest concentration tested ( 1,000 m ) . ic50 values for cell elongation were about 140 m for the volatilization method and 30 m for the direct - addition method . these results demonstrate that ( i ) the direct addition method was more effective than the volatilization method , and ( ii ) in a by-2 protoplast culture system , cell elongation was more sensitive to 1,8-cineole than cell proliferation . four - day - old by-2 cells were converted to protoplasts and cultured in either 2,4-d medium a or ba - naa medium b , and the changes in the mitotic index ( ) and cell length ( ) were followed during the culture . each data point for mitotic index each point for the length of cells / protoplasts represents the average value from 30 ( for 2,4-d medium ) or 60 ( for ba - naa medium ) cells / protoplasts . vertical bars represent standard errors effects of 1,8-cineole and its application methods on the proliferation and elongation of by-2 protoplasts / cells . in direct - addition method , the initial concentrations of 1,8-cineole in the culture medium ( liquid - phase ) were 50 times higher than the calculated final concentrations in the airspace ( gas - phase ) . ( b ) effects of 1,8-cineole on the proliferation of by-2 protoplasts grown in 2,4-d medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and the mitotic index was examined on d 2 . c effects of 1,8-cineole on the elongation of by-2 protoplasts grown in ba - naa medium . various doses of 1,8-cineole were applied through either the volatilization method ( left ) or the direct - addition method ( right ) , and cell length was examined on day 0 and day 7 . ic50 values estimated from the data are shown in the respective graphs effects of 1,8-cineole on proliferation , elongation , and starch accumulation of by-2 cells we examined the effects of 1,8-cineole on by-2 cells that retained intact cell walls . by-2 cells proliferate actively without significant increase of cell volume when cultured in d - medium that contains 0.2 mg / l 2,4-d , whereas they increase in cell volume ( mostly due to cell elongation ) and accumulate a large amount of starch without proliferation when cultured in b - medium that contains 1 mg / l ba ( sakai et al . accordingly , by-2 cells cultured in d - medium were used to examine the effects of 1,8-cineole on cell proliferation , while those cultured in b - medium were used to examine the effects on cell elongation and starch accumulation . , 1996 ) , based on the results of the experiments with the protoplast culture system , only the direct - addition method was used to apply 1,8-cineole.1,8-cineole lowered the mitotic index in a dose - dependent manner , and the ic50 value for proliferation was about 140 m ( fig . 5b ) , and at high concentrations ( 120 or 240 m ) , the cell size did not increase as compared to the value at the start of the culture ( 105 m ) . the ic50 value for cell elongation was about 30 m , and was lower than the ic50 value for cell proliferation . ic50 value for starch accumulation was about 80 m and was also lower than the ic50 value for cell proliferation . these results demonstrate that the inhibition by 1,8-cineole is not specific to cell proliferation . 5effects of 1,8-cineole on the proliferation and elongation of by-2 cells . a the effects of a range of doses of 1,8-cineole on the proliferation of by-2 cells cultured in d - medium are shown . b the effects of a range of doses of 1,8-cineole on elongation ( top ) and starch accumulation ( bottom ) in by-2 cells cultured in b - medium . ic50 values estimated from the data are shown in respective graphs effects of 1,8-cineole on the proliferation and elongation of by-2 cells . a the effects of a range of doses of 1,8-cineole on the proliferation of by-2 cells cultured in d - medium are shown . b the effects of a range of doses of 1,8-cineole on elongation ( top ) and starch accumulation ( bottom ) in by-2 cells cultured in b - medium . we found here that the effects of 1,8-cineole on n. tabacum seedlings were essentially the same as those on b. campestris seedlings ; 1,8-cineole preferentially inhibited root growth over hypocotyl growth ( fig . in the affected roots , cell size in the elongation zone ( upper region of the root ) was largely unaffected , whereas cell proliferation in the root apical region was severely inhibited ( fig . these results indicate that the mode of action of growth inhibition by 1,8-cineole is common within the two plant species . thus , we consider that tobacco , especially cultured tobacco cells , can be used as experimental material to examine the mode of action of 1,8-cineole at the cellular level . in the volatilization method , thus , the concentrations of 1,8-cineole that the cells experience should gradually increase and finally reach the highest values determined by equilibrium between the liquid and gas phase . in the direct - addition method , the cells should experience the highest concentrations of 1,8-cineole immediately after application of the compound . thus , in the latter method , the actual concentration that the cells experience should be higher and the time at which the cells experience the highest concentration should be earlier . in contrast to our initial expectation , 1,8-cineole inhibited cell elongation more efficiently than cell proliferation ; the ic50 value for cell proliferation was always higher than that for cell elongation , irrespective of the application methods ( fig . moreover , 1,8-cineole also inhibited starch accumulation with an ic50 value lower than that for cell proliferation ( fig . these results demonstrated clearly that the inhibitory effect of 1,8-cineole was not specific to cell proliferation ; rather , 1,8-cineole appeared to affect a wide spectrum of cellular activities in an almost non - specific manner when target cells were efficiently exposed to the compound . despite numerous reports on inhibitory effects of monoterpenes , their mechanisms of action remain obscure . among all the possible effects on biological membranes , the deleterious effects on mitochondrial membranes should cause inhibition of mitochondrial energy metabolism and result in disturbances in a wide range of physiological and biochemical processes within the cell . our results rule out the possibility that preferential inhibition by 1,8-cineole of root growth is explained by preferential inhibition of cell proliferation ; 1,8-cineole inhibited multiple distinct biological processes ( cell proliferation , cell elongation , and starch synthesis ) in cultured cells , and the sensitivity of cell proliferation to 1,8-cineole was , contrary to our initial expectation , relatively low ( figs . however , root growth was more sensitive to 1,8-cineole than hypocotyl growth even when the seedlings were grown under conditions in which both hypocotyls and roots were in equal contact with the filter paper , and therefore , the concentration of 1,8-cineole surrounding the two organs should have been equal ( nishida et al . , 2007 ) , demonstrated differential permeability ( and thus differential development of a cuticle layer ) within a root ; in various plant species , the root tip region that includes the root apical meristem exhibited higher permeability than that of the upper region of the same root , which corresponded to elongation / differentiation zone ( unpublished data ) . thus , differential permeability due to differential development of a cuticle layer might be involved in not only preferential inhibition of root growth over hypocotyl growth but also preferential inhibition of cell proliferation in the root apical region over cell elongation in the upper region of the same root . the relationship between development of the surface barriers , such as cuticle layer , and sensitivity to monoterpenes is now under further investigation .

glucocorticoids are the stress hormones secreted from the adrenals after activation of the hypothal - amus - pituitary - adrenal ( hpa ) axis ; that is , corticosterone in rats and mice , cortisol in humans . the effect on synaptic plasticity and memory formation is mediated by two types of nuclear receptors : mr ( mineralocorticoid receptor ) and gr ( glucocorticoid receptor ) which are located in areas involved in emotion , learning , and memory . while mr is present in the hippocampus and to lesser extent in the prefrontal cortex , amygdale , and paraventricular nucleus [ 25 ] , gr can be found throughout the brain with high levels in the hippocampus and paraventricular nucleus . other characteristics are the differential affinities for corticosterone : mr has a tenfold higher affinity than gr , resulting in predominant mr occupation during low basal levels and additional gr activation during increased corticosterone concentration due to stress or circadian peak activity of the hypothalamic - pituitary - adrenal ( hpa ) axis . the precise involvement of mr and gr in emotion and cognition is still debated . animal studies have shown that activation or blockade of either receptor influences behavior related to anxiety , exploration , and memory . these behaviors are linked to the limbic system and are part of the behavioral repertoire tested in spatial memory tasks and also in fear conditioning . with respect to unconditioned fear - related behavior , smythe et al . have described that mr modulates anxiety - like behavior of rats in the light / dark box . have shown that intracerebroventricular injection of a rather selective mr antagonist in rats influenced corticosterone - induced behavioral reactivity to spatial novelty . recent findings in mutant mice with inactivated mr in the forebrain ( cre - loxp recombination ) support the pharmacologically detected role of mr on the modulation of behavioral strategies . loss of the limbic mr impaired behavioral plasticity , evidenced by a differential performance during the first exposure to learning tasks , that is , their behavioral reactivity to novelty . in contrast , learning slopes in the water and radial arm maze were not affected . this increased behavioral reactivity to novel objects was observed in the face of normal anxiety - like behavior in the open field and elevated - o - maze . indeed , it should be clarified whether mr affects anxiety or appropriate context - dependent behavioral reactivity . others suggest that adaptive behavior is modulated by a combined mr / gr mediated action . an example is the inhibition of corticosterone production and thus prevention of gr activation in the face of full mr activation : this led to decreased fear - induced immobility and fear - related anxiety in rats . complementary , exogenous corticosterone application or prior social defeat increased anxiogenic behavior in rats tested in the elevated plus maze 24 hours later . indeed , gr is implicated in memory consolidation processes , demonstrated by using gr - agonists and gr - antagonists in rats , chickens , as well as gr mutant mice [ 1318 ] . calvo and volosin have shown that corticosterone - induced effects on anxiety after restraint stress require both mr and gr . taken together , mr appears to be responsible for the immediate facilitative effects of corticosterone on memory acquisition , while the modulation of spatial and fear memory relies on the presence of a functional gr . to disentangle the combined contribution of mr and gr to most adequate performance , we will study the functions of these receptors in a task that allows simultaneous registration of emotional and memory parameters . forgas and george suggested that a stimulus first needs to be identified before the appropriate emotional response will follow . others focus more on the neurobiological process of emotion and cognition , which can be functionally , anatomically , and even pharmacologically separated . we hypothesize that emotion and cognition are interdependent and both will be affected by differential mr and gr activations : we propose that the two corticosteroid receptors mr and gr contribute differentially but in a coordinated way to information processing . the aim of this study was to examine how mr and gr interact in information processing presented by emotional and learning / memory elements of a task . next to the well - known use of mr and gr antagonists , mr / gr activation ratios can be endocrinologically and pharmacologically adjusted by removal of the adrenals ( adrenalectomy ( adx ) ) and additional subcutaneous corticosterone pellet implantation . in contrast to rats , mice that undergo adrenalectomy remain to produce low concentrations of corticosterone from scattered cell groups in the vicinity of the adrenals [ 2325 ] . different degrees of continuous gr activation can be achieved via corticosterone released from implanted pellets . we used this approach and tested mice in the modified hole board measuring behaviors that define general activity , emotions , motivation , and learning and memory . subsequent principal component analysis will allow to determine the correlation between emotions and cognition . forty eight 12-week - old male c57bl/6 mice were obtained from charles river ( maastricht , the netherlands ) . after arrival , the mice were housed individually in the experimental room with sawdust bedding , water and food ad libitum , at 20c with controlled humidity under a 12 h : 12 h light / dark cycle ( lights on at 08.00 am ) for at least one week . to familiarize with the bait used in the modified hole board task , all mice received a few pieces of almonds daily in the week before surgery . all experiments were approved by the committee on animal health and care from the leiden university , the netherlands , and were performed in strict compliance with the eec recommendations for the care and use of laboratory animals . mice were randomly selected for one of the following groups and operated accordingly : ( i ) sham - operated ( sham ) , ( ii ) adrenalectomized mice ( adx ) , ( iii ) adrenalectomized mice with an additional low corticosterone pellet ( alc ) , or ( iv ) adrenalectomized mice with an additional high corticosterone pellet ( ahc ) . mice were gas anaesthetized with a mixture of isoflurane / nitrous oxide ( 4% isoflurane bolus followed by 2% isoflurane ) . adrenals were removed ( adx ) using the dorsal approach followed by subcutaneous pellet implantation on the flank of the animal . while in rats adx removes the endogenous source of corticosterone , in mice it clamps corticosterone to low concentrations comparable to the circadian trough of adrenally intact mice . accessory adrenocortical cells secrete stable amounts of corticosterone [ 2325 , 27 ] that maintain extensive occupation of mr . stress or circadian rhythm does not lead to a rise in corticosterone in adx mice . high circulating levels of acth indicate the lack of gr activation ; that is , no negative feedback . sham operation involved the same procedures as adrenalectomy except for the removal of the adrenals . surgery was performed between 10.00 and 12.00 am and lasted maximally 10 minutes per mouse . after surgery , all mice received an additional bottle containing 0.9% salt solution . behavioral testing started 3 days after surgery . to confirm effectiveness of the adrenalectomy and pellet implantation , plasma corticosterone levels were measured 2 days after surgery , on day 0 of the experiment , and one day after the last behavioral test on day 11 . mice with abnormal corticosterone concentrations in the blood were excluded from further analysis . two types of pellets were made for subcutaneous implantation : ( i ) a 5% corticosterone ( icn biomedicals , inc . , calif , usa ) 95% cholesterol pellet for moderate mr / gr activation and ( ii ) a 20% corticosterone 80% cholesterol pellet for strong mr / gr activation . all pellets weighed 100 mg , with a diameter of 7 mm and thickness of 2 mm and were homemade . corticosterone dose was chosen following a pilot experiment in which plasma corticosterone concentrations of about 100 and 150 ng / ml for the 5% and 20% pellets , respectively , were measured two days after implantation . the modified hole board consisted of an opaque grey pvc box ( 50 50 50 cm ) with a center board ( 37 20 cm ) on which 10 grey cylinders ( 4 cm height ) were staggered in two lines . always the same three cylinders were baited with a small piece of almond on top of a grid , and were marked with a white ring . seven other cylinders contained a nonobtainable almond underneath the grid and were marked with a black ring . the mice were placed in the modified hole board for 3 trials per day with changing start positions . one trial lasted maximally 5 minutes , or until the mouse had found the three baits . the behavior of the mice was observed , recorded , and analyzed with a semiautomatic scoring system ( the observer mobile 4.1 , noldus information technology , wageningen , the netherlands ) . all measured behavioral parameters are represented in table 1 . as indication for ( i ) working memory , the number of repeated holevisits was calculated and ( ii ) reference memory , the number of visits to nonbaited holes was taken . in addition , a camera was installed above the setup to measure distance moved and velocity of the mice with an automatic tracking system ( ethovision 1.95 , noldus information technology , wageningen , the netherlands ) . mice were tested in the modified hole board over 10 days . on days 1 to 5 and 8 , the three baited cylinders were marked with a white ring as visual cue while the remaining cylinders were marked with a black ring . all rings were removed from the cylinders , but the bait remained in the same cylinders . this allowed to estimate if the mice used a spatial strategy or visual discrimination to solve the task . a trial lasted maximally 5 minutes and was ended when the mouse had eaten all three baits . on days 0 and 11 , blood was collected via a tail incision or after decapitation . blood plasma was used to measure corticosterone concentrations ( icn biomedicals , inc . , calif , usa ) . because exposure to high concentrations of corticosterone results in shrinkage of the thymus , thymus weight was estimated as well . differences in corticosterone concentrations between groups and days were analyzed by two - way anova ( spss 11.5.0 ) with tukey 's post - hoc analysis . to analyze thymus and body - weight differences , the behavioral data are presented as mean of 3 trials per day sem . data were subjected to general linear model ( glm- ) repeated measures with tukey as post - hoc test to analyze progression over days and group differences per day . furthermore , factor analysis ( principal component analysis ( pca ) ) was performed over groups and days to obtain a more comprehensive analysis of emotional and cognitive parameters . this analysis uses cross - mouse comparisons to distinguish the relation between behavioral parameters . it includes as much data as possible in each factor to minimize residual variance from the original dataset . the pca was performed with a varimax rotation on variables with communalities over 0.7 , that is , of which 70% of the variance is explained by the factors extracted . the number of extracted factors was not predefined ; factors with an eigenvalue > 1 were accepted . factor scores were subjected to a two - way anova to determine differences between groups and days . figure 1 shows the results for some of the emotional and explorative parameters during all days of testing in the modified hole board . figure 1(a ) illustrates that adx followed by alc mice have a high percentage of time spent on the center board , indicative of low anxiety [ 26 , 2830 ] during the first few days . in contrast , ahc and sham mice spent little time on the center board during this period . from day 4 on , few significant differences were found between groups . glm from day 1 to 10 revealed a significant group / day interaction f(21,588 ) 2.355 , p = .001 . figure 1(b ) shows that ahc mice display twofold more defecation compared to other groups , indicating high arousal . with repeated testing , alc mice display less defecation compared to adx and ahc mice . glm revealed a significant progressive decrease over days f(21,588 ) 7.629 , p < .0001 , just passing statistical significance between groups ( f(21,588 ) 1.524 , p = .063 ) . the number of rearings was taken as measure for general exploration ( figure 1(c ) ) . comparing the first and the last days of testing , no differences were found between groups while on days 2 , 3 , and 4 adx mice displayed the lowest number of rearings . glm showed a significant change over days ( f(21,588 ) 11.439 , p < .0001 ) although not significant between groups ( f(21,588 ) 1.25 , p = .203 ) . adx mice display highly directed exploration / behavioral reactivity on all days of testing , reaching statistical significance on days 1 and 2 as indicated by the number of hole visits ( figure 1(d ) ) . sham , ahc , and alc mice start off with few hole visits which increase over time . glm supported this by significant group / day interaction f(21,588 ) 1.983 , p = .006 . total distance moved and velocity were comparable between groups over all days of testing ( data not shown ) . figure 2 shows the results for three cognitive parameters on all days of testing in the modified hole board . figure 2(a ) illustrates increased repeated hole visits ( working memory ) in adx mice on day 8 of testing compared to sham mice . we consider the low repeated hole visits on days 1 and 2 of sham , alc , and ahc mice as not reliable , because the total number of hole visits is also very low on these days . over time , sham , alc , and ahc mice show increased repeats in parallel with increased total hole visits . glm showed a significant group / day interaction ( f(21,532 ) 2.029 , p = .005 ) . figure 2(b ) shows no significant differences in nonbaited hole visits ( reference memory ) between sham , adx , alc , and ahc mice during all days of testing . the time to finish the task is an additional learning parameter ( figure 2(c ) ) . removal of the rings on days 9 and 10 did not influence the time to finish the task , indicating the use of a spatial learning strategy at that time of training . at the last day of testing , performance of sham mice was still poor although progression over days proved to be significant ( f(21,532 ) 18.327 , p = .000 ) . principal component analysis ( pca ) over all behavioral data resulted in the extraction of four factors ( table 2 ) which explain 81% of total variance . factor 1 ( 41% ) combines behavioral parameters that can be classified as anxiety , motivation , and good learning , factor 2 ( 19% ) represents directed exploration , behavioral reactivity , and working memory , factor 3 ( 11% ) represents general activity and factor 4 ( 10% ) includes behavioral parameters that can be classified as impaired learning . one - way anova between groups on factor loadings for factor 1 ( anxiety , motivation , good learning ) revealed significant differences between sham mice compared to adx , alc , and ahc mice ( f(3,279 ) 11.562 , p = .000 ) . significant group differences were also found between adx mice compared to sham , alc , and ahc mice for factor 3 ( general activity ; f(3,279 ) 8.362 , p = .000 ) . furthermore , when comparing the factor loadings over days , significant differences were found for factor 1 between days 3 and 4 compared to days 9 and 10 , ( f(7,279 ) 4.460 , p = .000 ) . this indicates low anxiety , more motivation , and better learning at the end of testing in all groups . factor 3 was significantly different between day 2 and days 1 , 8 , and 9 ( f(7,279 ) 2.522 , p = .016 ) , which indicates that general activity was decreased at the end of testing . both low and high corticosterone pellet groups , alc and ahc , had higher plasma corticosterone concentrations on day 0 ( f(3,31 ) 29.540 , p = .0001 ) than the sham and adx mice . on day 11 of the experiment , only ahc mice showed significantly increased corticosterone levels ( f(3,31 ) 28.977 , p = .0001 ) , compared to sham , adx , and alc mice . plasma corticosterone in sham and adx mice remained at the same low basal morning level throughout the experiment , while corticosterone concentrations of alc and ahc mice decreased in the course of the study ( f(1,15 ) 7.835 , p = .014 and f(1,15 ) 13.344 , p = .003 ) . thymus weights on day 11 supported the exposure to elevated corticosterone during the experiment with significantly lower thymus weights for alc and ahc mice compared to sham and adx mice ( f(3,31 ) 22.332 , p = .000 ) . alc mice had a less shrunken thymus than ahc mice , indicating exposure to lower corticosterone concentrations than ahc . body weight on day 11 was comparable between groups f(24,27 ) 1.731 , p = .187 . four groups of mice were generated by endocrine manipulation , resulting in different amounts of circulating corticosterone concentrations in the blood . given the different affinities of the receptors for the hormone , we expect a differential mr / gr activation in these groups : ( i ) sham mice with an intact hpa axis , ( ii ) adx mice with residual stable low corticosterone levels and thus continuous mr activation , ( iii ) alc mice with moderate elevated circulating corticosterone concentrations allowing extensive mr and moderate gr activations , and ( iv ) ahc mice with a full mr and a substantial gr activation due to high circulating levels of corticosterone . we found emotional expressions and cognitive performance related to differential corticosteroid receptor activation . continuous predominant mr activation directed emotional components indicative for less anxiety to the benefit of cognition , while continuous additional gr activation was associated with impaired learning . mice with stable predominant mr activation ( adx ) show increased directed exploration / behavioral reactivity towards the cylinders ( hole visits ) and low anxiety during the first days of testing , that is , when the setting is novel . this corresponds to the observation that transgenic mice with low gr , and rats with icv injection of gr antagonist express low - anxiety - related behavior [ 31 , 32 ] . however , it contrasts previous findings that gr blockade by single infusion of ru38486 into the hippocampus has no anxiolytic effect in rats in the light / dark box . of course , the methods to achieve predominant mr activation differ in the history of inactivated gr , species , stressed state of the animals , and behavioral task . also a differentiation between context - related behavioral reactivity and anxiety is not possible . factor analysis reveals that the variables time on center board ( anxiety , motivation , good learning ; factor 1 ) and hole visits ( directed exploration and behavioral reactivity ; factor 2 ) are not correlated . thus , the general idea that mice which are more prone to go to the unprotected center area are likely to display more cylinder directed behavior is not supported . in contrast , anxiety is correlated with motivation ( latency to first hole visit , latency eat bait ) : mice with a low anxiety approach the unprotected area faster . overall , low anxiety and high directed exploration / behavioral reactivity could be beneficial for the onset of learning , especially during the first days of testing . we observed an apparent fast onset of learning in these predominantly mr mice . high directed exploration towards the cylinders will eventually result in finding all baits , without any necessary learning of the task . indeed , mice of this group show an increase in working memory errors ( revisits ) after the two - day break without testing . gr is expected to promote the consolidation of mr - related adaptive behavior , leaving the lack of gr activation as the most likely explanation for the memory deficit . the results of the berger study can be interpreted the other way round : the lack of forebrain mr resulted in working memory deficits in the water maze task because a functional gr facilitated the consolidation of nonadaptive behavior . we conclude that the observed behavior of animals with differential mr and gr conditions will only be understood in relation to the contribution of both receptors . alc mice with mr and moderate gr activations display low anxiety during the first days of testing , general low arousal , and fast learning . corticosterone levels in the alc mice were continuously elevated in the range of the circadian rise , thus it would not be expected to cause damage to neurons , downregulation of mr and gr , or alterations in neurotransmitters implied in cognitive impairments . in fact , alc mice with mr and moderate gr activations showed the best cognitive performance . part of this improved learning and memory ability could be explained by the emotional state of the mice . like adx mice , alc mice have low anxiety ( and arousal ) during the first days of learning which is correlated with increased motivation and good learning . supporting our argument is the most recent finding of herrero , that rats with low anxiety showed faster spatial learning together with increased hippocampal mr ; opposite results were found in high - anxiety rats . stronger mr availability and activation might underlie the fast onset of learning , while gr are responsible for the consolidation of this context - related information . therefore , it is not surprising that alc mice with a moderately activated gr display improved or normal cognitive performance compared to adx mice with little or no gr activation throughout testing . for optimal coordination of cognition and emotion , both mr and a moderate activation of gr are necessary [ 39 , 40 ] . as described by many others , chronic strong gr activation caused by , for example , severe stressors or pharmacological modulation of the hpa axis results in impaired learning and memory [ 4143 ] , reduced synaptic plasticity in the hippocampus , increased anxiety , and even depression - like symptomatology . in patients suffering form depression or cushing 's disease , elevated levels of cortisol have been associated with poorer cognitive performance in verbal memory , working memory , and post - encoding tasks [ 4648 ] . furthermore , an association between cortisol level and increased fear perception has been found in patients suffering from recurring depression , which also indicates a modulatory role of glucocorticoids in emotional processes . we find similar results for emotions and cognition : ahc mice with mr and continuous high gr activation have a slow onset of learning together with increased arousal and anxiety - like behaviors and suppression of directed exploration . it is not surprising that these mice display a slower onset of learning ( opposite to low anxiety and fast learning as described above ) . at first glance , it seems surprising that when learning starts to occur , the magnitude of learning ( figure 2(c ) : time to finish task , slope of the learning curve ) is the same in alc and ahc mice . the change in corticosterone availability , due to the encapsulation of the pellet , is most likely responsible for the altered behavior . corticosterone levels decreased over the days to concentrations in the normal range , that is , comparable to circadian peak secretion and the amount of corticosterone measured in alc mice at the beginning of testing . thus , in ahc mice we deal with memory impairments and high emotional arousal only during specific stages of learning , namely during the first days of testing that coincide with really high exposure to corticosterone . we used sham - operated mice that have an intact hpa axis as control group . unexpectedly , these mice were characterized as highly anxious and with little motivation , with high arousal and a slow onset and little progress of learning . factor 1 was significantly different over time between sham and all other groups tested : low motivation and high anxiety throughout testing days . we got the impression that the behavioral setting remained anxiogenic to these mice . lack of exploration of the centre board might also prevent learning basic rules , for example , that cylinders are baited with almonds . this and the possible role of a prolonged effect of surgery on the hpa system resulted in a follow - up experiment . we used three groups of mice ( n = 6 per group ) : ( 1 ) sham - operated mice and ( 2 ) nave , nonoperated mice received almonds in the homecage to familiarize with the bait , like the experimental groups , ( 3 ) nave mice received almonds in the cylinders four times in the week before the modified hole board task . sham and nave mice without preexposure to the cylinders displayed similar high anxiety and slow learning as we saw before . however , after pretraining with baited cylinders anxiety decreased , motivation increased and learning improved ( figure 3 ) . since surgery did not influence behavior on the modified hole board , incomplete recovery from the surgery is unlikely to affect performance . using a somewhat different experimental design , comparably long times to finish the task have been reported for c57bl/6 mice ( ohl 2003 ; still 280 to 300 seconds after eight days of training ) . in contrast , prior familiarization to items of the test condition reduced anxiety - like behavior and increased motivation , which could ( in part ) increase cognitive performance like it was observed in adx and alc mice . it is remarkable that mice without adrenals dysregulated hpa - axis activity and additional pellet implantation did better compared to the relative intact sham and nave control groups . these findings even more underscore that ( i ) high anxiety and arousal have negative consequences for cognition while ( ii ) less anxiety , increased motivation , and goal - directed exploration have a positive influence on behavior ( see also ) . we consider the role of mr in the integration of sensory information and behavioral strategies central for reduced anxiety - related behavior . the adrenalectomy - induced deficit in corticosterone secretion results in the disinhibition of hpa activity , and thus enhanced release of corticotrophin - releasing hormone ( crh ) and vasopressin ( avp ) from the hypothalamus . crh , avp , and adrenaline , all might play a role in emotional expressions and cognitive performance of adx mice , with and without supplementary corticosterone . considering the function of the gr in the negative feedback , we may expect that adx mice ( predominant mr activation ) and alc mice ( mr and moderate gr ) have a deficient suppression of crh and avp activities [ 51 , 52 ] . mice with elevated levels of crh that acts predominantly via crh receptor 1 are expected to display increased anxiety . mutant mice with a deficient crh receptor 1 either by genetic deletion or pharmacological blockade are less anxious . clearly , crh is involved in anxiety - related behavior . however in the present study , adx and alc mice show low anxiety - related behavior , while ahc mice ( predominant gr activation ) are highly anxious . these findings do not support a role of hypothalamus - related crh activity in anxiety behavior in the present study . the same argument holds true for avp . in response to stress , noradrenalin release increases . this is thought to contribute to the anxiogenic effects of stress [ 50 , 54 ] , in which the amygdala plays an important role . ahc and sham mice showed the strongest arousal ( defecation ) and were characterized as most anxious : a participation of catecholamines in these responses can not be excluded . although food was present ad libitum throughout the experiment and body weight did not differ between the groups , motivation to go for the almond - bait might have been increased in adx and alc mice . factor analysis also underlines the role of motivation in relation to anxiety for the performance . anxiety - related behavior in rodents is generally deduced from the avoidance of an open , bright , and unprotected area . for example , rats that are specifically selected for their avoidance of open arms of the elevated plus maze , and thus classified as high anxiety rats , do not avoid the center ( open ) area of a hole board task . complexity and duration of the task , as well as motivational aspects might overcome state anxiety . directed exploration or behavioral reactivity is expressed by approach to certain stimuli , for example , the number of visits to a specific location in the testing area . does directed exploration rely on reduced anxiety ? in the present study , animals with low directed exploration would spend little time near the cylinders on the centre board . although it is likely that anxiety interacts with directed exploration , this does not necessarily has to be the case . it could be that our interpretation of high anxiety is characteristic for a more passive exploration strategy [ 57 , 58 ] without a dominant role for anxiety - related behavior . the setting of our task and subsequent factorial analysis allowed us to differentiate anxiety - like behavior from directed exploration : they did not coincide into one factor , indicating no correlation between the two . anxiety and motivation are important factors for the onset of learning , a process in which mr and gr and their coordinated activation play a crucial role . continuous predominant mr activation appears to be beneficial for the emotional state , resulting in low anxiety , high motivation , and high directed exploration and behavioral reactivity , but does not result in better learning and memory . additional moderate gr activation also results in low anxiety and high motivation , with the advantage of improved cognition expressed as a decrease in working memory errors . in contrast , mr with additional substantial gr activation results in a slow onset of learning together with high anxiety , showing similarities with patients suffering from depression and cushing 's disease . we conclude that optimal performance is bound to continuous mr activation together with moderate gr activation . further increase in corticosterone , and therefore substantial gr activation , will increase emotional arousal with impairing effects for learning and memory .

corticosteroids regulate stress response and influence emotion , learning , and memory via two receptors in the brain , the high - affinity mineralocorticoid ( mr ) and low - affinity glucocorticoid receptor ( gr ) . we test the hypothesis that mr- and gr - mediated effects interact in emotion and cognition when a novel situation is encountered that is relevant for a learning process . by adrenalectomy and additional constant corticosterone supplement we obtained four groups of male c57bl/6j mice with differential chronic mr and gr activations . using a hole board task , we found that mice with continuous predominant mr and moderate gr activations were fast learners that displayed low anxiety and arousal together with high directed explorative behavior . progressive corticosterone concentrations with predominant action via gr induced strong emotional arousal at the expense of cognitive performance . these findings underline the importance of a balanced mr / gr system for emotional and cognitive functioning that is critical for mental health .

1. INTRODUCTION 2. MATERIAL AND METHODS 3. RESULTS 4. DISCUSSION 5. CONCLUSION

the effect on synaptic plasticity and memory formation is mediated by two types of nuclear receptors : mr ( mineralocorticoid receptor ) and gr ( glucocorticoid receptor ) which are located in areas involved in emotion , learning , and memory . while mr is present in the hippocampus and to lesser extent in the prefrontal cortex , amygdale , and paraventricular nucleus [ 25 ] , gr can be found throughout the brain with high levels in the hippocampus and paraventricular nucleus . other characteristics are the differential affinities for corticosterone : mr has a tenfold higher affinity than gr , resulting in predominant mr occupation during low basal levels and additional gr activation during increased corticosterone concentration due to stress or circadian peak activity of the hypothalamic - pituitary - adrenal ( hpa ) axis . the precise involvement of mr and gr in emotion and cognition is still debated . animal studies have shown that activation or blockade of either receptor influences behavior related to anxiety , exploration , and memory . have described that mr modulates anxiety - like behavior of rats in the light / dark box . have shown that intracerebroventricular injection of a rather selective mr antagonist in rats influenced corticosterone - induced behavioral reactivity to spatial novelty . recent findings in mutant mice with inactivated mr in the forebrain ( cre - loxp recombination ) support the pharmacologically detected role of mr on the modulation of behavioral strategies . in contrast , learning slopes in the water and radial arm maze were not affected . others suggest that adaptive behavior is modulated by a combined mr / gr mediated action . complementary , exogenous corticosterone application or prior social defeat increased anxiogenic behavior in rats tested in the elevated plus maze 24 hours later . indeed , gr is implicated in memory consolidation processes , demonstrated by using gr - agonists and gr - antagonists in rats , chickens , as well as gr mutant mice [ 1318 ] . calvo and volosin have shown that corticosterone - induced effects on anxiety after restraint stress require both mr and gr . to disentangle the combined contribution of mr and gr to most adequate performance , we will study the functions of these receptors in a task that allows simultaneous registration of emotional and memory parameters . others focus more on the neurobiological process of emotion and cognition , which can be functionally , anatomically , and even pharmacologically separated . we hypothesize that emotion and cognition are interdependent and both will be affected by differential mr and gr activations : we propose that the two corticosteroid receptors mr and gr contribute differentially but in a coordinated way to information processing . the aim of this study was to examine how mr and gr interact in information processing presented by emotional and learning / memory elements of a task . next to the well - known use of mr and gr antagonists , mr / gr activation ratios can be endocrinologically and pharmacologically adjusted by removal of the adrenals ( adrenalectomy ( adx ) ) and additional subcutaneous corticosterone pellet implantation . we used this approach and tested mice in the modified hole board measuring behaviors that define general activity , emotions , motivation , and learning and memory . subsequent principal component analysis will allow to determine the correlation between emotions and cognition . forty eight 12-week - old male c57bl/6 mice were obtained from charles river ( maastricht , the netherlands ) . after arrival , the mice were housed individually in the experimental room with sawdust bedding , water and food ad libitum , at 20c with controlled humidity under a 12 h : 12 h light / dark cycle ( lights on at 08.00 am ) for at least one week . to familiarize with the bait used in the modified hole board task , all mice received a few pieces of almonds daily in the week before surgery . all experiments were approved by the committee on animal health and care from the leiden university , the netherlands , and were performed in strict compliance with the eec recommendations for the care and use of laboratory animals . to confirm effectiveness of the adrenalectomy and pellet implantation , plasma corticosterone levels were measured 2 days after surgery , on day 0 of the experiment , and one day after the last behavioral test on day 11 . mice with abnormal corticosterone concentrations in the blood were excluded from further analysis . , calif , usa ) 95% cholesterol pellet for moderate mr / gr activation and ( ii ) a 20% corticosterone 80% cholesterol pellet for strong mr / gr activation . the modified hole board consisted of an opaque grey pvc box ( 50 50 50 cm ) with a center board ( 37 20 cm ) on which 10 grey cylinders ( 4 cm height ) were staggered in two lines . always the same three cylinders were baited with a small piece of almond on top of a grid , and were marked with a white ring . the mice were placed in the modified hole board for 3 trials per day with changing start positions . the behavior of the mice was observed , recorded , and analyzed with a semiautomatic scoring system ( the observer mobile 4.1 , noldus information technology , wageningen , the netherlands ) . as indication for ( i ) working memory , the number of repeated holevisits was calculated and ( ii ) reference memory , the number of visits to nonbaited holes was taken . in addition , a camera was installed above the setup to measure distance moved and velocity of the mice with an automatic tracking system ( ethovision 1.95 , noldus information technology , wageningen , the netherlands ) . mice were tested in the modified hole board over 10 days . blood plasma was used to measure corticosterone concentrations ( icn biomedicals , inc . to analyze thymus and body - weight differences , the behavioral data are presented as mean of 3 trials per day sem . furthermore , factor analysis ( principal component analysis ( pca ) ) was performed over groups and days to obtain a more comprehensive analysis of emotional and cognitive parameters . figure 1 shows the results for some of the emotional and explorative parameters during all days of testing in the modified hole board . comparing the first and the last days of testing , no differences were found between groups while on days 2 , 3 , and 4 adx mice displayed the lowest number of rearings . sham , ahc , and alc mice start off with few hole visits which increase over time . figure 2 shows the results for three cognitive parameters on all days of testing in the modified hole board . over time , sham , alc , and ahc mice show increased repeats in parallel with increased total hole visits . removal of the rings on days 9 and 10 did not influence the time to finish the task , indicating the use of a spatial learning strategy at that time of training . at the last day of testing , performance of sham mice was still poor although progression over days proved to be significant ( f(21,532 ) 18.327 , p = .000 ) . factor 1 ( 41% ) combines behavioral parameters that can be classified as anxiety , motivation , and good learning , factor 2 ( 19% ) represents directed exploration , behavioral reactivity , and working memory , factor 3 ( 11% ) represents general activity and factor 4 ( 10% ) includes behavioral parameters that can be classified as impaired learning . one - way anova between groups on factor loadings for factor 1 ( anxiety , motivation , good learning ) revealed significant differences between sham mice compared to adx , alc , and ahc mice ( f(3,279 ) 11.562 , p = .000 ) . significant group differences were also found between adx mice compared to sham , alc , and ahc mice for factor 3 ( general activity ; f(3,279 ) 8.362 , p = .000 ) . this indicates low anxiety , more motivation , and better learning at the end of testing in all groups . factor 3 was significantly different between day 2 and days 1 , 8 , and 9 ( f(7,279 ) 2.522 , p = .016 ) , which indicates that general activity was decreased at the end of testing . plasma corticosterone in sham and adx mice remained at the same low basal morning level throughout the experiment , while corticosterone concentrations of alc and ahc mice decreased in the course of the study ( f(1,15 ) 7.835 , p = .014 and f(1,15 ) 13.344 , p = .003 ) . four groups of mice were generated by endocrine manipulation , resulting in different amounts of circulating corticosterone concentrations in the blood . given the different affinities of the receptors for the hormone , we expect a differential mr / gr activation in these groups : ( i ) sham mice with an intact hpa axis , ( ii ) adx mice with residual stable low corticosterone levels and thus continuous mr activation , ( iii ) alc mice with moderate elevated circulating corticosterone concentrations allowing extensive mr and moderate gr activations , and ( iv ) ahc mice with a full mr and a substantial gr activation due to high circulating levels of corticosterone . we found emotional expressions and cognitive performance related to differential corticosteroid receptor activation . continuous predominant mr activation directed emotional components indicative for less anxiety to the benefit of cognition , while continuous additional gr activation was associated with impaired learning . mice with stable predominant mr activation ( adx ) show increased directed exploration / behavioral reactivity towards the cylinders ( hole visits ) and low anxiety during the first days of testing , that is , when the setting is novel . this corresponds to the observation that transgenic mice with low gr , and rats with icv injection of gr antagonist express low - anxiety - related behavior [ 31 , 32 ] . however , it contrasts previous findings that gr blockade by single infusion of ru38486 into the hippocampus has no anxiolytic effect in rats in the light / dark box . of course , the methods to achieve predominant mr activation differ in the history of inactivated gr , species , stressed state of the animals , and behavioral task . factor analysis reveals that the variables time on center board ( anxiety , motivation , good learning ; factor 1 ) and hole visits ( directed exploration and behavioral reactivity ; factor 2 ) are not correlated . thus , the general idea that mice which are more prone to go to the unprotected center area are likely to display more cylinder directed behavior is not supported . in contrast , anxiety is correlated with motivation ( latency to first hole visit , latency eat bait ) : mice with a low anxiety approach the unprotected area faster . overall , low anxiety and high directed exploration / behavioral reactivity could be beneficial for the onset of learning , especially during the first days of testing . we conclude that the observed behavior of animals with differential mr and gr conditions will only be understood in relation to the contribution of both receptors . alc mice with mr and moderate gr activations display low anxiety during the first days of testing , general low arousal , and fast learning . corticosterone levels in the alc mice were continuously elevated in the range of the circadian rise , thus it would not be expected to cause damage to neurons , downregulation of mr and gr , or alterations in neurotransmitters implied in cognitive impairments . in fact , alc mice with mr and moderate gr activations showed the best cognitive performance . like adx mice , alc mice have low anxiety ( and arousal ) during the first days of learning which is correlated with increased motivation and good learning . supporting our argument is the most recent finding of herrero , that rats with low anxiety showed faster spatial learning together with increased hippocampal mr ; opposite results were found in high - anxiety rats . therefore , it is not surprising that alc mice with a moderately activated gr display improved or normal cognitive performance compared to adx mice with little or no gr activation throughout testing . for optimal coordination of cognition and emotion , both mr and a moderate activation of gr are necessary [ 39 , 40 ] . as described by many others , chronic strong gr activation caused by , for example , severe stressors or pharmacological modulation of the hpa axis results in impaired learning and memory [ 4143 ] , reduced synaptic plasticity in the hippocampus , increased anxiety , and even depression - like symptomatology . in patients suffering form depression or cushing 's disease , elevated levels of cortisol have been associated with poorer cognitive performance in verbal memory , working memory , and post - encoding tasks [ 4648 ] . we find similar results for emotions and cognition : ahc mice with mr and continuous high gr activation have a slow onset of learning together with increased arousal and anxiety - like behaviors and suppression of directed exploration . it is not surprising that these mice display a slower onset of learning ( opposite to low anxiety and fast learning as described above ) . at first glance , it seems surprising that when learning starts to occur , the magnitude of learning ( figure 2(c ) : time to finish task , slope of the learning curve ) is the same in alc and ahc mice . corticosterone levels decreased over the days to concentrations in the normal range , that is , comparable to circadian peak secretion and the amount of corticosterone measured in alc mice at the beginning of testing . thus , in ahc mice we deal with memory impairments and high emotional arousal only during specific stages of learning , namely during the first days of testing that coincide with really high exposure to corticosterone . we used three groups of mice ( n = 6 per group ) : ( 1 ) sham - operated mice and ( 2 ) nave , nonoperated mice received almonds in the homecage to familiarize with the bait , like the experimental groups , ( 3 ) nave mice received almonds in the cylinders four times in the week before the modified hole board task . using a somewhat different experimental design , comparably long times to finish the task have been reported for c57bl/6 mice ( ohl 2003 ; still 280 to 300 seconds after eight days of training ) . it is remarkable that mice without adrenals dysregulated hpa - axis activity and additional pellet implantation did better compared to the relative intact sham and nave control groups . these findings even more underscore that ( i ) high anxiety and arousal have negative consequences for cognition while ( ii ) less anxiety , increased motivation , and goal - directed exploration have a positive influence on behavior ( see also ) . the adrenalectomy - induced deficit in corticosterone secretion results in the disinhibition of hpa activity , and thus enhanced release of corticotrophin - releasing hormone ( crh ) and vasopressin ( avp ) from the hypothalamus . crh , avp , and adrenaline , all might play a role in emotional expressions and cognitive performance of adx mice , with and without supplementary corticosterone . considering the function of the gr in the negative feedback , we may expect that adx mice ( predominant mr activation ) and alc mice ( mr and moderate gr ) have a deficient suppression of crh and avp activities [ 51 , 52 ] . mice with elevated levels of crh that acts predominantly via crh receptor 1 are expected to display increased anxiety . however in the present study , adx and alc mice show low anxiety - related behavior , while ahc mice ( predominant gr activation ) are highly anxious . these findings do not support a role of hypothalamus - related crh activity in anxiety behavior in the present study . ahc and sham mice showed the strongest arousal ( defecation ) and were characterized as most anxious : a participation of catecholamines in these responses can not be excluded . for example , rats that are specifically selected for their avoidance of open arms of the elevated plus maze , and thus classified as high anxiety rats , do not avoid the center ( open ) area of a hole board task . directed exploration or behavioral reactivity is expressed by approach to certain stimuli , for example , the number of visits to a specific location in the testing area . in the present study , animals with low directed exploration would spend little time near the cylinders on the centre board . it could be that our interpretation of high anxiety is characteristic for a more passive exploration strategy [ 57 , 58 ] without a dominant role for anxiety - related behavior . anxiety and motivation are important factors for the onset of learning , a process in which mr and gr and their coordinated activation play a crucial role . continuous predominant mr activation appears to be beneficial for the emotional state , resulting in low anxiety , high motivation , and high directed exploration and behavioral reactivity , but does not result in better learning and memory . additional moderate gr activation also results in low anxiety and high motivation , with the advantage of improved cognition expressed as a decrease in working memory errors . in contrast , mr with additional substantial gr activation results in a slow onset of learning together with high anxiety , showing similarities with patients suffering from depression and cushing 's disease . we conclude that optimal performance is bound to continuous mr activation together with moderate gr activation . further increase in corticosterone , and therefore substantial gr activation , will increase emotional arousal with impairing effects for learning and memory .

case selection : all horses that were presented to the racehorse hospital , ritto training center for lameness evaluation in 2014 and diagnosed with mri abnormalities of one of the odsls were included in this study . radiography and ultrasonography : radiographs were taken using a computed digital radiography system ( fcr speedia cs , fujifilm , tokyo , japan ) . in all cases , radiographic projections included standing lateromedial and dorsopalmar views of the mcp joint , and dorsolateral palmaromedial 45 oblique and dorsomedial palmarolateral 45 oblique views of the mcp joint . ultrasonographic evaluation of the palmar pastern region was performed using an ultrasound system ( hi vision avius , hitachi , tokyo , japan ) with a 146 mhz linear probe . standing mri : a 0.27-tesla dedicated equine mri system ( equine limb scanner , hallmarq veterinary imaging , ltd . , guilford , u.k . ) was used to obtain mri images of the pastern regions . t1-weighted gradient echo ( gre ) [ echo tome ( te ) : 8 msec / repetition time ( tr ) : 50 msec , flip angle 55 ) , t2-weighted fast spin echo ( fse ) ( te : 88 msec / tr : 1681 msec ) and short tau inversion recovery ( stir ) fse [ te : 22 msec / tr : 2,460 msec , inversion time ( ti)=110 msec ] were acquired in the frontal , transverse and sagittal planes . mri images were acquired and reviewed by several clinicians experienced in the interpretation of smri images of thoroughbred racehorses , using the digital imaging and communications in medicine ( dicom ) viewer osirix , version 3.9.2 ( osirix project , geneva , switzerland ) . in all horses , medetomidine ( domitor , meiji seika co. , tokyo , japan ) was administered at an initial dose of 5 g / kg prior to the onset of positioning within the magnet . thereafter , small doses of medetomidnine ( 0.2 g / kg ) were administered periodically to maintain sufficient tranquilization for the scan . follow - up was conducted through interviews of the trainers or evaluation of the horses upon return to the ritto training center . a 5-year - old thoroughbred mare exhibited acute - onset left foreleg lameness following a race on a flat turf course . a 3-day course of oral diclofenac sodium ( 1 mg / kg ; blesin tablets , sawai pharmaceutical co. , ltd , osaka , japan ) , leg cooling and boxed rest did not improve the heat and pain in the pastern . on day 4 radiography revealed new periosteal bone formation at the mid - palmaromedial surface of the proximal phalanx ( fig . periosteal osteoproliferation is visible at the mid - palmaromedial surface of the proximal phalanx ( arrowheads ) . ) . a marked increase in fluid and small amount of fibrin in the digital flexor tendon sheath ( dfts ) were observed via ultrasonography ( fig . 2.(a ) transverse ultrasonographic image of case 1 , obtained in a lateral oblique position over the proximal pastern region . the palmar is at the top , and the lateral direction is at left . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . ( b ) transverse ultrasonographic image of case 1 , obtained in a medial oblique position over the proximal pastern region . in addition , the medial odsl appeared to be hypoechoic . to obtain a further diagnosis , smri images revealed high signal intensity in the enlarged medial odsl on t1-weighted gre , t2-weighted fse and stir fse images from the proximal pastern to mid - pastern that likely represented desmitis of the medial odsl ( fig . 4.(a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . an increased amount of fluid is present within the dfts ( arrowheads ) . ) . the enlarged dfts exhibited a hypointense signal on t1-weighted gre images , and hyperintense signals on t2-weighted fse and stir fse images ( fig . periosteal osteoproliferation is visible at the mid - palmaromedial surface of the proximal phalanx ( arrowheads ) . ( a ) transverse ultrasonographic image of case 1 , obtained in a lateral oblique position over the proximal pastern region . the palmar is at the top , and the lateral direction is at left . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . ( b ) transverse ultrasonographic image of case 1 , obtained in a medial oblique position over the proximal pastern region . ( a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . the dorsal and medial directions are at the top and left , respectively . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . following the smri examination , the mare was sent to a farm for rest . a 3-year - old thoroughbred filly exhibited right foreleg lameness after exercise . dexamethasone ( corson p injection , nihon zenyaku kogyo , fukushima , japan ) was administered intravenously at a dose of 40 g / kg to reduce swelling over the right fore pastern , and the filly was placed on boxed rest for 1 day , followed by 3 days of light exercise . on day 5 , the lameness and swelling recurred , and the filly was admitted to the hospital for further diagnosis . a distended dfts was identified via ultrasonography , and the medial odsl appeared to be enlarged ( fig . 5fig . 5.transverse ultrasonographic image obtained in the medial oblique direction over the proximal pastern region of case 2 . note the enlargement of the medial oblique distal sesamoidean ligament ( arrows ) . ) . no abnormality was observed on the other dsl or the branches of superficial digital flexor tendon . in addition , the marked pain response over the medial pastern did not resolve . on day 6 the medial odsl exhibited increased signal intensity on t1-weighted gre , t2-weighted fse and stir fse images ( fig . 6.(a ) transverse t1-weighted gradient echo image of the proximal pastern of case 2 . the dorsal and medial directions are at the top and right , respectively . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . moreover , a focal hyperintense signal in the lateral odsl was observed on t1-weighted gre and t2-weighted fse images . effusion of the dfts was also visible as areas of low signal intensity on t1-weighted gre images and of high signal intensity on t2-weighted fse and stir fse images . transverse ultrasonographic image obtained in the medial oblique direction over the proximal pastern region of case 2 . . the dorsal and medial directions are at the top and right , respectively . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . based on the above diagnosis , the filly was prescribed 4 months of rest . following this rest period , the filly resumed exercise and returned to racing 5 months after the injury . a 6-year - old thoroughbred colt was referred to the hospital with a sustained increased digital pulse and persistent swelling over the palmar aspect of the pastern on the left forelimb . although the colt did not exhibit signs of lameness , the increased digital pulse became more remarkable as the training intensity increased . an area of high signal intensity was observed within the medial odsl on t1-weighted gre and t2-weighted fse images ( fig . 7fig . 7.(a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . an increased amount of fluid is present within the dfts ( arrowhead ) . ) . based on these findings , a reduction in training intensity was advised , and eventually , the colt was removed from the ritto training center . ( a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . the dorsal and medial directions are at the top and left , respectively . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . the colt resumed training at the ritto training center approximately 3 months after the smri . subsequently , the colt returned to racing 4 months after the injury and won the second race . a 5-year - old thoroughbred mare exhibited acute - onset left foreleg lameness following a race on a flat turf course . a 3-day course of oral diclofenac sodium ( 1 mg / kg ; blesin tablets , sawai pharmaceutical co. , ltd , osaka , japan ) , leg cooling and boxed rest did not improve the heat and pain in the pastern . on day 4 radiography revealed new periosteal bone formation at the mid - palmaromedial surface of the proximal phalanx ( fig . periosteal osteoproliferation is visible at the mid - palmaromedial surface of the proximal phalanx ( arrowheads ) . ) . a marked increase in fluid and small amount of fibrin in the digital flexor tendon sheath ( dfts ) were observed via ultrasonography ( fig . 2.(a ) transverse ultrasonographic image of case 1 , obtained in a lateral oblique position over the proximal pastern region . the palmar is at the top , and the lateral direction is at left . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . ( b ) transverse ultrasonographic image of case 1 , obtained in a medial oblique position over the proximal pastern region . in addition , the medial odsl appeared to be hypoechoic . to obtain a further diagnosis , smri images revealed high signal intensity in the enlarged medial odsl on t1-weighted gre , t2-weighted fse and stir fse images from the proximal pastern to mid - pastern that likely represented desmitis of the medial odsl ( fig . 4.(a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . an increased amount of fluid is present within the dfts ( arrowheads ) . ) . the enlarged dfts exhibited a hypointense signal on t1-weighted gre images , and hyperintense signals on t2-weighted fse and stir fse images ( fig . periosteal osteoproliferation is visible at the mid - palmaromedial surface of the proximal phalanx ( arrowheads ) . ( a ) transverse ultrasonographic image of case 1 , obtained in a lateral oblique position over the proximal pastern region . the palmar is at the top , and the lateral direction is at left . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . ( b ) transverse ultrasonographic image of case 1 , obtained in a medial oblique position over the proximal pastern region . ( a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . the dorsal and medial directions are at the top and left , respectively . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . dexamethasone ( corson p injection , nihon zenyaku kogyo , fukushima , japan ) was administered intravenously at a dose of 40 g / kg to reduce swelling over the right fore pastern , and the filly was placed on boxed rest for 1 day , followed by 3 days of light exercise . on day 5 , the lameness and swelling recurred , and the filly was admitted to the hospital for further diagnosis . a distended dfts was identified via ultrasonography , and the medial odsl appeared to be enlarged ( fig . 5fig . 5.transverse ultrasonographic image obtained in the medial oblique direction over the proximal pastern region of case 2 . note the enlargement of the medial oblique distal sesamoidean ligament ( arrows ) . ) . no abnormality was observed on the other dsl or the branches of superficial digital flexor tendon . in addition , the marked pain response over the medial pastern did not resolve . on day 6 the medial odsl exhibited increased signal intensity on t1-weighted gre , t2-weighted fse and stir fse images ( fig . 6.(a ) transverse t1-weighted gradient echo image of the proximal pastern of case 2 . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . moreover , a focal hyperintense signal in the lateral odsl was observed on t1-weighted gre and t2-weighted fse images . effusion of the dfts was also visible as areas of low signal intensity on t1-weighted gre images and of high signal intensity on t2-weighted fse and stir fse images . transverse ultrasonographic image obtained in the medial oblique direction over the proximal pastern region of case 2 . ( a ) transverse t1-weighted gradient echo image of the proximal pastern of case 2 . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . hyperintense fluid is observed within the dfts ( arrowheads ) . based on the above diagnosis , the filly was prescribed 4 months of rest . following this rest period , the filly resumed exercise and returned to racing 5 months after the injury . a 6-year - old thoroughbred colt was referred to the hospital with a sustained increased digital pulse and persistent swelling over the palmar aspect of the pastern on the left forelimb . although the colt did not exhibit signs of lameness , the increased digital pulse became more remarkable as the training intensity increased . an area of high signal intensity was observed within the medial odsl on t1-weighted gre and t2-weighted fse images ( fig . 7fig . 7.(a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . an increased amount of fluid is present within the dfts ( arrowhead ) . ) . based on these findings , a reduction in training intensity was advised , and eventually , the colt was removed from the ritto training center . ( a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . the colt resumed training at the ritto training center approximately 3 months after the smri . subsequently , the colt returned to racing 4 months after the injury and won the second race . previous studies have described the etiology of dsl desmitis [ 6 , 16 , 21 ] . concurrent injuries to odsl and sdsl have also been reported . in the present study , injuries to odsl usually occur uniaxially , likely as a result of asymmetric loading caused by abnormal conformation , foot imbalance , a misstep or poor footing . most horses with minor dsl desmitis exhibit moderate lameness that quickly subsides with rest . however , persistent lameness has been attributed to severe desmitis in some horses , and in some cases , this lameness persists or recurs with exercise . in the present study , case 1 and case 2 presented with lameness . case 1 exhibited acute - onset forelimb lameness that gradually improved to a degree that allowed hand - walking within a week . in case 2 , the lameness initially improved with rest and medication , but recurred after 3 days of light exercise . the degrees of lameness in the present cases corresponded with the cases of odsl injury described in previous studies . in the present study , the primary importance of radiography in cases of lameness is to rule out fracture and to examine bone changes , such as periosteal osteoproliferation and bone resorption . in case 1 , radiography revealed periosteal osteoproliferation at the mid - palmaromedial aspect of the proximal phalanx , which likely indicated enthesiophytosis at the distal attachment of odsl . dyson reported that enthesiophytosis at the attachment of the odsl is a relatively common and possibly incidental finding . simultaneously , the presence of these lesions might also suggest chronic stress remodeling at the insertion site of one of the odsls , because periosteal osteoproliferation only gradually becomes visible after mechanical irritation . the periosteal osteoproliferation observed in case 1 indicated that the horse might have accumulated minor damage to the medial odsl without exhibiting any clinical symptoms . in contrast , periosteal osteoproliferation was not present in the radiographic evaluation of case 3 , despite the fact that this horse had a history of persistent digital pulse increase , which likely represented chronic inflammation of the lower limb . based on these findings , radiographic findings were presumed to be inconclusive for the diagnosis of odsl injury . in case 1 and the digital flexor tendons were clearly visualized using ultrasonography , and no abnormalities were observed in any case . in contrast , it was difficult to obtain good quality ultrasonographic images of dsl , because of the challenge in maintaining good surface contact in the distal direction , as well as acoustic shadowing caused by other surrounding tissues . dyson reported that it could be difficult to determine the extent of desmitis and whether the damage extended distally using ultrasonography alone . in case 2 , the medial odsl appeared to be enlarged , although echogenicity was homogeneous . a previous report described the diagnosis of severe dsl desmitis based on the echogenicity changes observed with ultrasonography . hypoechoic changes were observed around the medial odsl in the ultrasonographic images of case 1 . the smri images of case 1 revealed that damage to the medial odsl was present from the proximal attachment to the distal end of the ligament . as suggested in the previous study , ultrasonography could detect changes only when the damage was significant and present proximally , given the limited viewing capability of ultrasonography . in the present study , the following sequences were used in the smri examination : t1-weighted gre , t2-weighted fse , and stir fse . for mri , image quality is dependent on the resolution and signal - to - noise ratio ( snr ) , which can be affected by several factors including field strength , number of averaging , slice thickness and field of view ( fov ) . the t1-weighted gre sequence provides excellent anatomic detail , characterized by a relatively high resolution resulting from a high snr . in the present study , the t1-weighted gre sequence was particularly useful for evaluating tissues that require symmetrical imaging , such as the medial and lateral odsl . one advantage of the gre sequence over the spin echo sequence is that the former requires a shorter acquisition time to generate a comparable snr . a longer acquisition time increases the risk of motion artifacts during smri , as the images are obtained under sedation . the t2-weighted fse sequence and stir fse sequence produce images with prominent tissue contrast . in the present study , these sequences were used to identify fluid and pathologic changes within soft tissues and bones . the damaged odsl exhibited a hyperintense signal in all sequences . because odsl normally exhibits a uniform low to intermediate signal ( fig . 3fig . 3.transverse t1-weighted gradient echo ( a ) , t2-weighted fast spin echo ( fse ) ( b ) and short tau inversion recovery fse ( c ) images of a normal pastern at the level of the proximal third of the proximal phalanx . the medial oblique distal sesamoidean ligament ( odsl ) ( arrow ) and lateral odsl ( open arrowhead ) have fairly uniform shapes with low to intermediate signal intensity in all sequences . ) , the increased signal intensity within odsl likely indicates changes in the extracellular matrix and increased water content in the ligament . these signal changes corresponded well with the findings of a previous study by smith . injuries that affect ligaments could cause changes in the shapes and sizes of these ligaments . in the present study , high - contrast fse sequence images were useful for distinguishing the boarders of the enlarged ligament from the surrounding tissues . importantly , the signal differences between these tissues could be detected with relatively low - resolution smri . the fse sequence normally requires a longer acquisition time than gre sequences . with smri , it is important to recognize that resolution should be compromised in the fse sequence to avoid motion artifact . higher resolution can be achieved by adjusting fov . in the present study , the requirement of the low - field magnet to image the whole pastern placed a limitation on further reduction of fov . transverse t1-weighted gradient echo ( a ) , t2-weighted fast spin echo ( fse ) ( b ) and short tau inversion recovery fse ( c ) images of a normal pastern at the level of the proximal third of the proximal phalanx . the medial oblique distal sesamoidean ligament ( odsl ) ( arrow ) and lateral odsl ( open arrowhead ) have fairly uniform shapes with low to intermediate signal intensity in all sequences . the signal intensity of odsl is known to be influenced by the magic angle effect ( mae ) on images acquired with smri [ 13 , 22 ] . this phenomenon has been attributed to the variable fiber pattern within the proximal one - third of the ligament and the tendency for the medial odsl to exist at a more oblique angle to the vertical , relative to the lateral odsl [ 3 , 22 ] . the mae is particularly noticeable in sequences with a short te , such as the t1-weighted sequence . in contrast , long te sequences , such as the t2-weighted sequences , are the least susceptible to the mae . all cases in the present study exhibited a high signal within the medial odsl on t2-weighted fse images . accordingly , these areas were considered to be true lesions . in the present study , distention of the dfts appeared as areas of low signal intensity on t1-weighted gre images , whereas the areas exhibited high signal intensity on t2-weighted fse and stir fse images . the results of the present study suggest that both t2-weighted fse and stir fse sequences could detect signals generated from fluid in the dfts . the stir fse sequence , which use a fat suppression technique , outweighs the t2-weighted fse sequence for evaluating fat - based tissues , such as the trabecular bone . in the present study , horses with acute desmitis often present with a sudden onset of lameness and swelling of the palmar surface of the pastern region , which are caused by dfts effusion . distention of the dfts was suggested to represent the inflammatory changes that occurred concurrently with dsl injury . in the three horses described in the present report , desmitis of dsl was considered to be severe , based on the required length of resting time and the smri image interpretation . in a previous report , the mean resting period required for dsl desmitis was 6 months . extracorporeal shock wave therapy , ligament splitting , injection of the dfts with corticosteroids and hyaluronan , and regenerative therapies using platelet - rich plasma or stem cells are common treatment options for dsl injury [ 11 , 20 ] . in the present study , a follow - up interview of the trainers revealed that none of the horses received these treatments . two cases in the present study successfully returned to racing following rehabilitation , in which the exercise intensity was controlled and gradually increased over time . in conclusion , odsl injury should be considered as a differential diagnosis in thoroughbred racehorses with swelling over the palmar surface of the proximal phalanx . the image quality yielded by this modality is suitable for a detailed evaluation of each tissue and exclusion of artifacts , such as the mae . however , given the limited number of cases , the correlation between prognosis and severity according to smri image interpretation remains to be investigated .

desmitis of the oblique distal sesamoidean ligaments ( odsl ) is caused by hyperextension of the metacarpophalangeal / metatarsophalangeal joint and has been described as a significant cause of lameness in racehorses . in this study , three thoroughbred racehorses ( age range : 36 years ) were diagnosed with desmitis of the forelimb odsl using standing low - field magnetic resonance imaging ( smri ) . radiography and ultrasonography were inconclusive with regard to a definitive diagnosis . for all horses , the smri characteristics included increased signal intensity within the medial odsl on t1-weighted gradient echo , t2-weighted fast spin echo and short tau inversion recovery fast spin echo images , which use a fat suppression technique . effusion of the digital flexor tendon sheath was also clearly visible on smri . following rest and controlled exercise for roughly 3 months , two horses successfully returned to racing within 5 months . our findings support the use of smri for diagnosing odsl injuries in thoroughbred racehorses .

MATERIALS AND METHODS RESULTS Case 1 Case 2 Case 3 DISCUSSION

case selection : all horses that were presented to the racehorse hospital , ritto training center for lameness evaluation in 2014 and diagnosed with mri abnormalities of one of the odsls were included in this study . radiography and ultrasonography : radiographs were taken using a computed digital radiography system ( fcr speedia cs , fujifilm , tokyo , japan ) . t1-weighted gradient echo ( gre ) [ echo tome ( te ) : 8 msec / repetition time ( tr ) : 50 msec , flip angle 55 ) , t2-weighted fast spin echo ( fse ) ( te : 88 msec / tr : 1681 msec ) and short tau inversion recovery ( stir ) fse [ te : 22 msec / tr : 2,460 msec , inversion time ( ti)=110 msec ] were acquired in the frontal , transverse and sagittal planes . mri images were acquired and reviewed by several clinicians experienced in the interpretation of smri images of thoroughbred racehorses , using the digital imaging and communications in medicine ( dicom ) viewer osirix , version 3.9.2 ( osirix project , geneva , switzerland ) . in all horses , medetomidine ( domitor , meiji seika co. , tokyo , japan ) was administered at an initial dose of 5 g / kg prior to the onset of positioning within the magnet . a marked increase in fluid and small amount of fibrin in the digital flexor tendon sheath ( dfts ) were observed via ultrasonography ( fig . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . in addition , the medial odsl appeared to be hypoechoic . to obtain a further diagnosis , smri images revealed high signal intensity in the enlarged medial odsl on t1-weighted gre , t2-weighted fse and stir fse images from the proximal pastern to mid - pastern that likely represented desmitis of the medial odsl ( fig . (a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . ( a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . following the smri examination , the mare was sent to a farm for rest . note the enlargement of the medial oblique distal sesamoidean ligament ( arrows ) . ) on day 6 the medial odsl exhibited increased signal intensity on t1-weighted gre , t2-weighted fse and stir fse images ( fig . (a ) transverse t1-weighted gradient echo image of the proximal pastern of case 2 . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . effusion of the dfts was also visible as areas of low signal intensity on t1-weighted gre images and of high signal intensity on t2-weighted fse and stir fse images . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . following this rest period , the filly resumed exercise and returned to racing 5 months after the injury . an area of high signal intensity was observed within the medial odsl on t1-weighted gre and t2-weighted fse images ( fig . (a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . ( a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . subsequently , the colt returned to racing 4 months after the injury and won the second race . a marked increase in fluid and small amount of fibrin in the digital flexor tendon sheath ( dfts ) were observed via ultrasonography ( fig . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . in addition , the medial odsl appeared to be hypoechoic . to obtain a further diagnosis , smri images revealed high signal intensity in the enlarged medial odsl on t1-weighted gre , t2-weighted fse and stir fse images from the proximal pastern to mid - pastern that likely represented desmitis of the medial odsl ( fig . (a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . marked increases in fluid ( arrow ) and fibrin ( arrowheads ) are visible in the digital flexor tendon sheath . ( a ) transverse t1-weighted gradient echo image acquired at the level of the proximal third of the proximal phalanx in case 1 . enlargement of and increased signal intensity in the medial oblique distal sesamoidean ligament ( odsl ) are visible ( arrow ) . an area of low signal intensity is visible in the digital flexor tendon sheath ( dfts ; arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal phalanx of case 1 , acquired at the same level as above . ( c ) transverse short tau inversion recovery fse image of the proximal phalanx from the case 1 , acquired at the same level as above . increased signal intensity is visible in the medial odsl ( arrow ) . a distended dfts was identified via ultrasonography , and the medial odsl appeared to be enlarged ( fig . note the enlargement of the medial oblique distal sesamoidean ligament ( arrows ) . ) on day 6 the medial odsl exhibited increased signal intensity on t1-weighted gre , t2-weighted fse and stir fse images ( fig . (a ) transverse t1-weighted gradient echo image of the proximal pastern of case 2 . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . effusion of the dfts was also visible as areas of low signal intensity on t1-weighted gre images and of high signal intensity on t2-weighted fse and stir fse images . ( a ) transverse t1-weighted gradient echo image of the proximal pastern of case 2 . high signal intensity is observed in the medial oblique distal sesamoidean ligament ( odsl ; arrow ) . a discrete area of increased signal intensity is visible within the lateral odsl ( open arrowheads ) . ( b ) transverse t2-weighted fast spin echo ( fse ) image of the proximal pastern of case 2 . increased signal intensity is visible in the medial odsl ( arrow ) . ( c ) transverse short tau inversion recovery fse image of the proximal pastern of case 2 . increased signal intensity is present in the medial odsl ( arrow ) and lateral odsl ( open arrowheads ) . following this rest period , the filly resumed exercise and returned to racing 5 months after the injury . an area of high signal intensity was observed within the medial odsl on t1-weighted gre and t2-weighted fse images ( fig . (a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . ( a ) transverse t1-weighted gradient echo image of case 3 showing high signal intensity ( arrow ) in the medial oblique distal sesamoidean ligament ( odsl ) at the proximal third of the proximal phalanx . fluid in the digital flexor tendon sheath ( dfts ) is observed as an area of low signal intensity ( arrowhead ) . ( b ) transverse t2-weighted fast spin echo image acquired at the level of the proximal third of the proximal phalanx in case 3 . the colt resumed training at the ritto training center approximately 3 months after the smri . subsequently , the colt returned to racing 4 months after the injury and won the second race . in the present study , injuries to odsl usually occur uniaxially , likely as a result of asymmetric loading caused by abnormal conformation , foot imbalance , a misstep or poor footing . in case 2 , the lameness initially improved with rest and medication , but recurred after 3 days of light exercise . the degrees of lameness in the present cases corresponded with the cases of odsl injury described in previous studies . in the present study , the primary importance of radiography in cases of lameness is to rule out fracture and to examine bone changes , such as periosteal osteoproliferation and bone resorption . in case 1 , radiography revealed periosteal osteoproliferation at the mid - palmaromedial aspect of the proximal phalanx , which likely indicated enthesiophytosis at the distal attachment of odsl . simultaneously , the presence of these lesions might also suggest chronic stress remodeling at the insertion site of one of the odsls , because periosteal osteoproliferation only gradually becomes visible after mechanical irritation . in case 1 and the digital flexor tendons were clearly visualized using ultrasonography , and no abnormalities were observed in any case . in case 2 , the medial odsl appeared to be enlarged , although echogenicity was homogeneous . the smri images of case 1 revealed that damage to the medial odsl was present from the proximal attachment to the distal end of the ligament . in the present study , the following sequences were used in the smri examination : t1-weighted gre , t2-weighted fse , and stir fse . in the present study , the t1-weighted gre sequence was particularly useful for evaluating tissues that require symmetrical imaging , such as the medial and lateral odsl . 3.transverse t1-weighted gradient echo ( a ) , t2-weighted fast spin echo ( fse ) ( b ) and short tau inversion recovery fse ( c ) images of a normal pastern at the level of the proximal third of the proximal phalanx . the medial oblique distal sesamoidean ligament ( odsl ) ( arrow ) and lateral odsl ( open arrowhead ) have fairly uniform shapes with low to intermediate signal intensity in all sequences . ) , the increased signal intensity within odsl likely indicates changes in the extracellular matrix and increased water content in the ligament . importantly , the signal differences between these tissues could be detected with relatively low - resolution smri . in the present study , the requirement of the low - field magnet to image the whole pastern placed a limitation on further reduction of fov . transverse t1-weighted gradient echo ( a ) , t2-weighted fast spin echo ( fse ) ( b ) and short tau inversion recovery fse ( c ) images of a normal pastern at the level of the proximal third of the proximal phalanx . the medial oblique distal sesamoidean ligament ( odsl ) ( arrow ) and lateral odsl ( open arrowhead ) have fairly uniform shapes with low to intermediate signal intensity in all sequences . this phenomenon has been attributed to the variable fiber pattern within the proximal one - third of the ligament and the tendency for the medial odsl to exist at a more oblique angle to the vertical , relative to the lateral odsl [ 3 , 22 ] . all cases in the present study exhibited a high signal within the medial odsl on t2-weighted fse images . in the present study , distention of the dfts appeared as areas of low signal intensity on t1-weighted gre images , whereas the areas exhibited high signal intensity on t2-weighted fse and stir fse images . the stir fse sequence , which use a fat suppression technique , outweighs the t2-weighted fse sequence for evaluating fat - based tissues , such as the trabecular bone . in the present study , horses with acute desmitis often present with a sudden onset of lameness and swelling of the palmar surface of the pastern region , which are caused by dfts effusion . in the present study , a follow - up interview of the trainers revealed that none of the horses received these treatments . two cases in the present study successfully returned to racing following rehabilitation , in which the exercise intensity was controlled and gradually increased over time . in conclusion , odsl injury should be considered as a differential diagnosis in thoroughbred racehorses with swelling over the palmar surface of the proximal phalanx .

in middle - aged to elderly patients , a coronary anomaly is often an incidental finding . the clinical presentation varies from asymptomatic up to presentation as an acute myocardial infarction or even sudden cardiac death ( scd ) . we present a case of an aberrant right coronary artery ( arca ) and reviewed the literature concerning the best diagnostic and therapeutic strategy . a 40-year - old male farmer attended the outpatient clinic complaining of chest pain during work . when he tilted heavy straw bales he felt a piercing and oppressive left - sided thoracic pain , which ceased at rest . cardiovascular risk factors consisted of a familial predisposition for coronary artery disease ( father had coronary bypass at age of 65 ) and hypercholesterolemia . resting electrocardiogram ( ecg ) showed a regular sinus rhythm at 65 bpm , without further abnormalities . during his first visit , a treadmill exercise test and echocardiography were performed . during the maximal treadmill test ( fig . 1 ) up to 200 w , he mentioned the chest pain , he felt when working and tilting heavy weights . this was associated with st depression up to 1.5 mm in leads v5 and v6 and some solitary monomorphic premature ventricular complexes . 2 ) revealed a normal left and right ventricular function with normal function of the heart valves . treadmill test showing 1.5 mm st - segment depression in leads v5 to v6 and a solitary ventricular extrasystole . transthoracic echocardiography apical 4-chamber view showing normal left ventricle function , and moderate right ventricle hypertrabeculation . a further diagnostic work - up was initiated with stress echocardiography and cardiac magnetic resonance ( cmr ) . supine bicycle stress echocardiography once again provoked recognizable chest pain , electrocardiographic st depression in leads v5 to v6 , and revealed severe hypokinesia in the inferoposterior region . angiography revealed nonsignificant atherosclerosis in the 3 main coronary arteries in a right dominant coronary system . however , an anomalous take off of the right coronary artery ( rca ) in the left sinus of valsalva was diagnosed ( fig . 3 ) . coronary angiography showing the anomalous origin of the right coronary artery from the left sinus of valsalva . cmr confirmed some hypertrabeculation of the right ventricle , without signs of arrhythmogenic right ventricular dysplasia or noncompaction syndrome . the left and right ventricular function was preserved and no delayed enhancement or myocardial fibrosis was visualized . this imaging technique confirmed the aberrant origin of the rca , originating from the left coronary cusp and with a course of the proximal rca between the pulmonary artery and aorta . in conclusion , our patient presented with recurrent stable angina and only an arca could explain the symptoms . this was clearly confirmed by the abnormal myocardial ischemia tests ( treadmill exercise ecg and stress echocardiography ) in the territory of the rca . the case was debated in the heart team , and the patient was referred for a surgical correction . medication at the time of discharge included low - dose aspirin , a statin , and a beta - blocker . after 4 weeks the patient was already slowly resuming daily life , including his work as a farmer . four months after the operation , the patient consulted because of exertional shortness of breath ( new york heart association class ii ) . several forms of anatomical variants in coronary arteries exist . some are believed to be potentially dangerous , others are benign . potential malignant coronary artery anomalies are an ectopic coronary origin from the pulmonary artery , an ectopic coronary origin from the opposite sinus of valsalva , a single coronary artery , and large coronary fistulae or muscular bridging . the proximal course of an arca may vary : preaortic , interarterial ( between aorta and pulmonary artery , as in our patient ) , retrocardiac , retroaortic , intraseptal , and precardiac ( prepulmonary ) . the hemodynamic important variant ( i.e. , interarterial course ) runs between the aorta and pulmonary artery , either intramural ( within the aortic wall ) or separated from the aortic wall ( like 2 distinct arteries ) . it is supposed that only this interarterial course can cause symptoms or is potentially dangerous . with newer imaging techniques , newer anatomical variants of the proximal course of an arca the coronary takeoff can be high ( above the level of the pulmonary valve with a course between the aorta and pulmonary artery ) or low ( below the level of the pulmonary valve with a course between the aorta and right ventricular outflow tract [ rvot ] ) . this high takeoff variant is believed to be more hostile than an arca with low takeoff . post hoc analysis of the cmr images of our case , revealed a low takeoff . during ca studies , an arca has a higher incidence than an anomalous left coronary artery ( alca ) ( 0.020.15% ) and is presumed to be the most common type of hemodynamically significant coronary anomalies . in mdct coronary studies , the incidence of an arca is comparable ( 0.54% ) . among 18,950 autopsy cases in a los angeles hospital , 54 cases of coronary anomalies were detected of whom 39 with anomalies of the coronary ostia , totaling to an incidence of 0.206% . in young athletes , 12% of scds is caused by an anomalous coronary artery from the opposite aortic sinus . in young athletes , however , with increasing age of athletes , coronary anomaly becomes a less frequent cause of scd . mortality data derived from autopsy studies ( risk on scd in arca 057% , in alca 30100% ) are far too high when converted to the general population . these numbers can obviously not be extrapolated from autopsy studies to the general population and are probably severely overestimated as mortality rates are biased as we are not aware of the exact prevalence of an arca in asymptomatic patients . notwithstanding , it is assumed that there is a 3- to 6-fold increased risk for scd in people with an arca doing physical activity . there are several hypotheses : a slit - like orifice caused by an acute angle of takeoff , which could cause reduced coronary flow during exercise ; compression of the interarterial segment caused by systolic compression between the aorta and pulmonary artery , aggravated during increased flow , for example , with exercise ; ventricular arrhythmias caused by ischemia ; acute or repetitive ischemia provoking myocardial fibrosis or reperfusion ; intramural proximal intussusception of the anomalous artery at the aortic - root wall , as proposed by angelini et al during intravascular ultrasound ( ivus ) studies . , studies showed that a coronary anomaly is not associated with an increased risk for development of coronary atherosclerosis . it would be interesting to identify specific anatomical or clinical risk factors for scd in order to predict the possible hemodynamic significance . logically , the dominance of an anomalous coronary artery is an important risk factor . in a large pathology study of 30 cases of anomalous arteries , it was not possible to identify a certain anatomical feature related to increased mortality . in an mdct study , clinical symptoms were not related to the relative luminal narrowing nor angle of takeoff . however , a significant difference in the prevalence of major adverse cardiac events ( mace ) and typical angina was observed in a retrospective review of 22,925 consecutive mdct scans , in which 124 cases with an interarterial anomalous coronary artery were found . they differentiated the anatomical takeoff in a high and a low takeoff ( above or below the level of pulmonary valve ) . the group with high takeoff ( coursing between aorta and pulmonary artery ) proved to have a significant higher prevalence of typical angina ( 43% vs 6% , p = 0.001 ) and mace ( 28% vs 6% , p = 0.012 ) compared to those with a low takeoff ( coursing between aorta and rvot ) . age is an important clinical parameter that is related with the risk of scd . under the age of 30 it has been hypothesized that the aortic wall stiffens with age , which reduces compression . as mentioned above , exercise is a risk factor for scd in people with an arca . in a necropsy study of 242 patients who died suddenly , 62% of the patients were asymptomatic until the event . in a japanese review of 56 patients with coronary anomalies of whom 44 with an arca and with a mean age of 55.9 years old of 22 patients with an arca who had undergone a treadmill test , 10 proved abnormal . three out of 4 had a positive myocardial perfusion single photon emission computed tomography ( spect ) exercise test . these latter results show a much higher rate of positive stress testing than seen in other studies . this is why a negative stress test does not exclude a potentially dangerous coronary anomaly . when a stress test proves negative but symptoms are suspicious ( e.g. , exertional syncope or chest pain ) anatomical examinations ( i.e. , mdct scan ) should be considered . in young patients , or echogenic patients one study showed significant 2d strain impairment in 25 patients with a coronary anomaly ( of whom 15 with arca ) , suggesting subtle left ventricular contractility disorder in these patients . one study evaluated the accuracy of coronary artery calcium scanning for detecting coronary anomalies , which was found out to have a great diagnostic accuracy . however , nowadays , mdct ca is accepted as the gold standard for the evaluation of coronary anomalies . magnetic resonance angiography ( mra ) is similar successful , but the identification of the distal coronary course can be more difficult . some disadvantages of mra are also to be considered : availability is less , mra is not useful in patients with pacemakers or claustrophobia , and total study time takes much longer . one large benefit of mra is that it can assess and locate scarred tissue and viability in the course of the affected artery , which could have an important prognostic value . it is not always possible to define the exact proximal anatomical course of the coronary anomaly . in a ca study , only in 53% of the cases the exact proximal course could be defined . ivus showed in some studies intussusception or lateral compression during systole , aggravated by saline , atropine , or dobutamine infusion . fractional flow reserve ( ffr ) was tested by angelini et al , and showed results within normal limits during adenosine provocation ( ffr > 0.9 , cutoff 0.8 ) , indicating that ffr is not a good parameter to diagnose hemodynamic significance in this setting . stress - rest myocardial perfusion spect can be used to detect reversible perfusion defects , although results are often negative , as it is for stress ecg . long - term holter monitoring can be helpful in screening for arrhythmia , although being an aspecific tool . in summary , mdct ca seems to be the best examination for anatomical diagnosis and ischemic testing proves often negative . the 2008 american heart association guidelines for the management of adults with congenital disease , recommend that surgical coronary revascularization should be performed when there is evidence of ischemia in an anomalous rca coursing between aorta and pulmonary artery ( level of evidence class i , b ) . indication to treat should be individualized , and depends on age , symptoms , the anatomical variant , and ischemic testing . in literature , patients are separated by age ( younger and older than 35 to 40 years old ) . new anatomical understandings separate high and low coronary takeoff of the arca ( above or under level of pulmonary valve ) as a possible prognostic finding . lee et al suggest to operate all patients , younger than 40 years old , with high takeoff , regardless of symptoms . symptomatic patients older than 40 years old and with high takeoff should be operated if symptomatic , following their ideas . in patients with a low takeoff , close observation is suggested , unless when related symptoms are present surgery might be considered . in a japanese retrospective study of 56 patients ( mean age 55.9 years old ) , with anomalous origin of coronary artery ( 78% arca ) and without coexisting atherosclerosis , a conservative treatment was applied ( nitrates , calcium channel blockers , beta - adrenergic antagonists , or antiarrhythmic drugs ) . during follow - up ( 2 months to 14.5 years ) , there were no cardiac - related deaths . the authors concluded that the prognosis of middle - aged to elderly patients with an anomalous origin of the coronary artery is relatively good . a few other studies proved comparable results , suggesting a conservative approach ( beta - blockers and physical restriction ) may be safe . this somehow conflicting evidence illustrates that further research is warranted , matching anatomical with clinical and functional test data . one case series of 14 patients with proximal coronary artery stenting showed a normalization of stress test results and angiographical patency 6 months after the percutaneous coronary intervention was demonstrated . there are also different surgical strategies : reimplantation of the rca ( as in our case ) , unroofing of the intramural course with creation of a neo - orifice , the modified unroofing technique , patch augmentation , and classical bypass grafting . unroofing of the anomalous artery can be applied when the proximal course runs intramural , and when there is no involvement of aortic valve commissures , which otherwise could create aortic insufficiency . this technique relieves the ostial stenosis , creates a large neo - orifice , and removes the intramural segment . in the modified unroofing technique , the anomalous orifice is closed , a neo - orificium is created in the appropriate sinus , without extensive unroofing of the proximal intramural part of the anomalous coronary artery . the proximal course of an arca may vary : preaortic , interarterial ( between aorta and pulmonary artery , as in our patient ) , retrocardiac , retroaortic , intraseptal , and precardiac ( prepulmonary ) . the hemodynamic important variant ( i.e. , interarterial course ) runs between the aorta and pulmonary artery , either intramural ( within the aortic wall ) or separated from the aortic wall ( like 2 distinct arteries ) . it is supposed that only this interarterial course can cause symptoms or is potentially dangerous . with newer imaging techniques , newer anatomical variants of the proximal course of an arca the coronary takeoff can be high ( above the level of the pulmonary valve with a course between the aorta and pulmonary artery ) or low ( below the level of the pulmonary valve with a course between the aorta and right ventricular outflow tract [ rvot ] ) . this high takeoff variant is believed to be more hostile than an arca with low takeoff . post hoc analysis of the cmr images of our case , revealed a low takeoff . an arca has a higher incidence than an anomalous left coronary artery ( alca ) ( 0.020.15% ) and is presumed to be the most common type of hemodynamically significant coronary anomalies . in mdct coronary studies , the incidence of an arca is comparable ( 0.54% ) . among 18,950 autopsy cases in a los angeles hospital , 54 cases of coronary anomalies were detected of whom 39 with anomalies of the coronary ostia , totaling to an incidence of 0.206% . in young athletes , 12% of scds is caused by an anomalous coronary artery from the opposite aortic sinus . in young athletes , however , with increasing age of athletes , coronary anomaly becomes a less frequent cause of scd . mortality data derived from autopsy studies ( risk on scd in arca 057% , in alca 30100% ) are far too high when converted to the general population . these numbers can obviously not be extrapolated from autopsy studies to the general population and are probably severely overestimated as mortality rates are biased as we are not aware of the exact prevalence of an arca in asymptomatic patients . notwithstanding , it is assumed that there is a 3- to 6-fold increased risk for scd in people with an arca doing physical activity . there are several hypotheses : a slit - like orifice caused by an acute angle of takeoff , which could cause reduced coronary flow during exercise ; compression of the interarterial segment caused by systolic compression between the aorta and pulmonary artery , aggravated during increased flow , for example , with exercise ; ventricular arrhythmias caused by ischemia ; acute or repetitive ischemia provoking myocardial fibrosis or reperfusion ; intramural proximal intussusception of the anomalous artery at the aortic - root wall , as proposed by angelini et al during intravascular ultrasound ( ivus ) studies . , studies showed that a coronary anomaly is not associated with an increased risk for development of coronary atherosclerosis . it would be interesting to identify specific anatomical or clinical risk factors for scd in order to predict the possible hemodynamic significance . logically , the dominance of an anomalous coronary artery is an important risk factor . in a large pathology study of 30 cases of anomalous arteries , it was not possible to identify a certain anatomical feature related to increased mortality . in an mdct study , clinical symptoms were not related to the relative luminal narrowing nor angle of takeoff . however , a significant difference in the prevalence of major adverse cardiac events ( mace ) and typical angina was observed in a retrospective review of 22,925 consecutive mdct scans , in which 124 cases with an interarterial anomalous coronary artery were found . they differentiated the anatomical takeoff in a high and a low takeoff ( above or below the level of pulmonary valve ) . the group with high takeoff ( coursing between aorta and pulmonary artery ) proved to have a significant higher prevalence of typical angina ( 43% vs 6% , p = 0.001 ) and mace ( 28% vs 6% , p = 0.012 ) compared to those with a low takeoff ( coursing between aorta and rvot ) . age is an important clinical parameter that is related with the risk of scd . under the age of 30 it has been hypothesized that the aortic wall stiffens with age , which reduces compression . as mentioned above , in a necropsy study of 242 patients who died suddenly , 62% of the patients were asymptomatic until the event . in a japanese review of 56 patients with coronary anomalies of whom 44 with an arca and with a mean age of 55.9 years old of 22 patients with an arca who had undergone a treadmill test , 10 proved abnormal . three out of 4 had a positive myocardial perfusion single photon emission computed tomography ( spect ) exercise test . these latter results show a much higher rate of positive stress testing than seen in other studies . this is why a negative stress test does not exclude a potentially dangerous coronary anomaly . when a stress test proves negative but symptoms are suspicious ( e.g. , exertional syncope or chest pain ) anatomical examinations ( i.e. , mdct scan ) should be considered . in young patients , or echogenic patients one study showed significant 2d strain impairment in 25 patients with a coronary anomaly ( of whom 15 with arca ) , suggesting subtle left ventricular contractility disorder in these patients . one study evaluated the accuracy of coronary artery calcium scanning for detecting coronary anomalies , which was found out to have a great diagnostic accuracy . however , nowadays , mdct ca is accepted as the gold standard for the evaluation of coronary anomalies . magnetic resonance angiography ( mra ) is similar successful , but the identification of the distal coronary course can be more difficult . some disadvantages of mra are also to be considered : availability is less , mra is not useful in patients with pacemakers or claustrophobia , and total study time takes much longer . one large benefit of mra is that it can assess and locate scarred tissue and viability in the course of the affected artery , which could have an important prognostic value . ca it is not always possible to define the exact proximal anatomical course of the coronary anomaly . in a ca study , only in 53% of the cases the exact proximal course could be defined . ivus showed in some studies intussusception or lateral compression during systole , aggravated by saline , atropine , or dobutamine infusion . fractional flow reserve ( ffr ) was tested by angelini et al , and showed results within normal limits during adenosine provocation ( ffr > 0.9 , cutoff 0.8 ) , indicating that ffr is not a good parameter to diagnose hemodynamic significance in this setting . stress - rest myocardial perfusion spect can be used to detect reversible perfusion defects , although results are often negative , as it is for stress ecg . long - term holter monitoring can be helpful in screening for arrhythmia , although being an aspecific tool . in summary , mdct ca seems to be the best examination for anatomical diagnosis and ischemic testing proves often negative . the 2008 american heart association guidelines for the management of adults with congenital disease , recommend that surgical coronary revascularization should be performed when there is evidence of ischemia in an anomalous rca coursing between aorta and pulmonary artery ( level of evidence class i , b ) . indication to treat should be individualized , and depends on age , symptoms , the anatomical variant , and ischemic testing . in literature , patients are separated by age ( younger and older than 35 to 40 years old ) . new anatomical understandings separate high and low coronary takeoff of the arca ( above or under level of pulmonary valve ) as a possible prognostic finding . lee et al suggest to operate all patients , younger than 40 years old , with high takeoff , regardless of symptoms . symptomatic patients older than 40 years old and with high takeoff should be operated if symptomatic , following their ideas . in patients with a low takeoff , close observation is suggested , unless when related symptoms are present surgery might be considered . in a japanese retrospective study of 56 patients ( mean age 55.9 years old ) , with anomalous origin of coronary artery ( 78% arca ) and without coexisting atherosclerosis , a conservative treatment was applied ( nitrates , calcium channel blockers , beta - adrenergic antagonists , or antiarrhythmic drugs ) . during follow - up ( 2 months to 14.5 years ) , there were no cardiac - related deaths . the authors concluded that the prognosis of middle - aged to elderly patients with an anomalous origin of the coronary artery is relatively good . a few other studies proved comparable results , suggesting a conservative approach ( beta - blockers and physical restriction ) may be safe . this somehow conflicting evidence illustrates that further research is warranted , matching anatomical with clinical and functional test data . one case series of 14 patients with proximal coronary artery stenting showed a normalization of stress test results and angiographical patency 6 months after the percutaneous coronary intervention was demonstrated . there are also different surgical strategies : reimplantation of the rca ( as in our case ) , unroofing of the intramural course with creation of a neo - orifice , the modified unroofing technique , patch augmentation , and classical bypass grafting . with patch augmentation , unroofing of the anomalous artery can be applied when the proximal course runs intramural , and when there is no involvement of aortic valve commissures , which otherwise could create aortic insufficiency . this technique relieves the ostial stenosis , creates a large neo - orifice , and removes the intramural segment . in the modified unroofing technique , the anomalous orifice is closed , a neo - orificium is created in the appropriate sinus , without extensive unroofing of the proximal intramural part of the anomalous coronary artery . the interarterial form of an anomalous rca ( coursing between the aorta and pulmonary artery ) can lead to symptoms or even scd . coronary anomaly is the second most common cause of cardiac sudden death in young athletes , which demonstrates that young age and vigorous exercise are risk factors for sudden death in patients with an arca . further ischemic testing can help to guide the therapy strategy , although functional tests often prove negative . in middle - aged patients , this anomaly is often an incidental finding . notwithstanding the lack of randomized trials , we consider it advisable to operate when an arca is seen in young patients ( < 40 years old ) or , in older patients with proven related symptoms or positive ischemic testing . reimplantation of the rca or unroofing the proximal course of the anomalous artery seems to be the best surgical strategies . when a conservative approach is proposed , avoidance of vigorous exercise and prescribing beta - blockers are advised .

abstractbackground : an anomalous right coronary artery originating from the left sinus of valsalva is a rare , but often incidental , finding in middle - aged to elderly people . prevalence is difficult to define , as well as determining potential harmful hemodynamic consequences . moreover , the optimal treatment remains debatable.case summary : the authors present a case of a middle - aged patient diagnosed with an anomalous right coronary artery causing ischemia , who was treated surgically.conclusion:by reviewing literature , the authors conclude that choice of treatment depends on age , symptoms , and certain anatomic features of this anomaly . however , there are no randomized trials available in this field .

Introduction Case Discussion Aberrant right coronary artery Prevalence and mortality Pathophysiology Clinical presentation and diagnosis Treatment Invasive management Conclusion

in middle - aged to elderly patients , a coronary anomaly is often an incidental finding . we present a case of an aberrant right coronary artery ( arca ) and reviewed the literature concerning the best diagnostic and therapeutic strategy . a 40-year - old male farmer attended the outpatient clinic complaining of chest pain during work . when he tilted heavy straw bales he felt a piercing and oppressive left - sided thoracic pain , which ceased at rest . cardiovascular risk factors consisted of a familial predisposition for coronary artery disease ( father had coronary bypass at age of 65 ) and hypercholesterolemia . resting electrocardiogram ( ecg ) showed a regular sinus rhythm at 65 bpm , without further abnormalities . treadmill test showing 1.5 mm st - segment depression in leads v5 to v6 and a solitary ventricular extrasystole . transthoracic echocardiography apical 4-chamber view showing normal left ventricle function , and moderate right ventricle hypertrabeculation . supine bicycle stress echocardiography once again provoked recognizable chest pain , electrocardiographic st depression in leads v5 to v6 , and revealed severe hypokinesia in the inferoposterior region . however , an anomalous take off of the right coronary artery ( rca ) in the left sinus of valsalva was diagnosed ( fig . coronary angiography showing the anomalous origin of the right coronary artery from the left sinus of valsalva . the left and right ventricular function was preserved and no delayed enhancement or myocardial fibrosis was visualized . this imaging technique confirmed the aberrant origin of the rca , originating from the left coronary cusp and with a course of the proximal rca between the pulmonary artery and aorta . this was clearly confirmed by the abnormal myocardial ischemia tests ( treadmill exercise ecg and stress echocardiography ) in the territory of the rca . the case was debated in the heart team , and the patient was referred for a surgical correction . medication at the time of discharge included low - dose aspirin , a statin , and a beta - blocker . four months after the operation , the patient consulted because of exertional shortness of breath ( new york heart association class ii ) . some are believed to be potentially dangerous , others are benign . potential malignant coronary artery anomalies are an ectopic coronary origin from the pulmonary artery , an ectopic coronary origin from the opposite sinus of valsalva , a single coronary artery , and large coronary fistulae or muscular bridging . the proximal course of an arca may vary : preaortic , interarterial ( between aorta and pulmonary artery , as in our patient ) , retrocardiac , retroaortic , intraseptal , and precardiac ( prepulmonary ) . the hemodynamic important variant ( i.e. , interarterial course ) runs between the aorta and pulmonary artery , either intramural ( within the aortic wall ) or separated from the aortic wall ( like 2 distinct arteries ) . it is supposed that only this interarterial course can cause symptoms or is potentially dangerous . with newer imaging techniques , newer anatomical variants of the proximal course of an arca the coronary takeoff can be high ( above the level of the pulmonary valve with a course between the aorta and pulmonary artery ) or low ( below the level of the pulmonary valve with a course between the aorta and right ventricular outflow tract [ rvot ] ) . post hoc analysis of the cmr images of our case , revealed a low takeoff . during ca studies , an arca has a higher incidence than an anomalous left coronary artery ( alca ) ( 0.020.15% ) and is presumed to be the most common type of hemodynamically significant coronary anomalies . in mdct coronary studies , the incidence of an arca is comparable ( 0.54% ) . among 18,950 autopsy cases in a los angeles hospital , 54 cases of coronary anomalies were detected of whom 39 with anomalies of the coronary ostia , totaling to an incidence of 0.206% . in young athletes , 12% of scds is caused by an anomalous coronary artery from the opposite aortic sinus . in young athletes , however , with increasing age of athletes , coronary anomaly becomes a less frequent cause of scd . notwithstanding , it is assumed that there is a 3- to 6-fold increased risk for scd in people with an arca doing physical activity . there are several hypotheses : a slit - like orifice caused by an acute angle of takeoff , which could cause reduced coronary flow during exercise ; compression of the interarterial segment caused by systolic compression between the aorta and pulmonary artery , aggravated during increased flow , for example , with exercise ; ventricular arrhythmias caused by ischemia ; acute or repetitive ischemia provoking myocardial fibrosis or reperfusion ; intramural proximal intussusception of the anomalous artery at the aortic - root wall , as proposed by angelini et al during intravascular ultrasound ( ivus ) studies . , studies showed that a coronary anomaly is not associated with an increased risk for development of coronary atherosclerosis . it would be interesting to identify specific anatomical or clinical risk factors for scd in order to predict the possible hemodynamic significance . logically , the dominance of an anomalous coronary artery is an important risk factor . in an mdct study , clinical symptoms were not related to the relative luminal narrowing nor angle of takeoff . however , a significant difference in the prevalence of major adverse cardiac events ( mace ) and typical angina was observed in a retrospective review of 22,925 consecutive mdct scans , in which 124 cases with an interarterial anomalous coronary artery were found . they differentiated the anatomical takeoff in a high and a low takeoff ( above or below the level of pulmonary valve ) . the group with high takeoff ( coursing between aorta and pulmonary artery ) proved to have a significant higher prevalence of typical angina ( 43% vs 6% , p = 0.001 ) and mace ( 28% vs 6% , p = 0.012 ) compared to those with a low takeoff ( coursing between aorta and rvot ) . age is an important clinical parameter that is related with the risk of scd . under the age of 30 it has been hypothesized that the aortic wall stiffens with age , which reduces compression . as mentioned above , exercise is a risk factor for scd in people with an arca . in a necropsy study of 242 patients who died suddenly , 62% of the patients were asymptomatic until the event . in a japanese review of 56 patients with coronary anomalies of whom 44 with an arca and with a mean age of 55.9 years old of 22 patients with an arca who had undergone a treadmill test , 10 proved abnormal . three out of 4 had a positive myocardial perfusion single photon emission computed tomography ( spect ) exercise test . these latter results show a much higher rate of positive stress testing than seen in other studies . this is why a negative stress test does not exclude a potentially dangerous coronary anomaly . when a stress test proves negative but symptoms are suspicious ( e.g. , mdct scan ) should be considered . one study evaluated the accuracy of coronary artery calcium scanning for detecting coronary anomalies , which was found out to have a great diagnostic accuracy . however , nowadays , mdct ca is accepted as the gold standard for the evaluation of coronary anomalies . magnetic resonance angiography ( mra ) is similar successful , but the identification of the distal coronary course can be more difficult . some disadvantages of mra are also to be considered : availability is less , mra is not useful in patients with pacemakers or claustrophobia , and total study time takes much longer . it is not always possible to define the exact proximal anatomical course of the coronary anomaly . in a ca study , only in 53% of the cases the exact proximal course could be defined . fractional flow reserve ( ffr ) was tested by angelini et al , and showed results within normal limits during adenosine provocation ( ffr > 0.9 , cutoff 0.8 ) , indicating that ffr is not a good parameter to diagnose hemodynamic significance in this setting . stress - rest myocardial perfusion spect can be used to detect reversible perfusion defects , although results are often negative , as it is for stress ecg . the 2008 american heart association guidelines for the management of adults with congenital disease , recommend that surgical coronary revascularization should be performed when there is evidence of ischemia in an anomalous rca coursing between aorta and pulmonary artery ( level of evidence class i , b ) . indication to treat should be individualized , and depends on age , symptoms , the anatomical variant , and ischemic testing . in literature , patients are separated by age ( younger and older than 35 to 40 years old ) . in a japanese retrospective study of 56 patients ( mean age 55.9 years old ) , with anomalous origin of coronary artery ( 78% arca ) and without coexisting atherosclerosis , a conservative treatment was applied ( nitrates , calcium channel blockers , beta - adrenergic antagonists , or antiarrhythmic drugs ) . during follow - up ( 2 months to 14.5 years ) , there were no cardiac - related deaths . the authors concluded that the prognosis of middle - aged to elderly patients with an anomalous origin of the coronary artery is relatively good . this somehow conflicting evidence illustrates that further research is warranted , matching anatomical with clinical and functional test data . one case series of 14 patients with proximal coronary artery stenting showed a normalization of stress test results and angiographical patency 6 months after the percutaneous coronary intervention was demonstrated . there are also different surgical strategies : reimplantation of the rca ( as in our case ) , unroofing of the intramural course with creation of a neo - orifice , the modified unroofing technique , patch augmentation , and classical bypass grafting . unroofing of the anomalous artery can be applied when the proximal course runs intramural , and when there is no involvement of aortic valve commissures , which otherwise could create aortic insufficiency . this technique relieves the ostial stenosis , creates a large neo - orifice , and removes the intramural segment . in the modified unroofing technique , the anomalous orifice is closed , a neo - orificium is created in the appropriate sinus , without extensive unroofing of the proximal intramural part of the anomalous coronary artery . the proximal course of an arca may vary : preaortic , interarterial ( between aorta and pulmonary artery , as in our patient ) , retrocardiac , retroaortic , intraseptal , and precardiac ( prepulmonary ) . , interarterial course ) runs between the aorta and pulmonary artery , either intramural ( within the aortic wall ) or separated from the aortic wall ( like 2 distinct arteries ) . with newer imaging techniques , newer anatomical variants of the proximal course of an arca the coronary takeoff can be high ( above the level of the pulmonary valve with a course between the aorta and pulmonary artery ) or low ( below the level of the pulmonary valve with a course between the aorta and right ventricular outflow tract [ rvot ] ) . this high takeoff variant is believed to be more hostile than an arca with low takeoff . an arca has a higher incidence than an anomalous left coronary artery ( alca ) ( 0.020.15% ) and is presumed to be the most common type of hemodynamically significant coronary anomalies . in mdct coronary studies , the incidence of an arca is comparable ( 0.54% ) . among 18,950 autopsy cases in a los angeles hospital , 54 cases of coronary anomalies were detected of whom 39 with anomalies of the coronary ostia , totaling to an incidence of 0.206% . in young athletes , 12% of scds is caused by an anomalous coronary artery from the opposite aortic sinus . in young athletes , however , with increasing age of athletes , coronary anomaly becomes a less frequent cause of scd . these numbers can obviously not be extrapolated from autopsy studies to the general population and are probably severely overestimated as mortality rates are biased as we are not aware of the exact prevalence of an arca in asymptomatic patients . notwithstanding , it is assumed that there is a 3- to 6-fold increased risk for scd in people with an arca doing physical activity . there are several hypotheses : a slit - like orifice caused by an acute angle of takeoff , which could cause reduced coronary flow during exercise ; compression of the interarterial segment caused by systolic compression between the aorta and pulmonary artery , aggravated during increased flow , for example , with exercise ; ventricular arrhythmias caused by ischemia ; acute or repetitive ischemia provoking myocardial fibrosis or reperfusion ; intramural proximal intussusception of the anomalous artery at the aortic - root wall , as proposed by angelini et al during intravascular ultrasound ( ivus ) studies . , studies showed that a coronary anomaly is not associated with an increased risk for development of coronary atherosclerosis . logically , the dominance of an anomalous coronary artery is an important risk factor . in an mdct study , clinical symptoms were not related to the relative luminal narrowing nor angle of takeoff . however , a significant difference in the prevalence of major adverse cardiac events ( mace ) and typical angina was observed in a retrospective review of 22,925 consecutive mdct scans , in which 124 cases with an interarterial anomalous coronary artery were found . age is an important clinical parameter that is related with the risk of scd . under the age of 30 it has been hypothesized that the aortic wall stiffens with age , which reduces compression . as mentioned above , in a necropsy study of 242 patients who died suddenly , 62% of the patients were asymptomatic until the event . in a japanese review of 56 patients with coronary anomalies of whom 44 with an arca and with a mean age of 55.9 years old of 22 patients with an arca who had undergone a treadmill test , 10 proved abnormal . three out of 4 had a positive myocardial perfusion single photon emission computed tomography ( spect ) exercise test . this is why a negative stress test does not exclude a potentially dangerous coronary anomaly . , mdct scan ) should be considered . in young patients , or echogenic patients one study showed significant 2d strain impairment in 25 patients with a coronary anomaly ( of whom 15 with arca ) , suggesting subtle left ventricular contractility disorder in these patients . one study evaluated the accuracy of coronary artery calcium scanning for detecting coronary anomalies , which was found out to have a great diagnostic accuracy . however , nowadays , mdct ca is accepted as the gold standard for the evaluation of coronary anomalies . magnetic resonance angiography ( mra ) is similar successful , but the identification of the distal coronary course can be more difficult . some disadvantages of mra are also to be considered : availability is less , mra is not useful in patients with pacemakers or claustrophobia , and total study time takes much longer . ca it is not always possible to define the exact proximal anatomical course of the coronary anomaly . fractional flow reserve ( ffr ) was tested by angelini et al , and showed results within normal limits during adenosine provocation ( ffr > 0.9 , cutoff 0.8 ) , indicating that ffr is not a good parameter to diagnose hemodynamic significance in this setting . stress - rest myocardial perfusion spect can be used to detect reversible perfusion defects , although results are often negative , as it is for stress ecg . long - term holter monitoring can be helpful in screening for arrhythmia , although being an aspecific tool . the 2008 american heart association guidelines for the management of adults with congenital disease , recommend that surgical coronary revascularization should be performed when there is evidence of ischemia in an anomalous rca coursing between aorta and pulmonary artery ( level of evidence class i , b ) . indication to treat should be individualized , and depends on age , symptoms , the anatomical variant , and ischemic testing . in literature , patients are separated by age ( younger and older than 35 to 40 years old ) . symptomatic patients older than 40 years old and with high takeoff should be operated if symptomatic , following their ideas . in a japanese retrospective study of 56 patients ( mean age 55.9 years old ) , with anomalous origin of coronary artery ( 78% arca ) and without coexisting atherosclerosis , a conservative treatment was applied ( nitrates , calcium channel blockers , beta - adrenergic antagonists , or antiarrhythmic drugs ) . during follow - up ( 2 months to 14.5 years ) , there were no cardiac - related deaths . the authors concluded that the prognosis of middle - aged to elderly patients with an anomalous origin of the coronary artery is relatively good . this somehow conflicting evidence illustrates that further research is warranted , matching anatomical with clinical and functional test data . one case series of 14 patients with proximal coronary artery stenting showed a normalization of stress test results and angiographical patency 6 months after the percutaneous coronary intervention was demonstrated . there are also different surgical strategies : reimplantation of the rca ( as in our case ) , unroofing of the intramural course with creation of a neo - orifice , the modified unroofing technique , patch augmentation , and classical bypass grafting . with patch augmentation , unroofing of the anomalous artery can be applied when the proximal course runs intramural , and when there is no involvement of aortic valve commissures , which otherwise could create aortic insufficiency . this technique relieves the ostial stenosis , creates a large neo - orifice , and removes the intramural segment . in the modified unroofing technique , the anomalous orifice is closed , a neo - orificium is created in the appropriate sinus , without extensive unroofing of the proximal intramural part of the anomalous coronary artery . the interarterial form of an anomalous rca ( coursing between the aorta and pulmonary artery ) can lead to symptoms or even scd . coronary anomaly is the second most common cause of cardiac sudden death in young athletes , which demonstrates that young age and vigorous exercise are risk factors for sudden death in patients with an arca . further ischemic testing can help to guide the therapy strategy , although functional tests often prove negative . in middle - aged patients , this anomaly is often an incidental finding . notwithstanding the lack of randomized trials , we consider it advisable to operate when an arca is seen in young patients ( < 40 years old ) or , in older patients with proven related symptoms or positive ischemic testing . reimplantation of the rca or unroofing the proximal course of the anomalous artery seems to be the best surgical strategies .

the overwhelming majority of enteric neuronal cell bodies lie , together with enteric glial cells , in ganglionated nerve networks , the myenteric and the submucosal plexus . . 1schematic drawing of the three ganglionated plexus of the human enteric nervous system , depicted in layer preparation ( red : cross - sectioned muscle layer profiles ) . the different network architectures were approached from numerous original small and large intestinal wholemount specimens . between the three ganglionated plexus , there are abundant interconnecting strands mp myenteric plexus , esp external submucosal plexus , isp internal submucosal plexus schematic drawing of the three ganglionated plexus of the human enteric nervous system , depicted in layer preparation ( red : cross - sectioned muscle layer profiles ) . the different network architectures were approached from numerous original small and large intestinal wholemount specimens . between the three ganglionated plexus , there are abundant interconnecting strands mp myenteric plexus , esp external submucosal plexus , isp internal submucosal plexus situated between the longitudinal and the circular muscle layer is the myenteric plexus ( auerbach 1862 ) . its main , primary network is commonly monolayered and extends from the upper oesophageal to the internal anal sphincter . most human myenteric neurons are either nitrergic or cholinergic ( murphy et al . 2007 ; beck et al . 2009 ) . like in other species ( best known from the guinea pig , furness 2006 ) , human nitrergic myenteric neurons are suggested to be either descending inter- and/or inhibitory motor neurons ( grider 1993 ) . they are uniaxonal with spiny shaped dendrites ( spiny type i morphology ; brehmer et al . 2004a ; lindig et al . 2009 ) . besides their reactivity for the nitrergic marker neuronal nitric oxide synthase ( nnos ) , some of them co - stain immunohistochemically for vasoactive intestinal peptide ( vip ; brehmer et al . in contrast , cholinergic myenteric neurons , immunoreactive for the common choline acetyltransferase ( cchat ) , include primary afferent , inter- , and excitatory motor neurons as mainly known from the guinea pig ( furness 2006 ) . although we have characterized morpho - chemically some non - nitrergic myenteric neuron types in human intestines ( brehmer et al . 2004a , b , 2005 ) , our knowledge of diverse enteric neuron types in the human gut is still fragmentary ( brehmer 2006 ) . the submucosa of larger mammals harbours a two- or even multilayered plexus ( meissner 1857 ) which is restricted to the small and large intestines . in human , two ganglionated submucosal plexus can be distinguished ( brehmer et al . 2010 ) , primarily based on their locations within the submucosal layer and their network architectures . the internal submucosal plexus ( reassigned to meissner ) lies in the inner half of the submucosa and is frequently multilayered . the external submucosal plexus ( schabadasch 1930 ) is situated near the inner border of the circular muscle layer and is rather mono-(occasionally two- ) layered . as to their neuronal compositions , we have hitherto found only quantitative differences between the human internal and external submucosal plexus ( see below ) . this is in contrast , e.g. , to the pig , where clear qualitative differences between the two submucosal plexus were described ( stach 1977 ; timmermans et al . 2001 ; kapp et al . 2006 ) . submucosal nitrergic neurons represent a small minority and are partly even absent from the submucosal plexus ( timmermans et al . 1994 ; beyer et al . 2013 ; beuscher et al . 2014 ) . among the cholinergic neurons , there are two separate populations that commonly amount for up to 80 % of human submucosal neurons . the larger population is , in the colon , co - immunoreactive for vip and calretinin ( calr ) , and the smaller one for somatostatin ( som ) and , partly , substance p ( sp ; kustermann et al . the two populations differ also morphologically : vip / calr - neurons are dendritic , whereas som / sp - neurons are non - dendritic and ( pseudo)uniaxonal . both submucosal neuron populations may have multiple functions ( see below ) . among these , vip / calr - neurons may act as secretomotor neurons ( karaki and kuwahara 2004 ; farthing 2006 ; margolis and gershon 2009 ; chandrasekharan et al . 2013 ; beuscher et al . 2014 ) , and som / sp - neurons may be primary afferent ( farthing 2006 ; kustermann et al . megacolon is the chronic dilation of a colonic segment , irrespective of the location of its origin ( fig . 2 ) . already , after its first description by hirschsprung ( 1888 ; cited after howard 1972 ) , several early authors suspected the cause of this congenital form not in the megacolon itself but in the absence of enteric neurons ( aganglionosis ) in the anally located zone ( bodian et al . subsequently , adaptive transformation of the orally located , originally healthy colonic segment toward a secondary megacolon ( bodian et al . a secondary megacolon ( e.g. in hirschsprung s disease ) is due to permanent stenosis of an aganglionic segment ( arrow ) followed by stasis and secondary inflation of an originally healthy segment . b primary megacolon ( e.g. in chagas disease ) is due to inflation of the originally diseased segment itself ( arrows indicate the zone of an acquired hypoganglionosis ) two alternate ways for the development of megacolon . a secondary megacolon ( e.g. in hirschsprung s disease ) is due to permanent stenosis of an aganglionic segment ( arrow ) followed by stasis and secondary inflation of an originally healthy segment . b primary megacolon ( e.g. in chagas disease ) is due to inflation of the originally diseased segment itself ( arrows indicate the zone of an acquired hypoganglionosis ) in contrast , enteric neurodegeneration of an originally healthy ( normoganglionic ) segment becomes manifest in at least 20 % of patients suffering from chagas disease ( kberle 1968 ) . during its chronic phase , ( my)enteric neuron loss and irreversible dilation occur in one and the same ( frequently colonic ) gut segment . since cause and effect are at the same place , this dilation may be termed as primary megacolon . at the latest since kberle , ( my)enteric neuron loss was commonly considered as the cause for chagasic megacolon : denervation is the absolutely indispensable element for the appearance of these ( chagasic megacolonic ) syndromes ( kberle 1968 ) . during the 1960s , when kberle and others searched for pathohistological alterations in colonic ( and other ) megasyndromes , the enteric nervous system ( ens ) awaited its conceptual renaissance ( brehmer et al . 1999 ) . primarily based on works in the guinea pig , the unique position of the ens within the peripheral nervous system as well as the complexity of its internal organization and external relationships with surrounding tissues and systems these advances in our understanding of the ens were scarcely accompanied by advances in the explanation of the development of ( chagasic ) megacolon . the above , classical explanations for hirschsprung s megacolon on the one hand ( regional absence of enteric neurons leads to permanent contraction of adjacent smooth muscle ) and chagas megacolon on the other hand ( regional absence of enteric neurons leads to permanent dilation of adjacent smooth muscle ) are contradictory . the ens regulates not only motor but also mucosal functions , a vital one being the maintenance of the epithelial barrier of the mucosa . in patients suffering from chagasic megacolon for decades , this barrier should be largely intact which indicates that certain components of the ens should be undamaged as well . this led us to analyse the fate of different subgroups of enteric neurons and of some of their related structures in chagasic megacolonic segments . the enteric nervous tissue consists of neuronal and glial components . to estimate quantitatively their representation in our wholemount and section specimens as marker for demonstrating neuronal cell bodies , the human neuronal protein hu c / d ( hu ; phillips et al . 2006 ) is well established . in contrast , an immunohistochemical pan - axonal marker is , to our knowledge , not generally established . here , we used synaptophysin ( syn ) which is specific for synaptic vesicles ( wiedenmann and franke 1985 ; dzienis - koronkiewicz et al . 2005 ) . furthermore , specific neuronal markers were applied ( see above : calr , cchat , nnos , som , vip ) . as marker for the evaluation of changes of the glial component of the enteric nervous tissue classical role as pure supporting cells , a number of specialized functions were recently attributed to enteric glial cells , e.g. support of the epithelial barrier function , modulation of neurotransmission , neurogenesis , etc . interstitial cells of cajal ( iccs ) have been demonstrated by immunolabelling of the c - kit receptor ( huizinga et al . 2012 ) , which is located in both iccs and mast cells but not in enteric neurons ( maeda et al . iccs are considered as pacemakers of motility and mediate the input of neurons to the muscularis propria ( thuneberg 1982 ; rumessen and thuneberg 1996 ; ward et al . 2004 ) although this role has also been disputed ( see below ) . to enable conclusions as to the amount of potentially contractile smooth muscle tissue within the thickened muscle layers , we applied -smooth muscle actin ( sma ) as marker for gut musculature ( wedel et al . , we estimated the smooth muscle density as stained sma - profile area related to the muscle layer area in cryosections through the gut wall . pathohistological changes obvious in myenteric wholemount specimens of chagasic / megacolonic tissues appear as prototypic signs of degeneration which were most distinct in the dilated megacolonic and the anal ( non - dilated ) region ( jabari et al . similar alterations have earlier been demonstrated , by applying various methods , in colonic specimens derived from quite different diseases and from aged colon ( de biscop 1949 ; smith 1972 ; schuffler et al . 1978 , 1985 ; schuffler and jonak 1982 ; krishnamurthy et al . 1985 ; phillips et al . 2003 ; hanani et al . 2004 ) . instead of myenteric ganglia containing numerous neurons , networks of bulky fibre bundles , comb - like plexus structures , and these general signs of neurodegeneration were paralleled by a decrease of s100-immunoreactive glial elements which may leave the nerve elements unprotected against inflammatory damage ( da silveira et al . neuron loss in chagas disease is thought to begin in the acute phase after infection with the protozoon trypanosoma cruzi by direct action of the pathogen , whereas it continues in the chronic phase by ( auto)immunologic reactions of the organism ( kberle 1968 ; hudson and hindmarsh 1985 ; dutra et al . however , the parasite does not disappear completely from the afflicted host in most cases ( clayton 2010 ) . during the decades of the chronic phase of chagas disease , functional motility dysbalance into a permanent megacolon , kberle ( 1968 ) and meneghelli ( 2004 ) suggested that loss of more than half of the colonic enteric neurons is required . meneghelli ( 2004 ) found a 50 % loss of myenteric neurons in small intestinal samples displaying no mega - syndrome . whereas megaesophagus occurs almost as frequently as megacolon in chronic chagas disease , [ by far the most frequently affected organ in chronic chagas disease is the heart ( kberle 1968 ) ] . extensive myenteric neuron loss in chagasic megacolon was shown in a number of studies ( kberle 1968 ; fernandez et al . own results generally confirmed these findings and additionally demonstrated parallel decrease of nerve fibre density in the circular and longitudinal muscle layers ( jabari et al . this was demonstrated both by applying general neuronal and glial ( syn , s 100 ) as well as specific neuronal markers ( see below ) . besides general signs of neurodegeneration ( see above ) , we additionally found the few , remaining neurons to be frequently deformed and hyper- or atrophic and to be mostly immunoreactive for nnos and/or vip ( fig . co - application of the pan - neuronal marker hu enabled us to discriminate the relative increase of the nitrergic / vip - ergic subpopulation from its absolute decrease ( jabari et al . 1998 ) . the fraction of cholinergic neurons and nerve fibres , however , was disproportionately decreased . different studies in non - chagasic / non - megacolonic samples revealed between 51 and 56 % nitrergic myenteric neurons ( beck et al . 2009 ; jabari et al . in contrast , in chagasic / megacolonic tissues , this proportion increased up to 86 % ( jabari et al . even more conspicuously , the fraction of nitrergic neurons co - localizing vip increased disproportionately . in non - chagasic / non - megacolonic colon , nnos / vip - neurons amounted to only 9 % ( schuy et al . 2011 ) , whereas in chagasic / megacolonic samples , up to 51 % ( jabari et al . generally , these changes in ganglionic composition were paralleled by changes in intramuscular nerve fibre spectrum . besides a general reduction in nerve fibre density , nerve fibres immunoreactive for nnos or vip or both were increased . for example , in control colonic circular muscle , 68 % of nerve fibres were nnos- and/or vip - reactive , and this proportion increased from 75 % ( in the oral transitional zone ) via 84 % ( megacolonic zone ) up to 87 % ( anal zone ; jabari et al . also , with ageing , a relative , although less pronounced increase of nitrergic neurons was described in both animals and humans ( santer 1994 ; phillips et al . 2009).fig . 3 a , b myenteric ganglia in wholemounts immunohistochemically triple stained for nitric oxide synthase ( nnos , red ) common choline acetyl transferase ( cchat , blue ) and the human neuronal protein hu c / d ( hu , green ) . a is from a non - chagasic / non - megacolonic ( control ) , note the numerical balance between red and blue neurons , both with green nuclei . b is from a chagasic / megacolonic specimen , note the loosened structured ganglionic architecture with few , red neurons . c , d circular muscle layers with myenteric ganglia ( asterisks ) in cross sections stained for vasoactive intestinal peptide ( vip , red ) , cchat ( blue ) , and nnos ( green ) . c control specimen , d chagasic / megacolonic specimen . note the increased layer thickness and the decreased nerve fibre density in ( d ) in contrast to ( c ) a , b myenteric ganglia in wholemounts immunohistochemically triple stained for nitric oxide synthase ( nnos , red ) common choline acetyl transferase ( cchat , blue ) and the human neuronal protein hu c / d ( hu , green ) . a is from a non - chagasic / non - megacolonic ( control ) , note the numerical balance between red and blue neurons , both with green nuclei . b is from a chagasic / megacolonic specimen , note the loosened structured ganglionic architecture with few , red neurons . c , d circular muscle layers with myenteric ganglia ( asterisks ) in cross sections stained for vasoactive intestinal peptide ( vip , red ) , cchat ( blue ) , and nnos ( green ) . c control specimen , d chagasic / megacolonic specimen . note the increased layer thickness and the decreased nerve fibre density in ( d ) in contrast to ( c ) reasons for this partial , selective survival of nitrergic and vip - ergic nerve elements were discussed ( jabari et al . consequences of the prevalence of neurons and nerve fibres containing these two inhibitory neurotransmitters ( grider 1989 , 1993 ) seem to allow a casual explanation of the development of a permanently dilated megacolon. however , analysing also the transitional zones directly oral and anal to the dilated , megacolonic segment , only the findings of the oral zone ( displaying a less pronounced destruction and loss of cholinergic nerve elements ) were in line with this explanation . in contrast , in the anal zone , the dysbalance between inhibitory ( vip , nnos containing ) and excitatory ( cholinergic ) nerve elements was most pronounced toward the former ; however , this region was not dilated . in other words , the extent of changes demonstrated immunohistochemically by neuronal and glial markers corresponded with the change of calibre along the surgically removed chagasic megacolon only in the oral transitional and the megacolonic zones . they did not correspond in the anal transitional zone . here , the change of neuro - immunohistochemical parameters toward inhibitory elements is not accompanied by dilation . this suggests that the neuronal dysbalance alone is not sufficient for explaining the chronic dilation , and additional factors must be taken into account . both the external and the mucosal muscle layers are remarkably thickened in chagasic megacolon ( kberle 1968 ) . permanent dilation of a ( colonic ) gut segment is accompanied by thickening of its wall . initially , thickening predominates over dilation , whereas later , as a sign of decompensation , the gut wall distends with the muscle layers becoming thinner ( kberle 1968 ) . ( 2012 ) described muscle area thickening in sections of the muscularis propria in chagasic megacolon increasing as much as five times compared to controls . these authors concluded that the development of the dilation is accompanied by muscular hypertrophy . but muscle layer thickening is not only due to an increase of muscle tissue but also to proliferation of connective tissue . consequently , in the thickened muscle layers of the dilated megacolonic zones , we found a decreased density of sma together with a loosened architecture of the sma - stained circular and longitudinal layers ( fig . 4 ; jabari et al . decreased density of sma has been found also in other motility disorders , together with a strikingly changed architecture of musculature ( knowles et al . the decreased density of sma and thereby the reduction of contractile elements in the megacolonic zone may be a sign of the massive impairment of motility . besides , there are also reports that no or non - significant deficiencies of smooth musculature morphology occur in intestinal pseudo - obstruction , hirschsprung s disease , and idiopathic intractable constipation ( gamba et al . 2004 ; knowles et al . 2004 ; van den berg et al . 4cross sections through the muscle coats ( adjusted to the plane of the myenteric plexus , asterisks ) immunohistochemically quadruple stained for synaptophysin ( syn , red ) , -smooth muscle actin ( sma , blue ) , c - kit ( green ) , and s100 ( yellow ) . a control specimen , b chagasic / megacolonic specimen . note in ( b ) in contrast to ( a ) : the increased thickness of both muscle layers ( blue ) , the loosened architecture of the muscle tissue ( blue ) , and the decreased density of the other three markers ( for details see jabari et al . 2013 ) cross sections through the muscle coats ( adjusted to the plane of the myenteric plexus , asterisks ) immunohistochemically quadruple stained for synaptophysin ( syn , red ) , -smooth muscle actin ( sma , blue ) , c - kit ( green ) , and s100 ( yellow ) . a control specimen , b chagasic / megacolonic specimen . note in ( b ) in contrast to ( a ) : the increased thickness of both muscle layers ( blue ) , the loosened architecture of the muscle tissue ( blue ) , and the decreased density of the other three markers ( for details see jabari et al . 2013 ) a decrease of iccs has been described in chagasic megacolon ( hagger et al . although these cells are seen as contributors in the development of numerous motility disorders ( streutker et al . 2010 ) , their function especially in human gastrointestinal motility is unproven ( sanders et al . 2012 ) . the contraction of gastrointestinal smooth muscle is supposed to be generated by the common action of at least three electrically coupled cell types , i.e. iccs , non - icc - interstitial cells ( being immunoreactive for the platelet - derived growth factor receptor ) , and smooth muscle cells . enteric neurons , hormones , and paracrine substances modulate their contraction ( sanders et al . , the balance between these components is disturbed in different ways ( see above and fig . 2 ) . however , in hirschsprung s ( gfroerer and rolle 2013 ) and chagas megacolon ( see above ) , most studies found reduced numbers of iccs in the hypo- or aganglionic segments . ( 1998 ) who found no difference in icc distribution between aganglionic ( hirschsprung ) and intact descending colon . in chagasic megacolon , a close topographical correlation between the change of calibre along the resected specimens and the changes of parameters was observed related to smooth muscle and icc densities ( jabari et al . values were lower in dilated and megacolonic , and were higher in non - dilated oral and anal zones ( jabari et al . that is , decrease in muscle tissue and icc densities is topographically directly linked with the extent of the mega - syndrome . thus , changes of these two parameters provide a more consistent explanation for the change of the colonic circumference . most studies dealing with chagasic megacolon focused on structures related to motility , and only few presented counts of submucosal neurons in chagasic gut segments ( costa and de lima filho 1964 ; meneghelli 2004 ; iantorno et al . 2007 ) . our own counts in chagasic / megacolonic submucosal plexus principally confirmed the results of the earliest authors ( costa and de lima filho 1964 ) , i.e. in contrast to the massive myenteric neuron loss , the submucosal neuron loss was rather moderate ( jabari et al . however , there is a selective loss of neurons also in the two submucosal plexus . here , , colonic external and internal submucosal plexus , at the most one - third of neurons is vip - negative and these neurons degenerated almost completely in chagasic megacolon . among these vip - negative neurons , the largest and best known population is the som ( chat / sp)-immunoreactive one ( beyer et al . 2013 ) . consequently , mucosal nerve fibres immunoreactive for som were hardly found , whereas vip(/calr)-positive nerve fibres were , though reduced in density , present throughout all megacolonic and transitional zones ( jabari et al . the muscularis mucosae and the lamina propria displayed diverging changes as to the megacolonic and the anal segments . the lamina propria showed highest layer thickness and lowest densities of nerve fibres , glia , and smooth muscle tissue in the dilated portion itself . in contrast , in the muscularis mucosae , the layer thickness was highest and the nerve / glia densities were lowest in the anal segment thus paralleling results of the ( external ) muscle coat ( jabari et al . one of these is the epithelium whose barrier function requires , e.g. cell proliferation and expression of tight junction proteins . both depend on the action of vip - ergic submucosal neurons ( neunlist et al . if these vip - neurons would have died in the course of chagas disease , we suggest that these patients could not have survived for decades . in contrast , considering the almost total loss of som - neurons in chagasic megacolon , this population seems not to be of vital importance . the difference in proportions of vip - neurons in the respective plexus reflects the difference in the numbers of surviving neurons in chronic chagas disease . few myenteric neurons survive in chagasic megacolon ( fig . 3 ) . in contrast , vip(/calr)-neurons are a majority in the healthy submucosal plexus a considerable number of submucosal neurons survive ( fig . 5 ) . above , we discussed our suggestions as to quite different consequences of the survival of myenteric and submucosal vip - neurons . the myenteric vip - neurons may contribute to the development of the megacolon , whereas the submucosal vip - neurons may enable the patients to survive by maintaining the epithelial barrier.fig . 5 a , b external submucosal ganglia in wholemounts immunohistochemically triple stained for somatostatin ( som , red ) , the human neuronal protein hu c / d ( hu , blue ) , and calretinin ( calr , green ) . a is from a non - chagasic / non - megacolonic ( control ) , b from a chagasic / megacolonic specimen . note that in both cases , the ganglia display quite similar appearance ; in ( b ) , there is no red ( som - positive ) neuron . c , d mucosal layers in cross sections ( with adjusted muscularis mucosae , asterisks ) triple stained for som ( red ) , -smooth muscle actin ( sma , blue ) , and vasoactive intestinal peptide ( vip , green ) . note the increased thickness and the decreased nerve fibre density with a complete loss of red ( som - reactive ) nerve fibres in ( d ) in contrast to ( c ) . som - positive enteroendocrine cells ( red patches ) are present in both ( c ) and ( d ) a , b external submucosal ganglia in wholemounts immunohistochemically triple stained for somatostatin ( som , red ) , the human neuronal protein hu c / d ( hu , blue ) , and calretinin ( calr , green ) . a is from a non - chagasic / non - megacolonic ( control ) , b from a chagasic / megacolonic specimen . note that in both cases , the ganglia display quite similar appearance ; in ( b ) , there is no red ( som - positive ) neuron . c , d mucosal layers in cross sections ( with adjusted muscularis mucosae , asterisks ) triple stained for som ( red ) , -smooth muscle actin ( sma , blue ) , and vasoactive intestinal peptide ( vip , green ) . note the increased thickness and the decreased nerve fibre density with a complete loss of red ( som - reactive ) nerve fibres in ( d ) in contrast to ( c ) . som - positive enteroendocrine cells ( red patches ) are present in both ( c ) and ( d ) one reason for survival of these neurons may be vip itself . it is well established that this peptide is neuroprotective ( ekblad and bauer 2004 ; pozo and delgado 2004 ; brenneman 2007 ; arranz et al . additionally , also nitric oxide was able to promote enteric neuronal survival in culture ( sandgren et al . we suggest that vip may protect both the submucosal vip - neurons , acting on epithelial cells , and the myenteric vip - neurons , acting together with nnos as inhibitory motor neurons . in conclusion , we suggest that during megacolon formation in chronic chagas disease , neuron loss alone does not explain completely the loss of motility and the subsequent calibre change . other cells and tissues ( e.g. iccs , smooth muscle cells ) may be primarily damaged as well . as to the survival of patients despite the obvious changes in the enteric nervous system , vip may play a key role due to its neuroprotective and neuroeffectory functions .

megacolon , the irreversible dilation of a colonic segment , is a structural sign associated with various gastrointestinal disorders . in its hereditary , secondary form ( e.g. in hirschsprung s disease ) , dilation occurs in an originally healthy colonic segment due to an anally located , aganglionic zone . in contrast , in chronic chagas disease , the dilated segment itself displays pathohistological changes , and the earliest and most prominent being found was massive loss of myenteric neurons . this neuron loss was partial and selective , i.e. some neurons containing neuronal nitric oxide synthase and/or vasoactive intestinal peptide ( vip ) were spared from neuron death . this disproportionate survival of inhibitory neurons , however , did not completely correlate with the calibre change along the surgically removed , megacolonic segments . a better correlation was observed as to potentially contractile muscle tissue elements and the interstitial cells of cajal . therefore , the decreased densities of -smooth muscle actin- and c - kit - immunoreactive profiles were estimated along resected megacolonic segments . their lowest values were observed in the megacolonic zones itself , whereas less pronounced decreases were found in the non - dilated , transitional zones ( oral and anal to dilation ) . in contrast to the myenteric plexus , the submucosal plexus displayed only a moderate neuron loss . neurons co - immunoreactive for vip and calretinin survived disproportionately . as a consequence , these neurons may have contributed to maintain the epithelial barrier and allowed the chagasic patients to survive for decades , despite their severe disturbance of colonic motility . due to its neuroprotective and neuroeffectory functions , vip may play a key role in the development and duration of chagasic megacolon .

Structural components of the human enteric nervous system Megacolon:secondary versus primary Evaluation of enteric nervous tissue, interstitial cells of Cajal, and smooth musculature Chagasic megacolon:myenteric neurons and intramuscular nerve fibres Chagasic megacolon:smooth muscle and interstitial cells of Cajal (ICC) Chagasic megacolon:submucosal neurons and mucosal nerve fibres Vasoactive intestinal peptide (VIP)

the overwhelming majority of enteric neuronal cell bodies lie , together with enteric glial cells , in ganglionated nerve networks , the myenteric and the submucosal plexus . between the three ganglionated plexus , there are abundant interconnecting strands mp myenteric plexus , esp external submucosal plexus , isp internal submucosal plexus schematic drawing of the three ganglionated plexus of the human enteric nervous system , depicted in layer preparation ( red : cross - sectioned muscle layer profiles ) . between the three ganglionated plexus , there are abundant interconnecting strands mp myenteric plexus , esp external submucosal plexus , isp internal submucosal plexus situated between the longitudinal and the circular muscle layer is the myenteric plexus ( auerbach 1862 ) . besides their reactivity for the nitrergic marker neuronal nitric oxide synthase ( nnos ) , some of them co - stain immunohistochemically for vasoactive intestinal peptide ( vip ; brehmer et al . in contrast , cholinergic myenteric neurons , immunoreactive for the common choline acetyltransferase ( cchat ) , include primary afferent , inter- , and excitatory motor neurons as mainly known from the guinea pig ( furness 2006 ) . 2010 ) , primarily based on their locations within the submucosal layer and their network architectures . this is in contrast , e.g. , to the pig , where clear qualitative differences between the two submucosal plexus were described ( stach 1977 ; timmermans et al . the larger population is , in the colon , co - immunoreactive for vip and calretinin ( calr ) , and the smaller one for somatostatin ( som ) and , partly , substance p ( sp ; kustermann et al . among these , vip / calr - neurons may act as secretomotor neurons ( karaki and kuwahara 2004 ; farthing 2006 ; margolis and gershon 2009 ; chandrasekharan et al . 2014 ) , and som / sp - neurons may be primary afferent ( farthing 2006 ; kustermann et al . megacolon is the chronic dilation of a colonic segment , irrespective of the location of its origin ( fig . already , after its first description by hirschsprung ( 1888 ; cited after howard 1972 ) , several early authors suspected the cause of this congenital form not in the megacolon itself but in the absence of enteric neurons ( aganglionosis ) in the anally located zone ( bodian et al . subsequently , adaptive transformation of the orally located , originally healthy colonic segment toward a secondary megacolon ( bodian et al . in hirschsprung s disease ) is due to permanent stenosis of an aganglionic segment ( arrow ) followed by stasis and secondary inflation of an originally healthy segment . b primary megacolon ( e.g. in chagas disease ) is due to inflation of the originally diseased segment itself ( arrows indicate the zone of an acquired hypoganglionosis ) two alternate ways for the development of megacolon . a secondary megacolon ( e.g. in hirschsprung s disease ) is due to permanent stenosis of an aganglionic segment ( arrow ) followed by stasis and secondary inflation of an originally healthy segment . b primary megacolon ( e.g. in chagas disease ) is due to inflation of the originally diseased segment itself ( arrows indicate the zone of an acquired hypoganglionosis ) in contrast , enteric neurodegeneration of an originally healthy ( normoganglionic ) segment becomes manifest in at least 20 % of patients suffering from chagas disease ( kberle 1968 ) . during its chronic phase , ( my)enteric neuron loss and irreversible dilation occur in one and the same ( frequently colonic ) gut segment . at the latest since kberle , ( my)enteric neuron loss was commonly considered as the cause for chagasic megacolon : denervation is the absolutely indispensable element for the appearance of these ( chagasic megacolonic ) syndromes ( kberle 1968 ) . primarily based on works in the guinea pig , the unique position of the ens within the peripheral nervous system as well as the complexity of its internal organization and external relationships with surrounding tissues and systems these advances in our understanding of the ens were scarcely accompanied by advances in the explanation of the development of ( chagasic ) megacolon . the above , classical explanations for hirschsprung s megacolon on the one hand ( regional absence of enteric neurons leads to permanent contraction of adjacent smooth muscle ) and chagas megacolon on the other hand ( regional absence of enteric neurons leads to permanent dilation of adjacent smooth muscle ) are contradictory . the ens regulates not only motor but also mucosal functions , a vital one being the maintenance of the epithelial barrier of the mucosa . in patients suffering from chagasic megacolon for decades , this barrier should be largely intact which indicates that certain components of the ens should be undamaged as well . furthermore , specific neuronal markers were applied ( see above : calr , cchat , nnos , som , vip ) . support of the epithelial barrier function , modulation of neurotransmission , neurogenesis , etc . interstitial cells of cajal ( iccs ) have been demonstrated by immunolabelling of the c - kit receptor ( huizinga et al . to enable conclusions as to the amount of potentially contractile smooth muscle tissue within the thickened muscle layers , we applied -smooth muscle actin ( sma ) as marker for gut musculature ( wedel et al . pathohistological changes obvious in myenteric wholemount specimens of chagasic / megacolonic tissues appear as prototypic signs of degeneration which were most distinct in the dilated megacolonic and the anal ( non - dilated ) region ( jabari et al . instead of myenteric ganglia containing numerous neurons , networks of bulky fibre bundles , comb - like plexus structures , and these general signs of neurodegeneration were paralleled by a decrease of s100-immunoreactive glial elements which may leave the nerve elements unprotected against inflammatory damage ( da silveira et al . neuron loss in chagas disease is thought to begin in the acute phase after infection with the protozoon trypanosoma cruzi by direct action of the pathogen , whereas it continues in the chronic phase by ( auto)immunologic reactions of the organism ( kberle 1968 ; hudson and hindmarsh 1985 ; dutra et al . however , the parasite does not disappear completely from the afflicted host in most cases ( clayton 2010 ) . during the decades of the chronic phase of chagas disease , functional motility dysbalance into a permanent megacolon , kberle ( 1968 ) and meneghelli ( 2004 ) suggested that loss of more than half of the colonic enteric neurons is required . meneghelli ( 2004 ) found a 50 % loss of myenteric neurons in small intestinal samples displaying no mega - syndrome . whereas megaesophagus occurs almost as frequently as megacolon in chronic chagas disease , [ by far the most frequently affected organ in chronic chagas disease is the heart ( kberle 1968 ) ] . extensive myenteric neuron loss in chagasic megacolon was shown in a number of studies ( kberle 1968 ; fernandez et al . in contrast , in chagasic / megacolonic tissues , this proportion increased up to 86 % ( jabari et al . even more conspicuously , the fraction of nitrergic neurons co - localizing vip increased disproportionately . for example , in control colonic circular muscle , 68 % of nerve fibres were nnos- and/or vip - reactive , and this proportion increased from 75 % ( in the oral transitional zone ) via 84 % ( megacolonic zone ) up to 87 % ( anal zone ; jabari et al . 3 a , b myenteric ganglia in wholemounts immunohistochemically triple stained for nitric oxide synthase ( nnos , red ) common choline acetyl transferase ( cchat , blue ) and the human neuronal protein hu c / d ( hu , green ) . a is from a non - chagasic / non - megacolonic ( control ) , note the numerical balance between red and blue neurons , both with green nuclei . c , d circular muscle layers with myenteric ganglia ( asterisks ) in cross sections stained for vasoactive intestinal peptide ( vip , red ) , cchat ( blue ) , and nnos ( green ) . note the increased layer thickness and the decreased nerve fibre density in ( d ) in contrast to ( c ) a , b myenteric ganglia in wholemounts immunohistochemically triple stained for nitric oxide synthase ( nnos , red ) common choline acetyl transferase ( cchat , blue ) and the human neuronal protein hu c / d ( hu , green ) . a is from a non - chagasic / non - megacolonic ( control ) , note the numerical balance between red and blue neurons , both with green nuclei . c , d circular muscle layers with myenteric ganglia ( asterisks ) in cross sections stained for vasoactive intestinal peptide ( vip , red ) , cchat ( blue ) , and nnos ( green ) . note the increased layer thickness and the decreased nerve fibre density in ( d ) in contrast to ( c ) reasons for this partial , selective survival of nitrergic and vip - ergic nerve elements were discussed ( jabari et al . consequences of the prevalence of neurons and nerve fibres containing these two inhibitory neurotransmitters ( grider 1989 , 1993 ) seem to allow a casual explanation of the development of a permanently dilated megacolon. however , analysing also the transitional zones directly oral and anal to the dilated , megacolonic segment , only the findings of the oral zone ( displaying a less pronounced destruction and loss of cholinergic nerve elements ) were in line with this explanation . in contrast , in the anal zone , the dysbalance between inhibitory ( vip , nnos containing ) and excitatory ( cholinergic ) nerve elements was most pronounced toward the former ; however , this region was not dilated . in other words , the extent of changes demonstrated immunohistochemically by neuronal and glial markers corresponded with the change of calibre along the surgically removed chagasic megacolon only in the oral transitional and the megacolonic zones . they did not correspond in the anal transitional zone . both the external and the mucosal muscle layers are remarkably thickened in chagasic megacolon ( kberle 1968 ) . permanent dilation of a ( colonic ) gut segment is accompanied by thickening of its wall . initially , thickening predominates over dilation , whereas later , as a sign of decompensation , the gut wall distends with the muscle layers becoming thinner ( kberle 1968 ) . but muscle layer thickening is not only due to an increase of muscle tissue but also to proliferation of connective tissue . consequently , in the thickened muscle layers of the dilated megacolonic zones , we found a decreased density of sma together with a loosened architecture of the sma - stained circular and longitudinal layers ( fig . the decreased density of sma and thereby the reduction of contractile elements in the megacolonic zone may be a sign of the massive impairment of motility . besides , there are also reports that no or non - significant deficiencies of smooth musculature morphology occur in intestinal pseudo - obstruction , hirschsprung s disease , and idiopathic intractable constipation ( gamba et al . 4cross sections through the muscle coats ( adjusted to the plane of the myenteric plexus , asterisks ) immunohistochemically quadruple stained for synaptophysin ( syn , red ) , -smooth muscle actin ( sma , blue ) , c - kit ( green ) , and s100 ( yellow ) . note in ( b ) in contrast to ( a ) : the increased thickness of both muscle layers ( blue ) , the loosened architecture of the muscle tissue ( blue ) , and the decreased density of the other three markers ( for details see jabari et al . 2013 ) cross sections through the muscle coats ( adjusted to the plane of the myenteric plexus , asterisks ) immunohistochemically quadruple stained for synaptophysin ( syn , red ) , -smooth muscle actin ( sma , blue ) , c - kit ( green ) , and s100 ( yellow ) . note in ( b ) in contrast to ( a ) : the increased thickness of both muscle layers ( blue ) , the loosened architecture of the muscle tissue ( blue ) , and the decreased density of the other three markers ( for details see jabari et al . iccs , non - icc - interstitial cells ( being immunoreactive for the platelet - derived growth factor receptor ) , and smooth muscle cells . enteric neurons , hormones , and paracrine substances modulate their contraction ( sanders et al . however , in hirschsprung s ( gfroerer and rolle 2013 ) and chagas megacolon ( see above ) , most studies found reduced numbers of iccs in the hypo- or aganglionic segments . in chagasic megacolon , a close topographical correlation between the change of calibre along the resected specimens and the changes of parameters was observed related to smooth muscle and icc densities ( jabari et al . values were lower in dilated and megacolonic , and were higher in non - dilated oral and anal zones ( jabari et al . that is , decrease in muscle tissue and icc densities is topographically directly linked with the extent of the mega - syndrome . our own counts in chagasic / megacolonic submucosal plexus principally confirmed the results of the earliest authors ( costa and de lima filho 1964 ) , i.e. in contrast to the massive myenteric neuron loss , the submucosal neuron loss was rather moderate ( jabari et al . however , there is a selective loss of neurons also in the two submucosal plexus . here , , colonic external and internal submucosal plexus , at the most one - third of neurons is vip - negative and these neurons degenerated almost completely in chagasic megacolon . among these vip - negative neurons , the largest and best known population is the som ( chat / sp)-immunoreactive one ( beyer et al . consequently , mucosal nerve fibres immunoreactive for som were hardly found , whereas vip(/calr)-positive nerve fibres were , though reduced in density , present throughout all megacolonic and transitional zones ( jabari et al . the muscularis mucosae and the lamina propria displayed diverging changes as to the megacolonic and the anal segments . the lamina propria showed highest layer thickness and lowest densities of nerve fibres , glia , and smooth muscle tissue in the dilated portion itself . in contrast , in the muscularis mucosae , the layer thickness was highest and the nerve / glia densities were lowest in the anal segment thus paralleling results of the ( external ) muscle coat ( jabari et al . if these vip - neurons would have died in the course of chagas disease , we suggest that these patients could not have survived for decades . in contrast , considering the almost total loss of som - neurons in chagasic megacolon , this population seems not to be of vital importance . the difference in proportions of vip - neurons in the respective plexus reflects the difference in the numbers of surviving neurons in chronic chagas disease . in contrast , vip(/calr)-neurons are a majority in the healthy submucosal plexus a considerable number of submucosal neurons survive ( fig . above , we discussed our suggestions as to quite different consequences of the survival of myenteric and submucosal vip - neurons . the myenteric vip - neurons may contribute to the development of the megacolon , whereas the submucosal vip - neurons may enable the patients to survive by maintaining the epithelial barrier.fig . 5 a , b external submucosal ganglia in wholemounts immunohistochemically triple stained for somatostatin ( som , red ) , the human neuronal protein hu c / d ( hu , blue ) , and calretinin ( calr , green ) . note that in both cases , the ganglia display quite similar appearance ; in ( b ) , there is no red ( som - positive ) neuron . c , d mucosal layers in cross sections ( with adjusted muscularis mucosae , asterisks ) triple stained for som ( red ) , -smooth muscle actin ( sma , blue ) , and vasoactive intestinal peptide ( vip , green ) . note the increased thickness and the decreased nerve fibre density with a complete loss of red ( som - reactive ) nerve fibres in ( d ) in contrast to ( c ) . som - positive enteroendocrine cells ( red patches ) are present in both ( c ) and ( d ) a , b external submucosal ganglia in wholemounts immunohistochemically triple stained for somatostatin ( som , red ) , the human neuronal protein hu c / d ( hu , blue ) , and calretinin ( calr , green ) . c , d mucosal layers in cross sections ( with adjusted muscularis mucosae , asterisks ) triple stained for som ( red ) , -smooth muscle actin ( sma , blue ) , and vasoactive intestinal peptide ( vip , green ) . note the increased thickness and the decreased nerve fibre density with a complete loss of red ( som - reactive ) nerve fibres in ( d ) in contrast to ( c ) . som - positive enteroendocrine cells ( red patches ) are present in both ( c ) and ( d ) one reason for survival of these neurons may be vip itself . we suggest that vip may protect both the submucosal vip - neurons , acting on epithelial cells , and the myenteric vip - neurons , acting together with nnos as inhibitory motor neurons . in conclusion , we suggest that during megacolon formation in chronic chagas disease , neuron loss alone does not explain completely the loss of motility and the subsequent calibre change . as to the survival of patients despite the obvious changes in the enteric nervous system , vip may play a key role due to its neuroprotective and neuroeffectory functions .

according to a single - blind , within - subject comparison , 15 healthy , normal - weight men ( mean age se , 28.5 1.0 years ; bmi , 23.1 0.5 kg / m ) participated in two hyperinsulinemic - euglycemic clamp experiments ( human insulin , detemir ) spaced apart at least 1 week . all subjects gave written informed consent to the experiments , which were approved by the local ethics committee . in both experimental conditions , an insulin bolus injection ( 17.75 mu / kg body wt [ = 0.1065 nmol / kg body wt ] human insulin , insulin actrapid ; novo nordisk , bagsvrd , denmark ; 90 mu / kg body wt [ = 2.16 nmol / kg body wt ] detemir , insulin levemir ; novo nordisk ) was given followed by a steady 90-min infusion of 1.0 mu kg min (= 0.006 nmol kg min ) human insulin versus 2.0 mu kg min (= 0.048 nmol kg min ) detemir . to compensate for the relatively slower onset of the action of detemir , a higher dosage was chosen ( 15,17 ) to induce comparable peripheral effects of both compounds as assessed by rates of glucose infusion necessary to keep blood glucose levels in the euglycemic range . volunteers reported to the laboratory at 0800 , after an overnight fast of 10 h , and were prepared for electroencephalographic recordings , insulin infusions , and blood samplings . we inserted venous cannulas into the subject 's arms and connected them to tubes enabling infusion and blood sampling from an adjacent room without awareness of the subject . the arm from which samples were taken was positioned in a heated box ( 55c ) to enable drawing of arterialized venous blood . during recordings , subjects sat in a reclining chair in a sound - attenuated room of constant temperature , with their heads stabilized by a cushion . they were instructed to relax and not to move during recordings and to fixate their gaze on the wall in front of them . subjects pressed a button every estimated 30 s to maintain a constant state of mental activity and to not doze off . recordings of dc potentials started at 0940 with a baseline phase of 20 min followed by the bolus injection of human insulin and detemir , respectively ( t = 0 ) . subsequent insulin infusion lasted for 90 min , ending at 1130 when eeg recordings were also stopped . arterialized blood was drawn at 15-min intervals during baseline , at 5-min intervals during insulin infusion , and at 15-min intervals thereafter to monitor blood glucose concentration ( hemocue b - glucose - analyzer , hemocue ab , angelholm , sweden ) . during and after insulin administration , subjects intravenously received a 20% glucose solution at a variable rate to maintain normal plasma glucose levels . blood samples for the determination of hormonal parameters were repeatedly collected , and routine assays were used to determine concentrations of serum c - peptide , plasma acth , serum cortisol ( all immulite ; dpc , los angeles , ca ) , plasma glucagon ( ria ; adaltis , montreal , quebec , canada ) , and serum leptin ( ria ; linco research , st . charles , mo ) . at 1150 ( i.e. , 20 min after insulin infusion had stopped ) , a standardized buffet of around 4,650 kcal was offered from which subjects were allowed to eat ad libitum during the subsequent 50 min ( table 1 ) . subjects were kept unaware of hypothesized treatment effects on food intake and were not aware that their food intake was measured by weighing buffet components before and after food intake . in addition , to prevent overeating , subjects were allowed to take with them any remaining food afterward . before ( at 0915 ) and after ( at 1135 ) recordings and at the end of the session at 1300 , subjects rated their hunger , thirst , and tiredness on 10-point scales and completed a questionnaire assessing alertness and autonomic symptoms on 5-point bipolar scales of 20 contrasting adjective pairs ( e.g. , activated - inert and sweating - shivery ) ( 22 ) . they also filled in a checklist of 161 adjectives assessing mood on 14 dimensions ( 23 ) . composition of the test buffet composition of the buffet offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . standard recordings of dc potentials , electro - oculogram , and electromyogram were performed as described previously ( 24,25 ) . dc - potential recordings were obtained from left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) electrodes referenced to linked electrodes at the mastoids . a brainamp dc amplifier ( brain vision , london , u.k . ; low - pass filter : 30 hz , sampling rate : 200 hz ) was used . dc - potential drifts with short - circuited input were constantly < 5 v / h , and electrode impedance , measured before and after recordings , never exceeded 5 k. average dc - potential values were determined offline for subsequent 5-s intervals . linear potential drifts during the 20-min baseline period extending into the 90-min insulin infusion period were removed using a linear regression method . periods where electromyogram or electro - oculogram indicated increased muscular activity or eye movements were excluded from analysis . the average dc potential during baseline was set to 0 v , and potential shifts during treatment were expressed as difference values . differences in dc - potential values between conditions were evaluated first on an exploratory basis by point - wise comparisons using t tests to identify time ranges with most consistent differences ( 26 ) . the time range selected for analysis covered 2190 min of insulin infusion . values for this time interval were then subjected to anova , including the repeated - measures factors treatment ( human insulin vs. detemir ) and topography ( electrode locations ) . post hoc contrasts were used to specify significant anova main effects and interactions . for the dc - potential analysis , data from four subjects had to be excluded because of technical failures and artifacts of apparent nonbiological origin . behavioral measures and hormonal parameters were analyzed with anova and paired t tests as appropriate . according to a single - blind , within - subject comparison , 15 healthy , normal - weight men ( mean age se , 28.5 1.0 years ; bmi , 23.1 0.5 kg / m ) participated in two hyperinsulinemic - euglycemic clamp experiments ( human insulin , detemir ) spaced apart at least 1 week . all subjects gave written informed consent to the experiments , which were approved by the local ethics committee . in both experimental conditions , an insulin bolus injection ( 17.75 mu / kg body wt [ = 0.1065 nmol / kg body wt ] human insulin , insulin actrapid ; novo nordisk , bagsvrd , denmark ; 90 mu / kg body wt [ = 2.16 nmol / kg body wt ] detemir , insulin levemir ; novo nordisk ) was given followed by a steady 90-min infusion of 1.0 mu kg min (= 0.006 nmol kg min ) human insulin versus 2.0 mu kg min (= 0.048 nmol kg min ) detemir . to compensate for the relatively slower onset of the action of detemir , a higher dosage was chosen ( 15,17 ) to induce comparable peripheral effects of both compounds as assessed by rates of glucose infusion necessary to keep blood glucose levels in the euglycemic range . volunteers reported to the laboratory at 0800 , after an overnight fast of 10 h , and were prepared for electroencephalographic recordings , insulin infusions , and blood samplings . we inserted venous cannulas into the subject 's arms and connected them to tubes enabling infusion and blood sampling from an adjacent room without awareness of the subject . the arm from which samples were taken was positioned in a heated box ( 55c ) to enable drawing of arterialized venous blood . during recordings , subjects sat in a reclining chair in a sound - attenuated room of constant temperature , with their heads stabilized by a cushion . they were instructed to relax and not to move during recordings and to fixate their gaze on the wall in front of them . subjects pressed a button every estimated 30 s to maintain a constant state of mental activity and to not doze off . recordings of dc potentials started at 0940 with a baseline phase of 20 min followed by the bolus injection of human insulin and detemir , respectively ( t = 0 ) . subsequent insulin infusion lasted for 90 min , ending at 1130 when eeg recordings were also stopped . arterialized blood was drawn at 15-min intervals during baseline , at 5-min intervals during insulin infusion , and at 15-min intervals thereafter to monitor blood glucose concentration ( hemocue b - glucose - analyzer , hemocue ab , angelholm , sweden ) . during and after insulin administration , subjects intravenously received a 20% glucose solution at a variable rate to maintain normal plasma glucose levels . blood samples for the determination of hormonal parameters were repeatedly collected , and routine assays were used to determine concentrations of serum c - peptide , plasma acth , serum cortisol ( all immulite ; dpc , los angeles , ca ) , plasma glucagon ( ria ; adaltis , montreal , quebec , canada ) , and serum leptin ( ria ; linco research , st . at 1150 ( i.e. , 20 min after insulin infusion had stopped ) , a standardized buffet of around 4,650 kcal was offered from which subjects were allowed to eat ad libitum during the subsequent 50 min ( table 1 ) . subjects were kept unaware of hypothesized treatment effects on food intake and were not aware that their food intake was measured by weighing buffet components before and after food intake . in addition , to prevent overeating , subjects were allowed to take with them any remaining food afterward . before ( at 0915 ) and after ( at 1135 ) recordings and at the end of the session at 1300 , subjects rated their hunger , thirst , and tiredness on 10-point scales and completed a questionnaire assessing alertness and autonomic symptoms on 5-point bipolar scales of 20 contrasting adjective pairs ( e.g. , activated - inert and sweating - shivery ) ( 22 ) . they also filled in a checklist of 161 adjectives assessing mood on 14 dimensions ( 23 ) . composition of the test buffet composition of the buffet offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . standard recordings of dc potentials , electro - oculogram , and electromyogram were performed as described previously ( 24,25 ) . dc - potential recordings were obtained from left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) electrodes referenced to linked electrodes at the mastoids . a brainamp dc amplifier ( brain vision , london , u.k . ; low - pass filter : 30 hz , sampling rate : 200 hz ) was used . dc - potential drifts with short - circuited input were constantly < 5 v / h , and electrode impedance , measured before and after recordings , never exceeded 5 k. average dc - potential values were determined offline for subsequent 5-s intervals . linear potential drifts during the 20-min baseline period extending into the 90-min insulin infusion period were removed using a linear regression method . periods where electromyogram or electro - oculogram indicated increased muscular activity or eye movements were excluded from analysis . the average dc potential during baseline was set to 0 v , and potential shifts during treatment were expressed as difference values . differences in dc - potential values between conditions were evaluated first on an exploratory basis by point - wise comparisons using t tests to identify time ranges with most consistent differences ( 26 ) . the time range selected for analysis covered 2190 min of insulin infusion . values for this time interval were then subjected to anova , including the repeated - measures factors treatment ( human insulin vs. detemir ) and topography ( electrode locations ) . post hoc contrasts were used to specify significant anova main effects and interactions . for the dc - potential analysis , data from four subjects had to be excluded because of technical failures and artifacts of apparent nonbiological origin . behavioral measures and hormonal parameters were analyzed with anova and paired t tests as appropriate . 1a ; p > 0.14 for all comparisons ) , resulting in identical total amounts of energy supplied via glucose infusion ( human insulin , 240.8 23.3 kcal ; detemir , 239.5 25.0 kcal ; p > 0.93 ) . correspondingly , blood glucose concentrations were comparable throughout the experiment ( fig . 1b ; p > 0.34 for all comparisons ) as well as when restricting analysis to the period of insulin infusion ( p > 0.13 ) . serum c - peptide concentrations did not differ between conditions during insulin infusion ( p > 0.25 ) but rose to slightly elevated levels in the human insulin compared with the detemir condition at the end of experiments ( fig . 1d ) as well as leptin did not differ between conditions ( all comparisons , p > 0.40 ) . concentrations of plasma acth ( p > 0.57 ) and serum cortisol ( p > 0.71 ) were likewise similar in both conditions , with cortisol levels showing the expected meal - related increase ( f = 4.19 , p < 0.008 ) . a : rates of glucose infused to maintain euglycemia and concentrations of ( b ) blood glucose , ( c ) serum c - peptide , and ( d ) serum glucagon during intravenous infusion of human insulin ( ) and insulin detemir ( ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . at 20 min after the end of infusions , subjects ate ad libitum from a test buffet . the dc potential in the detemir condition showed a marked negative shift shortly after insulin injection that reached maximum values exceeding 600 v toward the end of the recording epoch ( fig . analyses of average dc - potential levels during the relevant interval from 21 min after the start until the end of insulin infusions confirmed a distinctly more negative potential level in the detemir than human insulin condition ( f = 7.03 , p = 0.02 ; table 2 ) , with this effect displaying an even topographic distribution ( f = 0.59 , p > 0.59 , for treatment electrode location ) . comparisons with preinjection baseline levels confirmed significance for the negative dc potential in the detemir condition during 2190 min of insulin infusion ( f = 8.53 , p = 0.02 ; f = 1.04 , p > 0.39 for electrode location ) . although dc potentials in the human insulin condition appeared to shift slightly toward positive values over central positions , respective analyses did not yield significant differences from baseline values ( f = 0.17 , p > 0.43 ; table 2 ) . average dc potentials recorded from left and right electrodes over frontal ( f3 , f4 , respectively ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas before and during intravenous infusion of human insulin ( thin lines ) and insulin detemir ( bold lines ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . in both conditions , rows of asterisks indicate significance ( p < 0.05 ; t tests ) for point - wise comparisons of the potential levels between conditions ( upper row ) and between the detemir condition and respective baseline levels ( lower row ) . dc - potential levels during euglycemic infusion of human insulin and detemir average dc - potential levels ( in v ) over left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas from 21 to 90 min of intravenous infusion of human insulin ( hi ) and insulin detemir ( det ) , respectively . the right three columns indicate significance for differences , respectively , between conditions and between the potential levels in the human insulin and detemir conditions and respective baseline levels ( t test ; n= 11 ) . detemir in comparison with human insulin significantly reduced food consumption by 303 135.7 kcal . macronutrient comparisons suggested this effect was particularly pronounced for protein and , to a lesser extent , for carbohydrate intake , but there was no significant statistical interaction between the factors treatment and macronutrients ( f = 1.63 , p > 0.22 ) . the reduction in total energy consumption and also carbohydrate intake by detemir in comparison with human insulin was also observed in analyses taking into account the energy received in the form of intravenous glucose ( table 3 ) . differences in carbohydrate and protein intake between conditions correlated significantly with respective differences in dc - potential shifts ( averaged over all recording sites ) from 21 to 90 min of infusions ( r = 0.74 , p < 0.04 , and r = 0.87 , p < 0.01 , respectively , bivariate pearson coefficients ) . the respective correlation with total energy intake failed to reach significance ( r = 0.61 , p = 0.11 ; r = 0.21 , p > 0.62 for fat intake ) . food intake after euglycemic infusion of human insulin and detemir food intake from a test buffet of 4,650 kcal offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . bottom lines indicate food consumption including the amount of energy infused as glucose to maintain euglycemia until the end of the test buffet . right column indicates significance for differences between conditions ( t test ; n = 15 ) . ten - point scale hunger ratings did not differ between conditions , showing the expected decline from baseline ( detemir , 4.87 0.68 ; human insulin , 5.93 0.54 ; p > 0.11 ) and post - eeg recording values ( 5.87 0.62 vs. 5.93 0.56 , p > 0.91 ) to low postingestion levels ( 1.47 0.43 vs. 1.00 0.24 , p > 0.33 ; p > 0.12 for treatment time ) . a comparable pattern without significant differences between conditions was observed for thirst ( all p > 0.12 ) and tiredness ( p > 0.33 ) ratings . according to the bipolar questionnaire , subjects in the detemir compared with the human insulin condition felt more critical ( critical - comfortable , 2.87 0.13 vs. 3.27 0.18 , p < 0.009 ) and reported increased wakefulness ( sleepy - awake , 3.33 0.23 vs. 2.73 0.23 , p < 0.03 ) and hunger ( full - hungry , 4.20 0.20 vs. 3.53 0.22 , p < 0.01 ) immediately after infusions . the mood adjective checklist did not yield significant differences between conditions for any of the subscales . 1a ; p > 0.14 for all comparisons ) , resulting in identical total amounts of energy supplied via glucose infusion ( human insulin , 240.8 23.3 kcal ; detemir , 239.5 25.0 kcal ; p > 0.93 ) . correspondingly , blood glucose concentrations were comparable throughout the experiment ( fig . 1b ; p > 0.34 for all comparisons ) as well as when restricting analysis to the period of insulin infusion ( p > 0.13 ) . serum c - peptide concentrations did not differ between conditions during insulin infusion ( p > 0.25 ) but rose to slightly elevated levels in the human insulin compared with the detemir condition at the end of experiments ( fig . 1d ) as well as leptin did not differ between conditions ( all comparisons , p > 0.40 ) . concentrations of plasma acth ( p > 0.57 ) and serum cortisol ( p > 0.71 ) were likewise similar in both conditions , with cortisol levels showing the expected meal - related increase ( f = 4.19 , p < 0.008 ) . a : rates of glucose infused to maintain euglycemia and concentrations of ( b ) blood glucose , ( c ) serum c - peptide , and ( d ) serum glucagon during intravenous infusion of human insulin ( ) and insulin detemir ( ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . at 20 min after the end of infusions , subjects ate ad libitum from a test buffet . the dc potential in the detemir condition showed a marked negative shift shortly after insulin injection that reached maximum values exceeding 600 v toward the end of the recording epoch ( fig . analyses of average dc - potential levels during the relevant interval from 21 min after the start until the end of insulin infusions confirmed a distinctly more negative potential level in the detemir than human insulin condition ( f = 7.03 , p = 0.02 ; table 2 ) , with this effect displaying an even topographic distribution ( f = 0.59 , p > 0.59 , for treatment electrode location ) . comparisons with preinjection baseline levels confirmed significance for the negative dc potential in the detemir condition during 2190 min of insulin infusion ( f = 8.53 , p = 0.02 ; f = 1.04 , p > 0.39 for electrode location ) . although dc potentials in the human insulin condition appeared to shift slightly toward positive values over central positions , respective analyses did not yield significant differences from baseline values ( f = 0.17 , p > 0.43 ; table 2 ) . average dc potentials recorded from left and right electrodes over frontal ( f3 , f4 , respectively ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas before and during intravenous infusion of human insulin ( thin lines ) and insulin detemir ( bold lines ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . in both conditions , rows of asterisks indicate significance ( p < 0.05 ; t tests ) for point - wise comparisons of the potential levels between conditions ( upper row ) and between the detemir condition and respective baseline levels ( lower row ) . dc - potential levels during euglycemic infusion of human insulin and detemir average dc - potential levels ( in v ) over left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas from 21 to 90 min of intravenous infusion of human insulin ( hi ) and insulin detemir ( det ) , respectively . the right three columns indicate significance for differences , respectively , between conditions and between the potential levels in the human insulin and detemir conditions and respective baseline levels ( t test ; n= 11 ) . detemir in comparison with human insulin significantly reduced food consumption by 303 135.7 kcal . macronutrient comparisons suggested this effect was particularly pronounced for protein and , to a lesser extent , for carbohydrate intake , but there was no significant statistical interaction between the factors treatment and macronutrients ( f = 1.63 , p > 0.22 ) . the reduction in total energy consumption and also carbohydrate intake by detemir in comparison with human insulin was also observed in analyses taking into account the energy received in the form of intravenous glucose ( table 3 ) . differences in carbohydrate and protein intake between conditions correlated significantly with respective differences in dc - potential shifts ( averaged over all recording sites ) from 21 to 90 min of infusions ( r = 0.74 , p < 0.04 , and r = 0.87 , p < 0.01 , respectively , bivariate pearson coefficients ) . the respective correlation with total energy intake failed to reach significance ( r = 0.61 , p = 0.11 ; r = 0.21 , p > 0.62 for fat intake ) . food intake after euglycemic infusion of human insulin and detemir food intake from a test buffet of 4,650 kcal offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . bottom lines indicate food consumption including the amount of energy infused as glucose to maintain euglycemia until the end of the test buffet . right column indicates significance for differences between conditions ( t test ; n = 15 ) . ten - point scale hunger ratings did not differ between conditions , showing the expected decline from baseline ( detemir , 4.87 0.68 ; human insulin , 5.93 0.54 ; p > 0.11 ) and post - eeg recording values ( 5.87 0.62 vs. 5.93 0.56 , p > 0.91 ) to low postingestion levels ( 1.47 0.43 vs. 1.00 0.24 , p > 0.33 ; p > 0.12 for treatment time ) . a comparable pattern without significant differences between conditions was observed for thirst ( all p > 0.12 ) and tiredness ( p > 0.33 ) ratings . according to the bipolar questionnaire , subjects in the detemir compared with the human insulin condition felt more critical ( critical - comfortable , 2.87 0.13 vs. 3.27 0.18 , p < 0.009 ) and reported increased wakefulness ( sleepy - awake , 3.33 0.23 vs. 2.73 0.23 , p < 0.03 ) and hunger ( full - hungry , 4.20 0.20 vs. 3.53 0.22 , p < 0.01 ) immediately after infusions . the remaining dimensions and the post food intake assessment were not affected . the mood adjective checklist did not yield significant differences between conditions for any of the subscales . administration of insulin to the central nervous system reduces food intake ( 3,5 ) and body weight ( 4,6 ) . we demonstrate that while eliciting comparable peripheral effects , euglycemic infusion of insulin detemir compared with human insulin triggers a distinct negative shift in eeg dc - potential recordings and reduces calorie uptake in healthy men , supporting our hypothesis that detemir affects brain functions to a greater extent than human insulin and induces stronger anorexigenic effects on central nervous networks that control food intake . this outcome suggests that enhanced catabolic insulin signaling to the brain may be an important mechanism behind the limitation of weight gain observed in diabetic patients receiving detemir treatment ( 9,1114 ) . in accordance with other investigators ( 15,17 ) , we administered insulin doses that were considerably higher in the detemir than in the human insulin condition to compensate for the delayed onset that human insulin accordingly , timing and strength of the effects of detemir and human insulin on systemic glucose homeostasis as reflected by the rates of glucose infusion as well as blood glucose and serum glucagon concentrations were identical . c - peptide concentrations likewise did not differ between conditions during insulin infusion , merely showing a slight increase in the human insulin condition after the test buffet that may have been due to greater food intake in this compared with the detemir condition . congruent peripheral effects were also indicated by comparable serum leptin concentrations that are known to respond to insulin infusion ( 27,28 ) . on the background of equipotent systemic effects , detemir elicited a marked brain response as indicated by a widespread negative shift of scalp - recorded dc potentials that started around 15 min after detemir bolus injection and exceeded potential levels in the control condition that remained unaffected by human insulin infusion . in the human insulin condition , subjects received roughly the same total amount of insulin that , when administered in single bolus form , induced a negative dc - potential shift in foregoing experiments ( 21 ) . slowly infused over the course of 90 min , this dose obviously was too weak a stimulus to evoke dc - potential responses to human insulin in the present experiments . in contrast , the 90-min infusion of a detemir dose equivalent in terms of systemic action triggered a sustained negative dc - potential shift comparable with the previously reported effect of human insulin administered in high - dose bolus form ( 21 ) . this pattern indicates that although central nervous detemir effects may be mimicked by disproportionally high doses of human insulin , the relative impact of detemir on brain functions is considerably greater when both insulins are administered at doses with similar peripheral impact . the mechanisms behind the strong effect of detemir on scalp - recorded dc potentials can not be derived from our data . brain dc - potential shifts of this amplitude most likely reflect changes in extracellular ionic concentrations stemming from potential shifts at glial membranes that are endowed with receptors for insulin and igf ( 21,2931 ) . the assumption of a widespread effect on cerebral cellular networks also fits with the global nature and long duration of the dc - potential shifts induced by detemir infusion . our observations corroborate previous findings of increased brain responses to detemir in comparison with human insulin in animals ( 32 ) and humans ( 1517 ) . superior central nervous efficacy of detemir may be due to improved permeation of the lipophilic molecule into the brain compartment , with enhanced receptor - mediated blood - brain barrier transport of albumin - bound detemir adding to this effect ( 15,32 ) . the detemir - induced negative dc - potential shift that is presumably of primary glial origin thus may be reinforced by electrical potentials generated in the course of receptor - mediated detemir transport across the blood - brain barrier ( 33 ) . a most remarkable finding of our study is the reduction of ad libitum food intake by around 300 kcal in the detemir compared with the human insulin condition in the presence of identical peripheral actions of both insulins . this pattern renders the contribution of systemic mediators to this effect highly unlikely , rather suggesting that enhanced central nervous insulin signaling in the detemir condition resulted in decreased caloric intake . the concept of insulin providing catabolic feedback on the body 's energy resources to brain networks that control energy homeostasis has been well established in animals ( 2,3 ) as well as in humans ( 5,6 ) . thus , the decrease in food intake after detemir compared with human insulin infusion suggests that the enhanced effect on brain functions indicated by the negative dc - potential shift particularly impacts the central nervous control of food intake . although because of methodological constraints , dc - eeg could not be recorded during food intake proper ( 34 ) , this interpretation is supported by the strong correlation between the reduction in calorie intake and the antecedent dc - potential effect elicited by detemir . it is also in line with animal experiments in which intravenous detemir compared with human insulin injections were associated with enhanced insulin receptor phosphorylation in hypothalamic and cerebrocortical tissue in conjunction with increased eeg - assessed cortical activity , whereas the activation of the insulin receptor signaling cascade was similar in muscle tissue and liver ( 32 ) . interestingly , detemir compared with human insulin infusion increased rather than decreased self - rated hunger before test buffet presentation , which may have been due to a biasing influence of enhanced wakefulness and activation after detemir infusion ( 35 ) . alternatively , this finding may also indicate that central nervous insulin exerts its anorexigenic effects via meal - related signals that contribute to the termination of a meal , but not by affecting hunger motivation per se ( 5 ) . weight gain is a frequent side effect when blood glucose levels of diabetic patients are normalized by insulin ( 36,37 ) , but is less pronounced in patients undergoing detemir therapy ( 9,1114 ) . the assumption that the use of detemir limits weight gain because its favorable safety profile decreases defensive snacking to prevent hypoglycemia was not supported by comparative clinical studies showing that insulin glargine , which like detemir reduces the risk of hypoglycemia , is associated with greater weight gain than detemir ( 38,39 ) . against this background , the anorexigenic brain impact of detemir found in our study rather suggests the contribution of central nervous mechanisms to the weight - sparing effect of detemir in the clinical context .

objectivein the treatment of diabetic patients , the long - acting insulin analog insulin detemir is less prone to induce weight gain than other insulin formulations . assuming that because of its pharmacologic properties , detemir displays stronger central nervous anorexigenic efficacy than human insulin , we compared acute effects of human insulin and detemir on electroencephalography ( eeg ) measures and food intake.research design and methodsfrontocortical eeg direct current ( dc ) potentials were recorded in 15 healthy men during two hyperinsulinemic - euglycemic clamps that included an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg1 min1 ) . a higher dosage was chosen for detemir to compensate for its delay in impact relative to human insulin and to elicit similar systemic effects . at 20 min after infusion , subjects were allowed to eat ad libitum from a test buffet.resultsmean glucose infusions to maintain euglycemia ( p > 0.93 ) and blood glucose concentrations ( p > 0.34 ) did not differ between conditions . detemir infusion induced a negative dc - potential shift , averaging 372.2 v from 21 to 90 min that was not observed during human insulin infusion ( 146.5 v , p = 0.02 ) . detemir , in comparison with human insulin , reduced subsequent food intake by 303 kcal ( 1,257 vs. 1,560 , p < 0.04).conclusionswhile inducing comparable peripheral effects , detemir exerts stronger acute effects on brain functions than human insulin and triggers a relative decrease in food consumption , suggesting an enhanced anorexigenic impact of detemir compared with human insulin on central nervous networks that control nutrient uptake .

RESEARCH DESIGN AND METHODS Subjects and design. Procedure. Food intake and mood assessment. Recordings. Statistical analysis. RESULTS Glucose infusion rates, blood glucose, and hormonal parameters. Negative DC-potential shift during detemir infusion. Reduction of food intake by detemir in comparison with human insulin. Rating scales. DISCUSSION

according to a single - blind , within - subject comparison , 15 healthy , normal - weight men ( mean age se , 28.5 1.0 years ; bmi , 23.1 0.5 kg / m ) participated in two hyperinsulinemic - euglycemic clamp experiments ( human insulin , detemir ) spaced apart at least 1 week . in both experimental conditions , an insulin bolus injection ( 17.75 mu / kg body wt [ = 0.1065 nmol / kg body wt ] human insulin , insulin actrapid ; novo nordisk , bagsvrd , denmark ; 90 mu / kg body wt [ = 2.16 nmol / kg body wt ] detemir , insulin levemir ; novo nordisk ) was given followed by a steady 90-min infusion of 1.0 mu kg min (= 0.006 nmol kg min ) human insulin versus 2.0 mu kg min (= 0.048 nmol kg min ) detemir . to compensate for the relatively slower onset of the action of detemir , a higher dosage was chosen ( 15,17 ) to induce comparable peripheral effects of both compounds as assessed by rates of glucose infusion necessary to keep blood glucose levels in the euglycemic range . recordings of dc potentials started at 0940 with a baseline phase of 20 min followed by the bolus injection of human insulin and detemir , respectively ( t = 0 ) . , 20 min after insulin infusion had stopped ) , a standardized buffet of around 4,650 kcal was offered from which subjects were allowed to eat ad libitum during the subsequent 50 min ( table 1 ) . composition of the test buffet composition of the buffet offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . according to a single - blind , within - subject comparison , 15 healthy , normal - weight men ( mean age se , 28.5 1.0 years ; bmi , 23.1 0.5 kg / m ) participated in two hyperinsulinemic - euglycemic clamp experiments ( human insulin , detemir ) spaced apart at least 1 week . in both experimental conditions , an insulin bolus injection ( 17.75 mu / kg body wt [ = 0.1065 nmol / kg body wt ] human insulin , insulin actrapid ; novo nordisk , bagsvrd , denmark ; 90 mu / kg body wt [ = 2.16 nmol / kg body wt ] detemir , insulin levemir ; novo nordisk ) was given followed by a steady 90-min infusion of 1.0 mu kg min (= 0.006 nmol kg min ) human insulin versus 2.0 mu kg min (= 0.048 nmol kg min ) detemir . to compensate for the relatively slower onset of the action of detemir , a higher dosage was chosen ( 15,17 ) to induce comparable peripheral effects of both compounds as assessed by rates of glucose infusion necessary to keep blood glucose levels in the euglycemic range . recordings of dc potentials started at 0940 with a baseline phase of 20 min followed by the bolus injection of human insulin and detemir , respectively ( t = 0 ) . , 20 min after insulin infusion had stopped ) , a standardized buffet of around 4,650 kcal was offered from which subjects were allowed to eat ad libitum during the subsequent 50 min ( table 1 ) . composition of the test buffet composition of the buffet offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . 1a ; p > 0.14 for all comparisons ) , resulting in identical total amounts of energy supplied via glucose infusion ( human insulin , 240.8 23.3 kcal ; detemir , 239.5 25.0 kcal ; p > 0.93 ) . 1b ; p > 0.34 for all comparisons ) as well as when restricting analysis to the period of insulin infusion ( p > 0.13 ) . serum c - peptide concentrations did not differ between conditions during insulin infusion ( p > 0.25 ) but rose to slightly elevated levels in the human insulin compared with the detemir condition at the end of experiments ( fig . 1d ) as well as leptin did not differ between conditions ( all comparisons , p > 0.40 ) . concentrations of plasma acth ( p > 0.57 ) and serum cortisol ( p > 0.71 ) were likewise similar in both conditions , with cortisol levels showing the expected meal - related increase ( f = 4.19 , p < 0.008 ) . a : rates of glucose infused to maintain euglycemia and concentrations of ( b ) blood glucose , ( c ) serum c - peptide , and ( d ) serum glucagon during intravenous infusion of human insulin ( ) and insulin detemir ( ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . at 20 min after the end of infusions , subjects ate ad libitum from a test buffet . analyses of average dc - potential levels during the relevant interval from 21 min after the start until the end of insulin infusions confirmed a distinctly more negative potential level in the detemir than human insulin condition ( f = 7.03 , p = 0.02 ; table 2 ) , with this effect displaying an even topographic distribution ( f = 0.59 , p > 0.59 , for treatment electrode location ) . comparisons with preinjection baseline levels confirmed significance for the negative dc potential in the detemir condition during 2190 min of insulin infusion ( f = 8.53 , p = 0.02 ; f = 1.04 , p > 0.39 for electrode location ) . although dc potentials in the human insulin condition appeared to shift slightly toward positive values over central positions , respective analyses did not yield significant differences from baseline values ( f = 0.17 , p > 0.43 ; table 2 ) . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . in both conditions , rows of asterisks indicate significance ( p < 0.05 ; t tests ) for point - wise comparisons of the potential levels between conditions ( upper row ) and between the detemir condition and respective baseline levels ( lower row ) . dc - potential levels during euglycemic infusion of human insulin and detemir average dc - potential levels ( in v ) over left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas from 21 to 90 min of intravenous infusion of human insulin ( hi ) and insulin detemir ( det ) , respectively . detemir in comparison with human insulin significantly reduced food consumption by 303 135.7 kcal . the reduction in total energy consumption and also carbohydrate intake by detemir in comparison with human insulin was also observed in analyses taking into account the energy received in the form of intravenous glucose ( table 3 ) . differences in carbohydrate and protein intake between conditions correlated significantly with respective differences in dc - potential shifts ( averaged over all recording sites ) from 21 to 90 min of infusions ( r = 0.74 , p < 0.04 , and r = 0.87 , p < 0.01 , respectively , bivariate pearson coefficients ) . the respective correlation with total energy intake failed to reach significance ( r = 0.61 , p = 0.11 ; r = 0.21 , p > 0.62 for fat intake ) . food intake after euglycemic infusion of human insulin and detemir food intake from a test buffet of 4,650 kcal offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . ten - point scale hunger ratings did not differ between conditions , showing the expected decline from baseline ( detemir , 4.87 0.68 ; human insulin , 5.93 0.54 ; p > 0.11 ) and post - eeg recording values ( 5.87 0.62 vs. 5.93 0.56 , p > 0.91 ) to low postingestion levels ( 1.47 0.43 vs. 1.00 0.24 , p > 0.33 ; p > 0.12 for treatment time ) . a comparable pattern without significant differences between conditions was observed for thirst ( all p > 0.12 ) and tiredness ( p > 0.33 ) ratings . according to the bipolar questionnaire , subjects in the detemir compared with the human insulin condition felt more critical ( critical - comfortable , 2.87 0.13 vs. 3.27 0.18 , p < 0.009 ) and reported increased wakefulness ( sleepy - awake , 3.33 0.23 vs. 2.73 0.23 , p < 0.03 ) and hunger ( full - hungry , 4.20 0.20 vs. 3.53 0.22 , p < 0.01 ) immediately after infusions . 1a ; p > 0.14 for all comparisons ) , resulting in identical total amounts of energy supplied via glucose infusion ( human insulin , 240.8 23.3 kcal ; detemir , 239.5 25.0 kcal ; p > 0.93 ) . 1b ; p > 0.34 for all comparisons ) as well as when restricting analysis to the period of insulin infusion ( p > 0.13 ) . serum c - peptide concentrations did not differ between conditions during insulin infusion ( p > 0.25 ) but rose to slightly elevated levels in the human insulin compared with the detemir condition at the end of experiments ( fig . 1d ) as well as leptin did not differ between conditions ( all comparisons , p > 0.40 ) . concentrations of plasma acth ( p > 0.57 ) and serum cortisol ( p > 0.71 ) were likewise similar in both conditions , with cortisol levels showing the expected meal - related increase ( f = 4.19 , p < 0.008 ) . a : rates of glucose infused to maintain euglycemia and concentrations of ( b ) blood glucose , ( c ) serum c - peptide , and ( d ) serum glucagon during intravenous infusion of human insulin ( ) and insulin detemir ( ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . at 20 min after the end of infusions , subjects ate ad libitum from a test buffet . analyses of average dc - potential levels during the relevant interval from 21 min after the start until the end of insulin infusions confirmed a distinctly more negative potential level in the detemir than human insulin condition ( f = 7.03 , p = 0.02 ; table 2 ) , with this effect displaying an even topographic distribution ( f = 0.59 , p > 0.59 , for treatment electrode location ) . comparisons with preinjection baseline levels confirmed significance for the negative dc potential in the detemir condition during 2190 min of insulin infusion ( f = 8.53 , p = 0.02 ; f = 1.04 , p > 0.39 for electrode location ) . although dc potentials in the human insulin condition appeared to shift slightly toward positive values over central positions , respective analyses did not yield significant differences from baseline values ( f = 0.17 , p > 0.43 ; table 2 ) . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . in both conditions , rows of asterisks indicate significance ( p < 0.05 ; t tests ) for point - wise comparisons of the potential levels between conditions ( upper row ) and between the detemir condition and respective baseline levels ( lower row ) . dc - potential levels during euglycemic infusion of human insulin and detemir average dc - potential levels ( in v ) over left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas from 21 to 90 min of intravenous infusion of human insulin ( hi ) and insulin detemir ( det ) , respectively . the right three columns indicate significance for differences , respectively , between conditions and between the potential levels in the human insulin and detemir conditions and respective baseline levels ( t test ; n= 11 ) . detemir in comparison with human insulin significantly reduced food consumption by 303 135.7 kcal . the reduction in total energy consumption and also carbohydrate intake by detemir in comparison with human insulin was also observed in analyses taking into account the energy received in the form of intravenous glucose ( table 3 ) . differences in carbohydrate and protein intake between conditions correlated significantly with respective differences in dc - potential shifts ( averaged over all recording sites ) from 21 to 90 min of infusions ( r = 0.74 , p < 0.04 , and r = 0.87 , p < 0.01 , respectively , bivariate pearson coefficients ) . the respective correlation with total energy intake failed to reach significance ( r = 0.61 , p = 0.11 ; r = 0.21 , p > 0.62 for fat intake ) . food intake after euglycemic infusion of human insulin and detemir food intake from a test buffet of 4,650 kcal offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . ten - point scale hunger ratings did not differ between conditions , showing the expected decline from baseline ( detemir , 4.87 0.68 ; human insulin , 5.93 0.54 ; p > 0.11 ) and post - eeg recording values ( 5.87 0.62 vs. 5.93 0.56 , p > 0.91 ) to low postingestion levels ( 1.47 0.43 vs. 1.00 0.24 , p > 0.33 ; p > 0.12 for treatment time ) . a comparable pattern without significant differences between conditions was observed for thirst ( all p > 0.12 ) and tiredness ( p > 0.33 ) ratings . according to the bipolar questionnaire , subjects in the detemir compared with the human insulin condition felt more critical ( critical - comfortable , 2.87 0.13 vs. 3.27 0.18 , p < 0.009 ) and reported increased wakefulness ( sleepy - awake , 3.33 0.23 vs. 2.73 0.23 , p < 0.03 ) and hunger ( full - hungry , 4.20 0.20 vs. 3.53 0.22 , p < 0.01 ) immediately after infusions . we demonstrate that while eliciting comparable peripheral effects , euglycemic infusion of insulin detemir compared with human insulin triggers a distinct negative shift in eeg dc - potential recordings and reduces calorie uptake in healthy men , supporting our hypothesis that detemir affects brain functions to a greater extent than human insulin and induces stronger anorexigenic effects on central nervous networks that control food intake . in accordance with other investigators ( 15,17 ) , we administered insulin doses that were considerably higher in the detemir than in the human insulin condition to compensate for the delayed onset that human insulin accordingly , timing and strength of the effects of detemir and human insulin on systemic glucose homeostasis as reflected by the rates of glucose infusion as well as blood glucose and serum glucagon concentrations were identical . c - peptide concentrations likewise did not differ between conditions during insulin infusion , merely showing a slight increase in the human insulin condition after the test buffet that may have been due to greater food intake in this compared with the detemir condition . on the background of equipotent systemic effects , detemir elicited a marked brain response as indicated by a widespread negative shift of scalp - recorded dc potentials that started around 15 min after detemir bolus injection and exceeded potential levels in the control condition that remained unaffected by human insulin infusion . in the human insulin condition , subjects received roughly the same total amount of insulin that , when administered in single bolus form , induced a negative dc - potential shift in foregoing experiments ( 21 ) . slowly infused over the course of 90 min , this dose obviously was too weak a stimulus to evoke dc - potential responses to human insulin in the present experiments . in contrast , the 90-min infusion of a detemir dose equivalent in terms of systemic action triggered a sustained negative dc - potential shift comparable with the previously reported effect of human insulin administered in high - dose bolus form ( 21 ) . this pattern indicates that although central nervous detemir effects may be mimicked by disproportionally high doses of human insulin , the relative impact of detemir on brain functions is considerably greater when both insulins are administered at doses with similar peripheral impact . our observations corroborate previous findings of increased brain responses to detemir in comparison with human insulin in animals ( 32 ) and humans ( 1517 ) . the detemir - induced negative dc - potential shift that is presumably of primary glial origin thus may be reinforced by electrical potentials generated in the course of receptor - mediated detemir transport across the blood - brain barrier ( 33 ) . a most remarkable finding of our study is the reduction of ad libitum food intake by around 300 kcal in the detemir compared with the human insulin condition in the presence of identical peripheral actions of both insulins . thus , the decrease in food intake after detemir compared with human insulin infusion suggests that the enhanced effect on brain functions indicated by the negative dc - potential shift particularly impacts the central nervous control of food intake . although because of methodological constraints , dc - eeg could not be recorded during food intake proper ( 34 ) , this interpretation is supported by the strong correlation between the reduction in calorie intake and the antecedent dc - potential effect elicited by detemir . interestingly , detemir compared with human insulin infusion increased rather than decreased self - rated hunger before test buffet presentation , which may have been due to a biasing influence of enhanced wakefulness and activation after detemir infusion ( 35 ) . weight gain is a frequent side effect when blood glucose levels of diabetic patients are normalized by insulin ( 36,37 ) , but is less pronounced in patients undergoing detemir therapy ( 9,1114 ) . against this background , the anorexigenic brain impact of detemir found in our study rather suggests the contribution of central nervous mechanisms to the weight - sparing effect of detemir in the clinical context .

brain metastases ( bms ) have been reported in up to 40% of patients with systemic cancer21 ) , and the incidence of bms is increasing due to the routine brain magnetic resonance imaging ( mri ) screening and an improved outcome of systemic therapy against primary cancers . management of bms depends on their size , number , and location together with patient factors such as age , performance status , and primary disease status12 ) . stereotactic radiosurgery ( srs ) , typically delivered in a single fraction , has been shown to be effective and safe in treating bms and , is generally indicated for a single or oligometastases <3 cm in diameter . however , the toxicity of srs given in a single fraction is considered to outweigh the benefits of local tumor control ( ltc ) for large bms > 3 cm and leads to increased risks of neurological morbidity from radiation necrosis ( rn)31823 ) . although large lesions are often amenable to microsurgical resection , surgery is infeasible in cases of critical location and/or poor patient medical status . recently , the concept of fractionated stereotactic radiosurgery ( fsrs ) has emerged , and it is reportedly an effective and safe way to treat bms , especially large lesions . since june 2011 , we adopted this approach in treating large bms . treatment outcomes were evaluated by the objective tumor response on mri , patient survival and functional improvement , and radiation necrosis . this study was approved by the institutional review board of the asan medical center . between june 2011 and december 2013 , a total of 37 patients with large bms were enrolled according to the inclusion and exclusion criteria indicated below . 1 ) age of 18 years , with histologically proven solid cancer and fewer than 6 brain metastases , one of which is > 3 cm in maximum diameter 2 ) intact cognitive function ; able to understand and sign a written informed consent 3 ) life expectancy > 3 months , as indicated by the medical oncologist 4 ) karnofsky performance status ( kps ) score 70 , or 50 - 60 with focal neurological deficits 1 ) suffering from significant mass effect or raised intracranial pressure for which surgical decompression is indicated 2 ) received any form of prior cranial irradiation 3 ) received prior surgical resection of the targeted lesion 4 ) primary hematologic malignancy , such as lymphoma or leukemia 5 ) pregnant or breast - feeding patients the baseline characteristics of the study patients are summarized in table 1 . of the 37 patients included in the analyses , bms originated from the lung ( n=19 , 51.4% ) , gastrointestinal tract ( n=10 , 27.0% ) , breast ( n=5 , 13.5% ) , and other tissues ( n=3 , 8.1% ) . at the time of fsrs , the primary cancer was under control in 21 patients ( 56.8% ) , and metastases to organs other than the brain were present in 26 patients ( 70.3% ) . the kps score was 70 in 30 patients ( 81.1% ) , and focal neurologic deficits were present in 17 patients ( 45.9% ) . there were 7 patients ( 18.9% ) of radiation therapy oncology group - recursive partitioning analysis ( rtog - rpa ) class i , 23 ( 62.2% ) of class ii , and 7 ( 18.9% ) of class iii . the diagnosis - specific graded prognostic assessment ( ds - gpa ) score was 1 in 14 patients ( 37.8% ) , 1.5 - 2.5 in 18 patients ( 48.6% ) , and 3 in 5 patients ( 13.5% ) . of 79 bms in 37 patients , lesions <3 cm were treated with single - fraction srs and were not included in our analysis . bms were located in the cerebral hemisphere in 26 lesions ( 68.4% ) , the cerebellum in 9 lesions ( 23.7% ) , and the basal ganglia or the diencephalon in 3 lesions ( 7.9% ) ( table 1 ) . mr images including axial / sagittal / coronal t2-weighted sequences ( 2 mm slices ) and 3d - t1-weighted gadolinium enhanced sequences ( 2 mm slices ) were also obtained . the mris were registered and manually fused with the planning ct images in the accuray multiplan treatment - planning system ( version 4.5 ) to facilitate the delineation of the gross tumor volume ( gtv ; equal to the planning target volume ) and the critical organ structures including the brainstem , the eyes , and the optic apparatus . additional hollow structures ( shells ) 3 mm and 30 mm away from the gtv were generated to optimize the conformity and dose compactness . a sequential optimization method was used along with the dose objectives to optimize minimum gtv dose and coverage . the prescription isodose percentage was applied to approximately 80% of the maximum dose with a conformity index ( ci , defined as the prescribed isodose volume divided by the volume of tumor encompassed by the prescription isodose volume ) < 1.2 and gtv coverage > 99% . the median prescription dose was 35 gy ( range , 30 - 41 gy ) . doses were administered in 3 to 5 daily fractions depending on the size of lesions ; lesions < 3.5 cm were treated in 3 fractions and lesions 3.5 cm in 5 fractions . tumor size was defined as the largest cross - sectional area at follow - up mri . each lesion was measured to evaluate local tumor response and graded using the macdonald criteria29 ) . complete response was indicated by a complete disappearance of all enhancing lesions on mri , no corticosteroid use , and clinical stability or improvement . partial response was indicated by > 50% decrease from the baseline in perpendicular diameter product sums of all measurable enhancing lesions on mri , elimination or reduction in corticosteroid dose , and clinical stability or improvement . progressive disease was indicated by > 25% increase in perpendicular diameter product sums of enhancing lesions on mri , appearance of a new lesion , or clinical deterioration . patients were considered to have stable disease if they did not meet the qualifications for complete response , partial response , or progression . the following criteria were considered for rn : 1 ) increased t1 contrast enhancement located in the irradiated area with central hypointensity and increased peripheral edema , 2 ) substantial regression or stability ( for at least 3 months ) of enhancing areas on serial follow - up mris without additional treatment , or 3 ) absence of perfusion within the contrast - enhancing lesion on dynamic susceptibility contrast perfusion mri19 ) . all radiation toxicities were graded using national cancer institute common terminology criteria for adverse events ( ctcae version 4.0 ) . ltc , overall survival ( os ) , progression - free survival ( pfs ) , and rn were estimated using the kaplan - meier method calculated from the treatment start date to the date of events or the last follow - up . factors possibly affecting the outcome were tested using the log - rank test for univariate analysis and the cox proportional hazards models with variable selection , which included age ( 65 years vs. < 65 years ) , gender , primary cancer type , tumor location , tumor volume ( < 22 cc vs. 22 cc ) , single vs. multiple bms , status of primary cancer , presence of extracranial metastases , pretreatment kps score ( 70 vs. < 70 ) , rotg - rpa class , ds - gpa score , and prescription dose . this study was approved by the institutional review board of the asan medical center . between june 2011 and december 2013 , a total of 37 patients with large bms were enrolled according to the inclusion and exclusion criteria indicated below . 1 ) age of 18 years , with histologically proven solid cancer and fewer than 6 brain metastases , one of which is > 3 cm in maximum diameter 2 ) intact cognitive function ; able to understand and sign a written informed consent 3 ) life expectancy > 3 months , as indicated by the medical oncologist 4 ) karnofsky performance status ( kps ) score 70 , or 50 - 60 with focal neurological deficits 1 ) suffering from significant mass effect or raised intracranial pressure for which surgical decompression is indicated 2 ) received any form of prior cranial irradiation 3 ) received prior surgical resection of the targeted lesion 4 ) primary hematologic malignancy , such as lymphoma or leukemia 5 ) pregnant or breast - feeding patients the baseline characteristics of the study patients are summarized in table 1 . of the 37 patients included in the analyses , bms originated from the lung ( n=19 , 51.4% ) , gastrointestinal tract ( n=10 , 27.0% ) , breast ( n=5 , 13.5% ) , and other tissues ( n=3 , 8.1% ) . at the time of fsrs , the primary cancer was under control in 21 patients ( 56.8% ) , and metastases to organs other than the brain were present in 26 patients ( 70.3% ) . the kps score was 70 in 30 patients ( 81.1% ) , and focal neurologic deficits were present in 17 patients ( 45.9% ) . there were 7 patients ( 18.9% ) of radiation therapy oncology group - recursive partitioning analysis ( rtog - rpa ) class i , 23 ( 62.2% ) of class ii , and 7 ( 18.9% ) of class iii . the diagnosis - specific graded prognostic assessment ( ds - gpa ) score was 1 in 14 patients ( 37.8% ) , 1.5 - 2.5 in 18 patients ( 48.6% ) , and 3 in 5 patients ( 13.5% ) of 79 bms in 37 patients , 38 lesions were > 3 cm in diameter and were treated with fsrs . lesions <3 cm were treated with single - fraction srs and were not included in our analysis . bms were located in the cerebral hemisphere in 26 lesions ( 68.4% ) , the cerebellum in 9 lesions ( 23.7% ) , and the basal ganglia or the diencephalon in 3 lesions ( 7.9% ) ( table 1 ) . mr images including axial / sagittal / coronal t2-weighted sequences ( 2 mm slices ) and 3d - t1-weighted gadolinium enhanced sequences ( 2 mm slices ) were also obtained . the mris were registered and manually fused with the planning ct images in the accuray multiplan treatment - planning system ( version 4.5 ) to facilitate the delineation of the gross tumor volume ( gtv ; equal to the planning target volume ) and the critical organ structures including the brainstem , the eyes , and the optic apparatus . additional hollow structures ( shells ) 3 mm and 30 mm away from the gtv were generated to optimize the conformity and dose compactness . a sequential optimization method was used along with the dose objectives to optimize minimum gtv dose and coverage . the prescription isodose percentage was applied to approximately 80% of the maximum dose with a conformity index ( ci , defined as the prescribed isodose volume divided by the volume of tumor encompassed by the prescription isodose volume ) < 1.2 and gtv coverage > 99% . the median prescription dose was 35 gy ( range , 30 - 41 gy ) . doses were administered in 3 to 5 daily fractions depending on the size of lesions ; lesions < 3.5 cm were treated in 3 fractions and lesions 3.5 cm in 5 fractions . tumor size was defined as the largest cross - sectional area at follow - up mri . each lesion was measured to evaluate local tumor response and graded using the macdonald criteria29 ) . complete response was indicated by a complete disappearance of all enhancing lesions on mri , no corticosteroid use , and clinical stability or improvement . partial response was indicated by > 50% decrease from the baseline in perpendicular diameter product sums of all measurable enhancing lesions on mri , elimination or reduction in corticosteroid dose , and clinical stability or improvement . progressive disease was indicated by > 25% increase in perpendicular diameter product sums of enhancing lesions on mri , appearance of a new lesion , or clinical deterioration . patients were considered to have stable disease if they did not meet the qualifications for complete response , partial response , or progression . the following criteria were considered for rn : 1 ) increased t1 contrast enhancement located in the irradiated area with central hypointensity and increased peripheral edema , 2 ) substantial regression or stability ( for at least 3 months ) of enhancing areas on serial follow - up mris without additional treatment , or 3 ) absence of perfusion within the contrast - enhancing lesion on dynamic susceptibility contrast perfusion mri19 ) . all radiation toxicities were graded using national cancer institute common terminology criteria for adverse events ( ctcae version 4.0 ) . ltc , overall survival ( os ) , progression - free survival ( pfs ) , and rn were estimated using the kaplan - meier method calculated from the treatment start date to the date of events or the last follow - up . factors possibly affecting the outcome were tested using the log - rank test for univariate analysis and the cox proportional hazards models with variable selection , which included age ( 65 years vs. < 65 years ) , gender , primary cancer type , tumor location , tumor volume ( < 22 cc vs. 22 cc ) , single vs. multiple bms , status of primary cancer , presence of extracranial metastases , pretreatment kps score ( 70 vs. < 70 ) , rotg - rpa class , ds - gpa score , and prescription dose . the maximum tumor response was evaluated for 36 lesions after exclusion of two lesions for which no follow - up images were available . the rates of complete response , partial response , stable disease , and progressive disease were 11.1% , 44.4% , 30.6% , and 13.9% , respectively . with a median follow - up of 10 months ( range , 1 - 37 months ) , the crude ltc rate was 86.8% and the estimated ltc rates at 12 and 24 months were 87.0% and 65.2% , respectively ( fig . 1 ) . prescription dose was the only factor affecting the ltc on univariate and multivariate analysis . both of the lesions treated with a prescription dose of 31 gy developed local failure , whereas only 3 of 36 lesions with a prescription dose of 35 gy developed local failure ( hazard ratio , 49.26 ; 95% confidence interval , 6.897 - 352.128 ; p<0.001 ) ( table 2 ) . the median os was 16 months , and the estimated os rates at 6 , 12 and 18 months were 81.1% , 56.8% , and 40.7% , respectively . of 21 patients who died , 10 ( 47.6% ) died from the progression of extracranial disease , 6 ( 28.6% ) from brain failure , and 5 ( 23.8% ) from unknown causes . on univariate analysis , kps score < 70 ( hazard ratio , 3.389 ; 95% confidence interval , 1.317 - 8.721 ; p=0.011 ) and rtog - rpa class iii ( hazard ratio , 5.26 ; 95% confidence interval , 1.328 - 20.886 ; p=0.018 ) indicated poor patient survival ( table 3 , fig . twenty - one patients ( 56.8% ) showed progression including distant failure in 20 patients , local failure in 5 patients , and both distant and local failure in 4 patients . the median pfs was 11 months and the estimated pfs rates at 6 , 12 , and 18 months were 65.5% , 44.9% , and 25.7% , respectively . the cumulative incidence function ( cif ) for progression is shown in fig . multiple bms were associated with poor pfs ( hazard ratio , 2.603 ; 95% confidence interval , 1.027 - 6.598 ; p=0.044 ) ( table 3 ) , as 14 of 19 patients ( 73.7% ) with multiple bms developed progression vs. 7 of 18 ( 38.8% ) with a single bm . preoperative focal neurologic deficits such as motor weakness and cerebellar dysfunction , improved in 12 of 17 patients ( 70.6% ) 3 months after treatment . the kps score improved in 20 of 35 patients ( 57.1% ) , with a mean preoperative kps score of 74 ( median , 70 ; range , 50 - 100 ) vs. a mean kps score of 80.6 ( median , 80 ; range , 50 - 100 ) 3 months after treatment ( p=0.001 ) ( fig . 5 ) . the median time to rn was 10.5 months ( range , 6 - 18 months ) . five patients with rn of toxicity grade 2 were controlled with corticosteroid medication and 1 patient with toxicity grade 3 was salvaged by surgery . the maximum tumor response was evaluated for 36 lesions after exclusion of two lesions for which no follow - up images were available . the rates of complete response , partial response , stable disease , and progressive disease were 11.1% , 44.4% , 30.6% , and 13.9% , respectively . with a median follow - up of 10 months ( range , 1 - 37 months ) , the crude ltc rate was 86.8% and the estimated ltc rates at 12 and 24 months were 87.0% and 65.2% , respectively ( fig . 1 ) . prescription dose was the only factor affecting the ltc on univariate and multivariate analysis . both of the lesions treated with a prescription dose of 31 gy developed local failure , whereas only 3 of 36 lesions with a prescription dose of 35 gy developed local failure ( hazard ratio , 49.26 ; 95% confidence interval , 6.897 - 352.128 ; p<0.001 ) ( table 2 ) . 2 . the median os was 16 months , and the estimated os rates at 6 , 12 and 18 months were 81.1% , 56.8% , and 40.7% , respectively . of 21 patients who died , 10 ( 47.6% ) died from the progression of extracranial disease , 6 ( 28.6% ) from brain failure , and 5 ( 23.8% ) from unknown causes . on univariate analysis , kps score < 70 ( hazard ratio , 3.389 ; 95% confidence interval , 1.317 - 8.721 ; p=0.011 ) and rtog - rpa class iii ( hazard ratio , 5.26 ; 95% confidence interval , 1.328 - 20.886 ; p=0.018 ) indicated poor patient survival ( table 3 , fig . twenty - one patients ( 56.8% ) showed progression including distant failure in 20 patients , local failure in 5 patients , and both distant and local failure in 4 patients . the median pfs was 11 months and the estimated pfs rates at 6 , 12 , and 18 months were 65.5% , 44.9% , and 25.7% , respectively . multiple bms were associated with poor pfs ( hazard ratio , 2.603 ; 95% confidence interval , 1.027 - 6.598 ; p=0.044 ) ( table 3 ) , as 14 of 19 patients ( 73.7% ) with multiple bms developed progression vs. 7 of 18 ( 38.8% ) with a single bm . preoperative focal neurologic deficits such as motor weakness and cerebellar dysfunction , improved in 12 of 17 patients ( 70.6% ) 3 months after treatment . the kps score improved in 20 of 35 patients ( 57.1% ) , with a mean preoperative kps score of 74 ( median , 70 ; range , 50 - 100 ) vs. a mean kps score of 80.6 ( median , 80 ; range , 50 - 100 ) 3 months after treatment ( p=0.001 ) ( fig . 5 ) . the median time to rn was 10.5 months ( range , 6 - 18 months ) . five patients with rn of toxicity grade 2 were controlled with corticosteroid medication and 1 patient with toxicity grade 3 was salvaged by surgery . although srs typically delivered in a single fraction has been proven to be effective and safe in treating bms , it is not feasible for large lesions , especially those > 3.0 cm , due to increased toxicity and local treatment failure3182324 ) . microsurgical resection is usually indicated for large bms , immediately decompressing the mass effect and alleviating neurological symptoms . however , not all patients with large bms are eligible for surgery when considering surgical accessibility , the number of lesions , and patient medical status1733 ) . moreover , systemic therapy against primary cancers should be withheld during perioperative periods , which can be further confounded by surgical morbidity in certain cases . alternatively , whole - brain radiotherapy ( wbrt ) remains palliative in nature and may influence cognitive function . currently , the first - line treatment for large bms has not been established and is usually determined by considering various factors , including tumor volume , number , location , and overall condition of patient12 ) . theoretically , fractionated administration of radiation dose potentially minimizes toxicity to late - responding healthy tissues , with a low / ratio compared to a single acute dose of radiation for a given level of tumor damage , according to the linear quadratic model of cellular survival826 ) . in addition , reoxygenation and redistribution of the cell cycle between dose fractions renders hypoxic tumor cells , which are abundant in large bms compared to small tumors , more radiosensitive6142225 ) . as expected , recently published studies on fsrs for large bms have demonstrated high ltc rates , ranging from 63 - 100% , at 1 year follow - up , with acceptable risks of toxicity1410141531 ) ( table 4 ) . consistent with these results , our present ltc rates were 87.0% and 65.2% at 1- and 2-years follow - up , respectively , and the median os was 16 months , which also compares well with the outcomes of single - fraction srs for small bms2111316 ) . furthermore , patient performance status and neurological function improved significantly , presumably benefitting the quality of life in these cases . the optimal dose fractionation protocol for fsrs in bms has not yet been established . in a recent systematic review on stereotactic radiotherapy dose and ltc probability , wiggenraad et al.30 ) reported that a biological effective dose , using an / ratio of 12 ( bed12 ) , of at least 40 gy , which correspond to a single fraction dose of 20 gy , was associated with a 1-year ltc rate of 70% or more . the high ltc rate at 1-year follow - up in our present study appears to accord with this observation . vogelbaum et al.27 ) reported a 1-year ltc rate of 45% for lesions of 3.1 - 4.0 cm in diameter vs. 85% for lesions 2.0 cm with a prescription dose of 15 gy . in our present analyses , which included only large bms , the overall ltc rates observed were comparable to historic single fraction srs for small bms . the ltc rates of even larger lesions in our current series ( 3.5 cm ) treated with more fractions were not inferior to those of lesions < 3.5 cm , indicating a promising role of fsrs in treating large bms . recently , murai et al.20 ) reported that dose fractionation of 27 - 30 gy in 3 fractions and 31 - 35 gy in 5 fractions on consecutive days was tolerable and effective in treating large bms . further studies are needed to determine the optimal dose fractionation protocol , especially in relation to tumor size . rn has been reported at a rate of 2 - 15% after fsrs1410141531 ) . in our current study , rn occurred in 6 out of the 38 lesions we examined ( 15.8% ) , which falls at the upper margin of this range . this can be explained in part by a slightly higher prescription dose employed for our present cases and lack of uniform criteria for rn in different studies . meanwhile , most of our patients with rn were controlled with corticosteroid medication , except for one instance salvaged by surgery . as the incidence of brain necrosis after srs increases with the size of the target volume , the volume of normal brain receiving a certain threshold dose has been implicated in the development of rn3181923 ) , with smaller volumes having a lower risk of rn . in line with previous studies , our multivariate analysis showed that good patient performance ( kps score 70 ) and lower rtog - rpa class significantly predicted a better survival outcome . gaspar et al.7 ) reported that rtog - rpa class i cases had the best survival outcomes ( median 7.1 months ) , whereas those of rotg - rpa class iii had the poorest survival results ( median 2.3 months ) . kim et al.14 ) reported that good kps ( 70 ) , controlled primary cancer , no extracranial metastases , lower rtog - rpa class , higher ds - gpa score , single brain metastasis , and absence of previous wbrt were significant predictors of longer survival , of these variables , only the number of extracranial metastatic organs was found to be a only significant predictor in our multivariate analysis . minniti et al.19 ) reported previously that stable extracranial disease and a good kps ( > 70 ) were associated with the most significant survival benefit . fsrs is now emerging , yet controversial and not a current standard of practice in treating large bms . this study presents additional clinical data that support the application of this approach as valid modality in terms of efficacy and safety . further studies are needed to determine the optimal dose fractionation protocol in relation to tumor size and identify reliable prognostic factors for large bms .

objectiveto investigate the efficacy and safety of fractionated stereotactic radiosurgery for large brain metastases ( bms).methodsbetween june 2011 and december 2013 , a total of 38 large bms > 3.0 cm in 37 patients were treated with fractionated cyberknife radiosurgery . these patients comprised 16 men ( 43.2% ) and 21 women , with a median age of 60 years ( range , 38 - 75 years ) . bms originated from the lung ( n=19 , 51.4% ) , the gastrointestinal tract ( n=10 , 27.0% ) , the breast ( n=5 , 13.5% ) , and other tissues ( n=3 , 8.1% ) . the median tumor volume was 17.6 cc ( range , 9.4 - 49.6 cc ) . for cyberknife treatment , a median peripheral dose of 35 gy ( range , 30 - 41 gy ) was delivered in 3 to 5 fractions.resultswith a median follow - up of 10 months ( range , 1 - 37 months ) , the crude local tumor control ( ltc ) rate was 86.8% and the estimated ltc rates at 12 and 24 months were 87.0% and 65.2% , respectively . the median overall survival ( os ) and progression - free survival ( pfs ) rates were 16 and 11 months , respectively . the estimated os and pfs rates at 6 , 12 , and 18 months were 81.1% and 65.5% , 56.8% and 44.9% , and 40.7% and 25.7% , respectively . patient performance status and preoperative focal neurologic deficits improved in 20 of 35 ( 57.1% ) and 12 of 17 patients ( 70.6% ) , respectively . radiation necrosis with a toxicity grade of 2 or 3 occurred in 6 lesions ( 15.8%).conclusionthese results suggest a promising role of fractionated stereotactic radiosurgery in treating large bms in terms of both efficacy and safety .

INTRODUCTION MATERIALS AND METHODS Eligibility Inclusion criteria Exclusion criteria Demographic data Tumor variables Fractionated stereotactic radiosurgery Follow-up, outcome measures, and statistics RESULTS Objective tumor response and local tumor control Survival Neurological and functional outcomes Radiation necrosis DISCUSSION CONCLUSION

brain metastases ( bms ) have been reported in up to 40% of patients with systemic cancer21 ) , and the incidence of bms is increasing due to the routine brain magnetic resonance imaging ( mri ) screening and an improved outcome of systemic therapy against primary cancers . stereotactic radiosurgery ( srs ) , typically delivered in a single fraction , has been shown to be effective and safe in treating bms and , is generally indicated for a single or oligometastases <3 cm in diameter . however , the toxicity of srs given in a single fraction is considered to outweigh the benefits of local tumor control ( ltc ) for large bms > 3 cm and leads to increased risks of neurological morbidity from radiation necrosis ( rn)31823 ) . recently , the concept of fractionated stereotactic radiosurgery ( fsrs ) has emerged , and it is reportedly an effective and safe way to treat bms , especially large lesions . since june 2011 , we adopted this approach in treating large bms . between june 2011 and december 2013 , a total of 37 patients with large bms were enrolled according to the inclusion and exclusion criteria indicated below . 1 ) age of 18 years , with histologically proven solid cancer and fewer than 6 brain metastases , one of which is > 3 cm in maximum diameter 2 ) intact cognitive function ; able to understand and sign a written informed consent 3 ) life expectancy > 3 months , as indicated by the medical oncologist 4 ) karnofsky performance status ( kps ) score 70 , or 50 - 60 with focal neurological deficits 1 ) suffering from significant mass effect or raised intracranial pressure for which surgical decompression is indicated 2 ) received any form of prior cranial irradiation 3 ) received prior surgical resection of the targeted lesion 4 ) primary hematologic malignancy , such as lymphoma or leukemia 5 ) pregnant or breast - feeding patients the baseline characteristics of the study patients are summarized in table 1 . of the 37 patients included in the analyses , bms originated from the lung ( n=19 , 51.4% ) , gastrointestinal tract ( n=10 , 27.0% ) , breast ( n=5 , 13.5% ) , and other tissues ( n=3 , 8.1% ) . at the time of fsrs , the primary cancer was under control in 21 patients ( 56.8% ) , and metastases to organs other than the brain were present in 26 patients ( 70.3% ) . the kps score was 70 in 30 patients ( 81.1% ) , and focal neurologic deficits were present in 17 patients ( 45.9% ) . there were 7 patients ( 18.9% ) of radiation therapy oncology group - recursive partitioning analysis ( rtog - rpa ) class i , 23 ( 62.2% ) of class ii , and 7 ( 18.9% ) of class iii . the diagnosis - specific graded prognostic assessment ( ds - gpa ) score was 1 in 14 patients ( 37.8% ) , 1.5 - 2.5 in 18 patients ( 48.6% ) , and 3 in 5 patients ( 13.5% ) . of 79 bms in 37 patients , lesions <3 cm were treated with single - fraction srs and were not included in our analysis . bms were located in the cerebral hemisphere in 26 lesions ( 68.4% ) , the cerebellum in 9 lesions ( 23.7% ) , and the basal ganglia or the diencephalon in 3 lesions ( 7.9% ) ( table 1 ) . the mris were registered and manually fused with the planning ct images in the accuray multiplan treatment - planning system ( version 4.5 ) to facilitate the delineation of the gross tumor volume ( gtv ; equal to the planning target volume ) and the critical organ structures including the brainstem , the eyes , and the optic apparatus . the median prescription dose was 35 gy ( range , 30 - 41 gy ) . doses were administered in 3 to 5 daily fractions depending on the size of lesions ; lesions < 3.5 cm were treated in 3 fractions and lesions 3.5 cm in 5 fractions . the following criteria were considered for rn : 1 ) increased t1 contrast enhancement located in the irradiated area with central hypointensity and increased peripheral edema , 2 ) substantial regression or stability ( for at least 3 months ) of enhancing areas on serial follow - up mris without additional treatment , or 3 ) absence of perfusion within the contrast - enhancing lesion on dynamic susceptibility contrast perfusion mri19 ) . ltc , overall survival ( os ) , progression - free survival ( pfs ) , and rn were estimated using the kaplan - meier method calculated from the treatment start date to the date of events or the last follow - up . factors possibly affecting the outcome were tested using the log - rank test for univariate analysis and the cox proportional hazards models with variable selection , which included age ( 65 years vs. < 65 years ) , gender , primary cancer type , tumor location , tumor volume ( < 22 cc vs. 22 cc ) , single vs. multiple bms , status of primary cancer , presence of extracranial metastases , pretreatment kps score ( 70 vs. < 70 ) , rotg - rpa class , ds - gpa score , and prescription dose . between june 2011 and december 2013 , a total of 37 patients with large bms were enrolled according to the inclusion and exclusion criteria indicated below . 1 ) age of 18 years , with histologically proven solid cancer and fewer than 6 brain metastases , one of which is > 3 cm in maximum diameter 2 ) intact cognitive function ; able to understand and sign a written informed consent 3 ) life expectancy > 3 months , as indicated by the medical oncologist 4 ) karnofsky performance status ( kps ) score 70 , or 50 - 60 with focal neurological deficits 1 ) suffering from significant mass effect or raised intracranial pressure for which surgical decompression is indicated 2 ) received any form of prior cranial irradiation 3 ) received prior surgical resection of the targeted lesion 4 ) primary hematologic malignancy , such as lymphoma or leukemia 5 ) pregnant or breast - feeding patients the baseline characteristics of the study patients are summarized in table 1 . of the 37 patients included in the analyses , bms originated from the lung ( n=19 , 51.4% ) , gastrointestinal tract ( n=10 , 27.0% ) , breast ( n=5 , 13.5% ) , and other tissues ( n=3 , 8.1% ) . at the time of fsrs , the primary cancer was under control in 21 patients ( 56.8% ) , and metastases to organs other than the brain were present in 26 patients ( 70.3% ) . the kps score was 70 in 30 patients ( 81.1% ) , and focal neurologic deficits were present in 17 patients ( 45.9% ) . there were 7 patients ( 18.9% ) of radiation therapy oncology group - recursive partitioning analysis ( rtog - rpa ) class i , 23 ( 62.2% ) of class ii , and 7 ( 18.9% ) of class iii . the diagnosis - specific graded prognostic assessment ( ds - gpa ) score was 1 in 14 patients ( 37.8% ) , 1.5 - 2.5 in 18 patients ( 48.6% ) , and 3 in 5 patients ( 13.5% ) of 79 bms in 37 patients , 38 lesions were > 3 cm in diameter and were treated with fsrs . bms were located in the cerebral hemisphere in 26 lesions ( 68.4% ) , the cerebellum in 9 lesions ( 23.7% ) , and the basal ganglia or the diencephalon in 3 lesions ( 7.9% ) ( table 1 ) . the mris were registered and manually fused with the planning ct images in the accuray multiplan treatment - planning system ( version 4.5 ) to facilitate the delineation of the gross tumor volume ( gtv ; equal to the planning target volume ) and the critical organ structures including the brainstem , the eyes , and the optic apparatus . the median prescription dose was 35 gy ( range , 30 - 41 gy ) . doses were administered in 3 to 5 daily fractions depending on the size of lesions ; lesions < 3.5 cm were treated in 3 fractions and lesions 3.5 cm in 5 fractions . partial response was indicated by > 50% decrease from the baseline in perpendicular diameter product sums of all measurable enhancing lesions on mri , elimination or reduction in corticosteroid dose , and clinical stability or improvement . the following criteria were considered for rn : 1 ) increased t1 contrast enhancement located in the irradiated area with central hypointensity and increased peripheral edema , 2 ) substantial regression or stability ( for at least 3 months ) of enhancing areas on serial follow - up mris without additional treatment , or 3 ) absence of perfusion within the contrast - enhancing lesion on dynamic susceptibility contrast perfusion mri19 ) . ltc , overall survival ( os ) , progression - free survival ( pfs ) , and rn were estimated using the kaplan - meier method calculated from the treatment start date to the date of events or the last follow - up . factors possibly affecting the outcome were tested using the log - rank test for univariate analysis and the cox proportional hazards models with variable selection , which included age ( 65 years vs. < 65 years ) , gender , primary cancer type , tumor location , tumor volume ( < 22 cc vs. 22 cc ) , single vs. multiple bms , status of primary cancer , presence of extracranial metastases , pretreatment kps score ( 70 vs. < 70 ) , rotg - rpa class , ds - gpa score , and prescription dose . the rates of complete response , partial response , stable disease , and progressive disease were 11.1% , 44.4% , 30.6% , and 13.9% , respectively . with a median follow - up of 10 months ( range , 1 - 37 months ) , the crude ltc rate was 86.8% and the estimated ltc rates at 12 and 24 months were 87.0% and 65.2% , respectively ( fig . both of the lesions treated with a prescription dose of 31 gy developed local failure , whereas only 3 of 36 lesions with a prescription dose of 35 gy developed local failure ( hazard ratio , 49.26 ; 95% confidence interval , 6.897 - 352.128 ; p<0.001 ) ( table 2 ) . the median os was 16 months , and the estimated os rates at 6 , 12 and 18 months were 81.1% , 56.8% , and 40.7% , respectively . of 21 patients who died , 10 ( 47.6% ) died from the progression of extracranial disease , 6 ( 28.6% ) from brain failure , and 5 ( 23.8% ) from unknown causes . on univariate analysis , kps score < 70 ( hazard ratio , 3.389 ; 95% confidence interval , 1.317 - 8.721 ; p=0.011 ) and rtog - rpa class iii ( hazard ratio , 5.26 ; 95% confidence interval , 1.328 - 20.886 ; p=0.018 ) indicated poor patient survival ( table 3 , fig . twenty - one patients ( 56.8% ) showed progression including distant failure in 20 patients , local failure in 5 patients , and both distant and local failure in 4 patients . the median pfs was 11 months and the estimated pfs rates at 6 , 12 , and 18 months were 65.5% , 44.9% , and 25.7% , respectively . multiple bms were associated with poor pfs ( hazard ratio , 2.603 ; 95% confidence interval , 1.027 - 6.598 ; p=0.044 ) ( table 3 ) , as 14 of 19 patients ( 73.7% ) with multiple bms developed progression vs. 7 of 18 ( 38.8% ) with a single bm . preoperative focal neurologic deficits such as motor weakness and cerebellar dysfunction , improved in 12 of 17 patients ( 70.6% ) 3 months after treatment . the kps score improved in 20 of 35 patients ( 57.1% ) , with a mean preoperative kps score of 74 ( median , 70 ; range , 50 - 100 ) vs. a mean kps score of 80.6 ( median , 80 ; range , 50 - 100 ) 3 months after treatment ( p=0.001 ) ( fig . the median time to rn was 10.5 months ( range , 6 - 18 months ) . the rates of complete response , partial response , stable disease , and progressive disease were 11.1% , 44.4% , 30.6% , and 13.9% , respectively . with a median follow - up of 10 months ( range , 1 - 37 months ) , the crude ltc rate was 86.8% and the estimated ltc rates at 12 and 24 months were 87.0% and 65.2% , respectively ( fig . both of the lesions treated with a prescription dose of 31 gy developed local failure , whereas only 3 of 36 lesions with a prescription dose of 35 gy developed local failure ( hazard ratio , 49.26 ; 95% confidence interval , 6.897 - 352.128 ; p<0.001 ) ( table 2 ) . the median os was 16 months , and the estimated os rates at 6 , 12 and 18 months were 81.1% , 56.8% , and 40.7% , respectively . of 21 patients who died , 10 ( 47.6% ) died from the progression of extracranial disease , 6 ( 28.6% ) from brain failure , and 5 ( 23.8% ) from unknown causes . on univariate analysis , kps score < 70 ( hazard ratio , 3.389 ; 95% confidence interval , 1.317 - 8.721 ; p=0.011 ) and rtog - rpa class iii ( hazard ratio , 5.26 ; 95% confidence interval , 1.328 - 20.886 ; p=0.018 ) indicated poor patient survival ( table 3 , fig . twenty - one patients ( 56.8% ) showed progression including distant failure in 20 patients , local failure in 5 patients , and both distant and local failure in 4 patients . the median pfs was 11 months and the estimated pfs rates at 6 , 12 , and 18 months were 65.5% , 44.9% , and 25.7% , respectively . multiple bms were associated with poor pfs ( hazard ratio , 2.603 ; 95% confidence interval , 1.027 - 6.598 ; p=0.044 ) ( table 3 ) , as 14 of 19 patients ( 73.7% ) with multiple bms developed progression vs. 7 of 18 ( 38.8% ) with a single bm . preoperative focal neurologic deficits such as motor weakness and cerebellar dysfunction , improved in 12 of 17 patients ( 70.6% ) 3 months after treatment . the kps score improved in 20 of 35 patients ( 57.1% ) , with a mean preoperative kps score of 74 ( median , 70 ; range , 50 - 100 ) vs. a mean kps score of 80.6 ( median , 80 ; range , 50 - 100 ) 3 months after treatment ( p=0.001 ) ( fig . the median time to rn was 10.5 months ( range , 6 - 18 months ) . although srs typically delivered in a single fraction has been proven to be effective and safe in treating bms , it is not feasible for large lesions , especially those > 3.0 cm , due to increased toxicity and local treatment failure3182324 ) . however , not all patients with large bms are eligible for surgery when considering surgical accessibility , the number of lesions , and patient medical status1733 ) . currently , the first - line treatment for large bms has not been established and is usually determined by considering various factors , including tumor volume , number , location , and overall condition of patient12 ) . theoretically , fractionated administration of radiation dose potentially minimizes toxicity to late - responding healthy tissues , with a low / ratio compared to a single acute dose of radiation for a given level of tumor damage , according to the linear quadratic model of cellular survival826 ) . as expected , recently published studies on fsrs for large bms have demonstrated high ltc rates , ranging from 63 - 100% , at 1 year follow - up , with acceptable risks of toxicity1410141531 ) ( table 4 ) . consistent with these results , our present ltc rates were 87.0% and 65.2% at 1- and 2-years follow - up , respectively , and the median os was 16 months , which also compares well with the outcomes of single - fraction srs for small bms2111316 ) . furthermore , patient performance status and neurological function improved significantly , presumably benefitting the quality of life in these cases . in a recent systematic review on stereotactic radiotherapy dose and ltc probability , wiggenraad et al.30 ) reported that a biological effective dose , using an / ratio of 12 ( bed12 ) , of at least 40 gy , which correspond to a single fraction dose of 20 gy , was associated with a 1-year ltc rate of 70% or more . the high ltc rate at 1-year follow - up in our present study appears to accord with this observation . vogelbaum et al.27 ) reported a 1-year ltc rate of 45% for lesions of 3.1 - 4.0 cm in diameter vs. 85% for lesions 2.0 cm with a prescription dose of 15 gy . in our present analyses , which included only large bms , the overall ltc rates observed were comparable to historic single fraction srs for small bms . the ltc rates of even larger lesions in our current series ( 3.5 cm ) treated with more fractions were not inferior to those of lesions < 3.5 cm , indicating a promising role of fsrs in treating large bms . recently , murai et al.20 ) reported that dose fractionation of 27 - 30 gy in 3 fractions and 31 - 35 gy in 5 fractions on consecutive days was tolerable and effective in treating large bms . in our current study , rn occurred in 6 out of the 38 lesions we examined ( 15.8% ) , which falls at the upper margin of this range . as the incidence of brain necrosis after srs increases with the size of the target volume , the volume of normal brain receiving a certain threshold dose has been implicated in the development of rn3181923 ) , with smaller volumes having a lower risk of rn . kim et al.14 ) reported that good kps ( 70 ) , controlled primary cancer , no extracranial metastases , lower rtog - rpa class , higher ds - gpa score , single brain metastasis , and absence of previous wbrt were significant predictors of longer survival , of these variables , only the number of extracranial metastatic organs was found to be a only significant predictor in our multivariate analysis . fsrs is now emerging , yet controversial and not a current standard of practice in treating large bms . this study presents additional clinical data that support the application of this approach as valid modality in terms of efficacy and safety .

pharmacogenetics refers to the influence of dna variants on drug response , the knowledge of which can facilitate selection of the optimal drug , dose , and treatment duration and avert adverse drug reactions . several demonstrations have been given on the differences in response to drugs between children and adults . these include differences in drug metabolism and gene expression , the latter being a highly dynamic process functioning from the neonatal period over childhood into adult life . though the number of studies specifically devoted to the pediatric population is still limited compared to adults , an increasing number of genes are being identified in which variants have an influence on pharmacological treatment of childhood diseases . the identification of variants in novel genes as well as the validation of their functional effects will further increase our ability to predict drug treatment response in children ; at the same time , the clinical implementation of this knowledge will demand an efficient diagnostic approach to first identify a pharmacogenomic profile in an individual patient in a short period of time , next to evidence - based clinical guidelines to facilitate decision making based on the genotype . the current golden standard for detecting pathogenic variants single nucleotide variations or small indels developed in the late 70s by frederick sanger , an english biochemist , the technique has currently been optimized to evaluate variations in pcr - amplified dna fragments with high sensitivity and specificity . the major disadvantages of sanger sequencing particularly in a domain such as pharmacogenetics where for a specific drug variants in multiple genes can be , either independent of or in interaction with each other , involved are that each novel genetic test needs optimization and turn - around times for each gene analysis can be relatively long , certainly if therapeutic decisions would be based on these results . together with the sometimes ambiguous evidence for the effect of certain variants and the lack of robust validation and clinical guidelines , this technical hurdle has been one of the reasons that genotyping to inform clinical decisions regarding pharmacological treatment is not widely practiced to date . the introduction of next generation sequencing ( ngs ) brought about a technological revolution among genetic screening tools , as it now becomes possible to screen the whole exome the coding regions of our dna and even the complete genome in a single experiment [ 79 ] . the increase of technological capacities and decrease of costs involved in such analysis have resulted in successful implementation of exome sequencing as a research tool , particularly to identify novel genes for rare disorders [ 10 , 11 ] . 147920 ) were identified by combining whole exome sequencing data from different patients with a typical phenotype of these conditions . they demonstrate that it is possible to capture exomic variation and identify pathogenic variants using bioinformatic tools . because of this success , these screening techniques are slowly starting to make their way as a diagnostic tool . the idea of sequencing all 23.000 genes in the exome in a single reaction is an alluring alternative . similarly , in a field such as pharmacogenetics , with different variants in different genes influencing the final drug response in an individual patient , such parallel sequencing techniques can provide the promptness which would be required in a clinical setting . this shift to the more extensive screening assays has induced an evolution from pharmacogenetics to pharmacogenomics . besides the excitement surrounding these technical innovations , these include not only the quantity of data which is generated , its analysis , and interpretation but also ethical and legal aspects . in the pediatric population , the latter have very particular properties as a consequence of the incapacity of the child to give informed consent himself and of the predictive character of the interpreted sequence data which may go beyond the initial clinical question . in this paper , we will consider the characteristics of ngs , the different means by which ngs technology can be applied , and set out a concept that we think would be feasible to use ngs - based pharmacogenetics in a present - day clinical pediatric setting . the human genome is the entirety of an individual 's hereditary information , including both the coding and noncoding regions of dna and rna , while the human exome encompasses the coding regions of the genes the exons equivalenting ~1%-2% of the total haploid genomic sequence [ 12 , 13 ] . since the establishment of the reference genome and subsequent sequencing of several individual genomes , insights have emerged on the significant variation present in the genome within and between different ethnicities . this variation can be roughly divided into simple nucleotide variations on one hand and structural variation on the other . the first include single nucleotide polymorphisms ( snps ) and small insertions / deletions ( indels ) which have been surveyed in large groups of individuals , resulting in , for example , dbsnp , a database of over 10 million common variants in different ethnic groups . increased knowledge on the architecture of the genome revealed , however ; the spectrum of variation was much broader than these nucleotide changes , referred to as structural variation . this includes not only inversions and copy number variations ( cnvs , i.e. , deletions and duplications ) but also , for example , the presence of stretches of megabases of dna unique to a single personal genome . it has become clear that structural variation in the genome is unexpectedly high and much more complex than previously anticipated ( table 1 ) . in pharmacogenomics , both single nucleotide variants and structural variation such as cnvs have been shown to contribute to the drug response of an individual [ 14 , 15 ] . the pleiotropy by which this variation occurs has an impact on the identification of functional variants for drug response and on the analysis and interpretation of genomic screening assays . following the human genome project , which set out to sequence the three billion nucleotides of the human genome , several high throughput technologies were developed . among these , ngs has known a rapid evolution in a few years time , increasing throughput and reducing costs by continuous improvement of several analysis platforms [ 7 , 9 , 16 , 17 ] . although all are based on the principle of massive parallel sequencing , the specific workflow of ngs depends on the platform that is being used ; one of these techniques which demonstrates excellently the meaning of massive parallel sequencing was developed by margulies et al . ( figure 1 ) [ 6 , 9 , 18 ] . in summary , after fragmentation of the genomic dna , these fragments are bound to tiny beads under specific conditions so that only one dna fragment can bind to a specific bead . these beads are encased in droplets of oil , containing all reactants necessary to amplify the dna via polymerase chain reaction ( pcr ) . in this way , each bead ends up with about 10 million copies of the initial dna fragment . for sequencing of these fragments , one bead per well and sequenced by the sequencing - by - synthesis method general limitations of massive parallel sequencing include error rate , which is higher than sanger sequencing , warranting confirmation of identified causal variants by conventional sequencing methods [ 6 , 11 , 19 ] . further , the quality of the sequence data or so - called coverage ( i.e. , to which extent is every nucleotide of the sequence of interest whether it is a selected set of genes or the whole genome reliably analyzed ) depends on the sequence depth . valid results can be obtained from 40- to 80-fold sequence depth , meaning that every nucleotide is sequenced 40 to 80 times [ 9 , 20 ] . the higher the coverage , the more reliable the result , but also the more expensive and laborious will the analysis be as more sequencing needs to be done . the importance of optimizing coverage of current ngs assays in pharmacogenetics was recently demonstrated in a meta - analysis which evaluated the efficiency of current platforms in the analysis of 253 pharmacogenes . it was shown that a maximum of 85% of coverage of these genes could be obtained , while maximally 30% of missense polymorphisms were covered . this underscores the limitations of genome - wide methods and the challenges and priorities for further optimizing ngs assays . ngs can be used in a targeted manner or can be applied as a whole exome of whole genome diagnostic tool . every one of these approaches has its advantages and weaknesses which will be discussed in the following , with respect to pharmacogenomics in children . in a targeted assay there are two ways to go about selecting the genes of interest , which determines the molecular technique that will be applied : either a microarray - based target enrichment approach or a targeted analysis of whole exome / genome sequencing ( wes and wgs , resp . ) can be used . in the first , direct hybridization of the patient 's dna to an oligonucleotide array , containing probes complementary to the selection of target genes , is performed and then analyzed by ngs , thus generating sequence data only of the genes of interest [ 16 , 21 ] . in the second approach , sequence data of the complete exome or genome is generated , but afterwards only the specific genes of interest are bioinformatically selected and analyzed further , while the remaining sequence is disregarded [ 9 , 17 ] . the major advantage of the array - based target enrichment assays is that these selective tests can be optimized to have full coverage of the genes of interest , hence reaching high sensitivity and specificity . moreover , for each gene it is possible to design the array to just cover the sequence ( exon , intron or promotor ) in which a particular snp is present . further , by generating sequence data only of the genes of your interest , the risk for incidental findings or ethical issues on generated but unanalyzed sequence data both discussed in the following is minimized . the drug response to warfarin a paradigm for drugs with a narrow therapeutic index is determined by variants in several genes , including vkorc1 , ggcx , cyp2c9 and , cyp4f2 [ 2224 ] . among these , vkorc1 and ggcx encode key enzymes ( vk - oxide reductase and gamma - glutamyl carboxylase ; resp . ) of the vitamin k ( vk ) cycle , the metabolic process essential for activation of vk - dependent coagulation factors ii , vii , ix , and x which is blocked by warfarin ( figure 2 ) . cytochromes p450 2c9 and p450 4f2 are important in the metabolization of the drug , catalyzing the warfarin s - enantiomer into its inactive metabolites ( cyp2c9 ) or oxidase vk1 , the essential cofactor of the vk cycle ( cyp4f2 ) [ 24 , 26 ] . an important observation is that the functional variants in , for example , the vkorc1 gene reside mostly in noncoding regions such as the introns and promotor of the gene . contrary to exome analysis , where only the coding regions are sequenced , a targeted array - based assay can be maximally optimized to cover both the coding and non - coding regions of these 4 genes , thus resulting in a maximum of relevant information in a single reaction . nevertheless , it must be remembered that drugs such as warfarin , where most of the variance in metabolism and clearance can be captured by analyzing a handful of genomic variants , represent only a proportion of drugs with a narrow therapeutic index ; for many other drugs , this will not be the case , confronting us with the main limitation of this type of targeted analysis which is the limited flexibility in design . it can be expected that for many drugs , novel genes and variants will be discovered which will have a pharmacogenomic effect in addition to the ones known to date . though this number may be rather small for a very specific topic such as warfarin biology , the pharmacogenetics of adhd or asthma treatment for both of which variants in non - coding regions were described ( table 2)will likely expand significantly in the years to come [ 3 , 2730 ] . this implies that with every newly identified gene , the assay needs to be adjusted or updated to obtain the highest yield of useful information . though novel generation arrays have already become more user friendly to expand the number of targeted genes , it still does not come near the ease by which additional genes can be analyzed in a prospective way using wes or wgs . when applying targeted exome analysis , whole exome sequencing is performed resulting in sequencing data of 23.000 genes . when a novel gene is identified , it is easy to go back to the initial sequence data , access the sequence of the new gene , and analyze variants . however , as mentioned , the major limitation of this approach is the lack of good sequence data of non - coding regions , making this technique only useful to obtain a pharmacogenetic profile for those drugs of which the relevant variants are in coding regions . one such example is the treatment of acute lymphoblastic leukemia ( all ) in children [ 2 , 31 ] . as chemotherapeutic agents are often given at a dose near the toxic range and significant interindividual variability can be seen in effect and adverse reactions , pharmacogenetics can aid in tailoring treatment to the specific needs of the patient . several polymorphisms in enzymes which metabolize chemotherapeutics have been shown to alter treatment response ( table 3 ) , all of which affect the coding regions of the respective genes . in all patients , targeted wes can be an option as all relevant variants will be covered . since the completion of the human genome project in 2001 , sequencing of the complete personal genome has become a technical reality [ 12 , 13 ] . since the initial assembly , the reference genome has been refined and provided us initial insights in to the complexity and extent of human genetic variation . since then , initiatives such as the 1000 genomes project aim to further characterize human variation of all types in different ethnic populations . similar to exome sequencing , the true challenge of whole genome analysis lies in the identification of disease causing mutations or functional snps among an average of 3.03.5 million snvs and 1000 cnvs in a human diploid genome . the current feasibility of pharmacogenetic implementation of wgs data has been shown in published personal genomes such as the lupski or the venter genome [ 7 , 12 ] . in both , another recent example on how wgs can aid therapeutic treatment came from a pair of twins suffering from dopa - responsive dystonia . one of these was sepiapterin ( spr ) , a gene previously reported in association with drd ( omim no . importantly , patients with spr mutations also have insufficient tetrahydrobiopterin ( bh4 ) , an important cofactor in the biosynthesis of dopamine and serotonin . treatment of these patients with l - dopa and 5-hydroxy - tryptophan resulted in marked clinical improvement . though in this particular case unrestricted wgs was applied , it can be conceived that targeted wgs focussing on all genes known to be related to drd could be used to improve diagnostics and choose the optimal treatment strategy . one of the issues that has risen recently is whether the characterization of the genomic sequence of an individual should become the standard of care . this issue has great relevance to the pediatric population , and several arguments can be conceived why this data should or should not be available as early on as possible , ideally in the neonatal period . there can be two rationales to gain knowledge of this genomic information : the first would be to improve preventive medicine by identifying causal mutations or risk alleles associated with so - called actionable diseases , that is , diseases for which preventive measurements of screening have been shown useful for improvement of prognosis . after sequencing , the whole genome data set is completely analyzed , with the intrinsic risk to also unveil risk alleles or mutations for nonactionable disorders such as , for example , neurodegenerative diseases . the second rationale would be to have rapid access to the genetic background of an individual if there is an acute disease episode , for diagnostic and pharmacogenetic purposes . this would imply that the uninterpreted data is stored , and when , for example , the patient develops symptoms of asthma the sequences of those genes known to be involved in drug response of beta - agonists can be quickly assembled and searched for variants that may guide treatment options . though both scenarios can be seen as the ultimate refinement of personalized medicine , there are several practical , ethical , and legal considerations that need to be made before this can be implemented , most of which also apply for wes . besides technical issues related to storage and access of the data and data analysis ( method and quality of analysis as well as the bioinformatic hard- and software capacities ) , the main practical issue remains the interpretation of the sequence data ( figure 3 ) [ 7 , 3335 ] . the human genome is highly variable , with a difference of each personal genome from the reference assembly in 3.5 million snps and 1000 large cnvs . moreover , it is considered that each personal genome contains 400.000 to 600.000 novel snps compared to databases such as dbsnp . moreover , not only the functional effect of each individual variant should be considered but also the interaction between different variants . this has , for example , been shown for copy number variants , many of which can be found across genes encoding proteins with known drug - metabolizing activity . these deletions and duplications can have a high prevalence in the general population , ranging from 2% to 34% , and individuals who are considered outlier metabolizers ( poor or ultra - metabolizers ) were shown to harbor a high amount of these cnvs ( deletions and duplications , resp . ) . however , the presence of such a cnv in a patient does not necessarily mean that it will be predictable for the metabolizer status of that patient . besides the fact that many of these particularly duplications may be nonfunctional , their effect may be compensated by other variants or regulatory mechanisms , in the end leading to little change in the drug metabolism of that individual . hence , decision - making for , for example , drug dosage based on this variant alone may result in under- or overdosing . this leads to the conclusion that extensive empirical evaluation of variants on drug metabolism in the general population will be needed before they can be applied in clinical routine and that postmarketing studies will also need to address this issue . several ethical considerations need to be made prior to routine clinical implementation , in adults but particularly also in children . the huge amount of personal medical data produced by ngs , the fact that some will be irrelevant , that some may be relevant for diseases beyond the primary reason for the test and that some may be difficult to interpret and hence unclear , make that all ethical issues raised before on genetic testing now come together in a single test because of its specific nature , certainly wes and wgs require a different kind of consent compared to the routine genetic tests . as mentioned previously , a potential benefit of these screening technologies for the patient may be the early detection of actionable diseases , the symptoms of which can occur in childhood . besides the obvious advantages for followup and prognosis , it must be taken into account that being confronted unexpectedly with the knowledge that the child may develop one or more diseases can bring about significant psychological burden for the child and the parents . while this will be so for variants associated with high risk to develop disease , -suddenly , an additional medical track needs to be established for this novel health problem- , the psychological effect may be even more pronounced when a variant is discovered with mediocre or low penetrance and hence increased uncertainty about the future of the child . therefore , it seems imperative that prior to wes or wgs for a given diagnostic question , the patient and/or his parents are informed about other diseases for which the test can reveal information and what the implications can be of each of these . the consent that is given will need to not only stipulate rigorously which diseases are being ( indirectly ) looked at , but also mention the possibility of variants of unknown significance , of which it remains uncertain what the effect may be . needless to say that this will require a much more extensive pretest counseling compared to the molecular testing that is routinely used to date as well as posttest ( psychological ) followup . to respect the right of the child not to know , it has been a standard policy not to perform presymptomatic tests in children , when the onset of the disease is in adulthood . a broad screening assay such as untargeted wes or wgs in the context of pharmacogenomics for a childhood disease will also reveal information on these specific late - onset diseases so that one can reflect on whether parents who give consent for this analysis have the right to disregard the right not to know of their child . in contrast , the situation may occur that a mutation of a late - onset disorder was inherited from a parent who does not have any symptoms yet at the moment of the test . when it concerns an actionable disease , this knowledge may improve treatment and prognosis of this individual and may play an important role in the decision - making for future pregnancies . mutation analysis in obligate or potential carriers is in most cases not performed in childhood , as there are no implications for the health of the carrier , and the child has the right to decide himself / herself whether he or she wants to know their carrier status . using untargeted wes or wgs , carriership of several autosomal recessive traits would be identified in every patient analyzed . one could argue to discard this information as it has no immediate benefit for the patient . on the other hand , this information may be important for the parents if they would have another child and possibly other family members , particularly if the carrier frequency in the general population is considerable . should the right not to know of the child overrule the potential benefit for the parents and other family members ? these situations can be seen as a plea for targeted analysis , as the risk for such incidental findings would be minimized . though this poses few problems when the sequence data that is generated is limited to the genes of interest , but when targeted analysis is performed on a larger sequence data set in the context of targeted wes or wgs it should be considered that all sequence data on actionable diseases , although uninterpreted , is available . does the child not have the right to know whether other genetic information in his genome is present for actionable diseases , the knowledge of which may influence his health in a significant manner ? or correspondingly , does the physician or the diagnostic lab not have the obligation to inform the patient ? though no conclusive answer can be given to these questions at this time , many uncertainties can be avoided if the informed consent form stipulates in detail not only which tests will be performed but also what will not be examined . all issues mentioned previously underline that a thorough debate addressing the medical , ethical , and psychological aspects of wes and wgs with respect to the child , the parents and the rest of the family is necessary prior to the diagnostic implementation of these techniques in , for example , pharmacogenomics . such debate has begun to occur within the genetic community , and international consensus and guidelines will need to be drafted regarding late - onset disorders , carriership of recessive diseases , and actionable or non - actionable childhood diseases . however , because of the extensive impact these screening strategies can have , a more global public debate is necessary to inform the public about these novel possibilities and their challenges and to think about what people really want to learn from such a genetic test when sufficiently informed . the main argument not to provide such details is that it does not give additional information to the patient at the time of consultation . however , as knowledge increases , more may become known about these variants ; this could imply that what was once a variant of unknown significance may turn out to be of immediate relevance to the patients health or that of his family members . knowledge of these variants , even when their meaning is initially unclear , may be useful in the followup . in this respect , the question whether the physician or diagnostic facility has the duty to recontact patients when the interpretation of their sequence data changes over the years has not been answered . if this were to be the case , a fully automated informatics system would be needed to regularly screen the stored genomic data of every patient and match all variants with the current literature . to our knowledge , such large - scale systems which are flawless are not yet available , making the duty to recontact for these large data sets nearly impossible at this time . legal issues that can arise around wes / wgs include the storage and access of genomic sequence data and the question of who can gain access : the individual himself , his treating physician(s ) , insurance companies , police , and so forth . the technical revolution in sequencing analysis tools has lead to new perspectives for personalized medicine in general and in pharmacogenetics / genomics specifically . next generation sequencing and its applications have increased our ability to unravel the genetic code of an individual with significant improvement of the speed of the analysis . on the other hand , the implementation of these assays brings about several considerations regarding sensitivity , data analysis , and interpretation as well as ethical aspects . of the current ngs technologies , the array - based approach seems to be the most feasible one for pharmacogenomic applications in childhood . its targeted nature avoids incidental findings while offering sufficient coverage of coding and noncoding regions of genes of interest . with little doubt , the future perspective will be the application of wgs as a diagnostic tool , also in pharmacogenomics . however , many questions need to be addressed before implementing this screening technique in the clinic , including technical challenges , interpretation difficulties , and ethical considerations . most importantly , the implementation of ngs requires the establishment of genotypes with clinical utility and guidelines on how to use them . though the topic of research in many areas , more effort will have to go to validating genotypic data and developing clinical algorithms using them .

pharmacogenetics is considered as a prime example of how personalized medicine nowadays can be put into practice . however , genotyping to guide pharmacological treatment is relatively uncommon in the routine clinical practice . several reasons can be found why the application of pharmacogenetics is less than initially anticipated , which include the contradictory results obtained for certain variants and the lack of guidelines for clinical implementation . however , more reproducible results are being generated , and efforts have been made to establish working groups focussing on evidence - based clinical guidelines . for another pharmacogenetic hurdle , the speed by which a pharmacogenetic profile for a certain drug can be obtained in an individual patient , there has been a revolution in molecular genetics through the introduction of next generation sequencing ( ngs ) , making it possible to sequence a large number of genes up to the complete genome in a single reaction . besides the enthusiasm due to the tremendous increase of our sequencing capacities , several considerations need to be made regarding quality and interpretation of the sequence data as well as ethical aspects of this technology . this paper will focus on the different ngs applications that may be useful for pharmacogenomics in children and the challenges that they bring on .

1. Introduction 2. Genomes, Exomes, and the Variation within 3. Next Generation Sequencing 4. Applications of NGS 5. Conclusion

pharmacogenetics refers to the influence of dna variants on drug response , the knowledge of which can facilitate selection of the optimal drug , dose , and treatment duration and avert adverse drug reactions . several demonstrations have been given on the differences in response to drugs between children and adults . though the number of studies specifically devoted to the pediatric population is still limited compared to adults , an increasing number of genes are being identified in which variants have an influence on pharmacological treatment of childhood diseases . the identification of variants in novel genes as well as the validation of their functional effects will further increase our ability to predict drug treatment response in children ; at the same time , the clinical implementation of this knowledge will demand an efficient diagnostic approach to first identify a pharmacogenomic profile in an individual patient in a short period of time , next to evidence - based clinical guidelines to facilitate decision making based on the genotype . the current golden standard for detecting pathogenic variants single nucleotide variations or small indels developed in the late 70s by frederick sanger , an english biochemist , the technique has currently been optimized to evaluate variations in pcr - amplified dna fragments with high sensitivity and specificity . the major disadvantages of sanger sequencing particularly in a domain such as pharmacogenetics where for a specific drug variants in multiple genes can be , either independent of or in interaction with each other , involved are that each novel genetic test needs optimization and turn - around times for each gene analysis can be relatively long , certainly if therapeutic decisions would be based on these results . together with the sometimes ambiguous evidence for the effect of certain variants and the lack of robust validation and clinical guidelines , this technical hurdle has been one of the reasons that genotyping to inform clinical decisions regarding pharmacological treatment is not widely practiced to date . the introduction of next generation sequencing ( ngs ) brought about a technological revolution among genetic screening tools , as it now becomes possible to screen the whole exome the coding regions of our dna and even the complete genome in a single experiment [ 79 ] . the increase of technological capacities and decrease of costs involved in such analysis have resulted in successful implementation of exome sequencing as a research tool , particularly to identify novel genes for rare disorders [ 10 , 11 ] . because of this success , these screening techniques are slowly starting to make their way as a diagnostic tool . the idea of sequencing all 23.000 genes in the exome in a single reaction is an alluring alternative . similarly , in a field such as pharmacogenetics , with different variants in different genes influencing the final drug response in an individual patient , such parallel sequencing techniques can provide the promptness which would be required in a clinical setting . besides the excitement surrounding these technical innovations , these include not only the quantity of data which is generated , its analysis , and interpretation but also ethical and legal aspects . in the pediatric population , the latter have very particular properties as a consequence of the incapacity of the child to give informed consent himself and of the predictive character of the interpreted sequence data which may go beyond the initial clinical question . in this paper , we will consider the characteristics of ngs , the different means by which ngs technology can be applied , and set out a concept that we think would be feasible to use ngs - based pharmacogenetics in a present - day clinical pediatric setting . the human genome is the entirety of an individual 's hereditary information , including both the coding and noncoding regions of dna and rna , while the human exome encompasses the coding regions of the genes the exons equivalenting ~1%-2% of the total haploid genomic sequence [ 12 , 13 ] . since the establishment of the reference genome and subsequent sequencing of several individual genomes , insights have emerged on the significant variation present in the genome within and between different ethnicities . this variation can be roughly divided into simple nucleotide variations on one hand and structural variation on the other . increased knowledge on the architecture of the genome revealed , however ; the spectrum of variation was much broader than these nucleotide changes , referred to as structural variation . , deletions and duplications ) but also , for example , the presence of stretches of megabases of dna unique to a single personal genome . in pharmacogenomics , both single nucleotide variants and structural variation such as cnvs have been shown to contribute to the drug response of an individual [ 14 , 15 ] . the pleiotropy by which this variation occurs has an impact on the identification of functional variants for drug response and on the analysis and interpretation of genomic screening assays . following the human genome project , which set out to sequence the three billion nucleotides of the human genome , several high throughput technologies were developed . although all are based on the principle of massive parallel sequencing , the specific workflow of ngs depends on the platform that is being used ; one of these techniques which demonstrates excellently the meaning of massive parallel sequencing was developed by margulies et al . further , the quality of the sequence data or so - called coverage ( i.e. , to which extent is every nucleotide of the sequence of interest whether it is a selected set of genes or the whole genome reliably analyzed ) depends on the sequence depth . valid results can be obtained from 40- to 80-fold sequence depth , meaning that every nucleotide is sequenced 40 to 80 times [ 9 , 20 ] . the higher the coverage , the more reliable the result , but also the more expensive and laborious will the analysis be as more sequencing needs to be done . the importance of optimizing coverage of current ngs assays in pharmacogenetics was recently demonstrated in a meta - analysis which evaluated the efficiency of current platforms in the analysis of 253 pharmacogenes . this underscores the limitations of genome - wide methods and the challenges and priorities for further optimizing ngs assays . ngs can be used in a targeted manner or can be applied as a whole exome of whole genome diagnostic tool . every one of these approaches has its advantages and weaknesses which will be discussed in the following , with respect to pharmacogenomics in children . in a targeted assay there are two ways to go about selecting the genes of interest , which determines the molecular technique that will be applied : either a microarray - based target enrichment approach or a targeted analysis of whole exome / genome sequencing ( wes and wgs , resp . ) in the first , direct hybridization of the patient 's dna to an oligonucleotide array , containing probes complementary to the selection of target genes , is performed and then analyzed by ngs , thus generating sequence data only of the genes of interest [ 16 , 21 ] . in the second approach , sequence data of the complete exome or genome is generated , but afterwards only the specific genes of interest are bioinformatically selected and analyzed further , while the remaining sequence is disregarded [ 9 , 17 ] . the major advantage of the array - based target enrichment assays is that these selective tests can be optimized to have full coverage of the genes of interest , hence reaching high sensitivity and specificity . moreover , for each gene it is possible to design the array to just cover the sequence ( exon , intron or promotor ) in which a particular snp is present . further , by generating sequence data only of the genes of your interest , the risk for incidental findings or ethical issues on generated but unanalyzed sequence data both discussed in the following is minimized . of the vitamin k ( vk ) cycle , the metabolic process essential for activation of vk - dependent coagulation factors ii , vii , ix , and x which is blocked by warfarin ( figure 2 ) . cytochromes p450 2c9 and p450 4f2 are important in the metabolization of the drug , catalyzing the warfarin s - enantiomer into its inactive metabolites ( cyp2c9 ) or oxidase vk1 , the essential cofactor of the vk cycle ( cyp4f2 ) [ 24 , 26 ] . an important observation is that the functional variants in , for example , the vkorc1 gene reside mostly in noncoding regions such as the introns and promotor of the gene . contrary to exome analysis , where only the coding regions are sequenced , a targeted array - based assay can be maximally optimized to cover both the coding and non - coding regions of these 4 genes , thus resulting in a maximum of relevant information in a single reaction . nevertheless , it must be remembered that drugs such as warfarin , where most of the variance in metabolism and clearance can be captured by analyzing a handful of genomic variants , represent only a proportion of drugs with a narrow therapeutic index ; for many other drugs , this will not be the case , confronting us with the main limitation of this type of targeted analysis which is the limited flexibility in design . it can be expected that for many drugs , novel genes and variants will be discovered which will have a pharmacogenomic effect in addition to the ones known to date . though this number may be rather small for a very specific topic such as warfarin biology , the pharmacogenetics of adhd or asthma treatment for both of which variants in non - coding regions were described ( table 2)will likely expand significantly in the years to come [ 3 , 2730 ] . this implies that with every newly identified gene , the assay needs to be adjusted or updated to obtain the highest yield of useful information . though novel generation arrays have already become more user friendly to expand the number of targeted genes , it still does not come near the ease by which additional genes can be analyzed in a prospective way using wes or wgs . when a novel gene is identified , it is easy to go back to the initial sequence data , access the sequence of the new gene , and analyze variants . however , as mentioned , the major limitation of this approach is the lack of good sequence data of non - coding regions , making this technique only useful to obtain a pharmacogenetic profile for those drugs of which the relevant variants are in coding regions . as chemotherapeutic agents are often given at a dose near the toxic range and significant interindividual variability can be seen in effect and adverse reactions , pharmacogenetics can aid in tailoring treatment to the specific needs of the patient . several polymorphisms in enzymes which metabolize chemotherapeutics have been shown to alter treatment response ( table 3 ) , all of which affect the coding regions of the respective genes . since the completion of the human genome project in 2001 , sequencing of the complete personal genome has become a technical reality [ 12 , 13 ] . since the initial assembly , the reference genome has been refined and provided us initial insights in to the complexity and extent of human genetic variation . similar to exome sequencing , the true challenge of whole genome analysis lies in the identification of disease causing mutations or functional snps among an average of 3.03.5 million snvs and 1000 cnvs in a human diploid genome . importantly , patients with spr mutations also have insufficient tetrahydrobiopterin ( bh4 ) , an important cofactor in the biosynthesis of dopamine and serotonin . though in this particular case unrestricted wgs was applied , it can be conceived that targeted wgs focussing on all genes known to be related to drd could be used to improve diagnostics and choose the optimal treatment strategy . one of the issues that has risen recently is whether the characterization of the genomic sequence of an individual should become the standard of care . this issue has great relevance to the pediatric population , and several arguments can be conceived why this data should or should not be available as early on as possible , ideally in the neonatal period . there can be two rationales to gain knowledge of this genomic information : the first would be to improve preventive medicine by identifying causal mutations or risk alleles associated with so - called actionable diseases , that is , diseases for which preventive measurements of screening have been shown useful for improvement of prognosis . the second rationale would be to have rapid access to the genetic background of an individual if there is an acute disease episode , for diagnostic and pharmacogenetic purposes . this would imply that the uninterpreted data is stored , and when , for example , the patient develops symptoms of asthma the sequences of those genes known to be involved in drug response of beta - agonists can be quickly assembled and searched for variants that may guide treatment options . though both scenarios can be seen as the ultimate refinement of personalized medicine , there are several practical , ethical , and legal considerations that need to be made before this can be implemented , most of which also apply for wes . besides technical issues related to storage and access of the data and data analysis ( method and quality of analysis as well as the bioinformatic hard- and software capacities ) , the main practical issue remains the interpretation of the sequence data ( figure 3 ) [ 7 , 3335 ] . this has , for example , been shown for copy number variants , many of which can be found across genes encoding proteins with known drug - metabolizing activity . these deletions and duplications can have a high prevalence in the general population , ranging from 2% to 34% , and individuals who are considered outlier metabolizers ( poor or ultra - metabolizers ) were shown to harbor a high amount of these cnvs ( deletions and duplications , resp . ) however , the presence of such a cnv in a patient does not necessarily mean that it will be predictable for the metabolizer status of that patient . besides the fact that many of these particularly duplications may be nonfunctional , their effect may be compensated by other variants or regulatory mechanisms , in the end leading to little change in the drug metabolism of that individual . this leads to the conclusion that extensive empirical evaluation of variants on drug metabolism in the general population will be needed before they can be applied in clinical routine and that postmarketing studies will also need to address this issue . several ethical considerations need to be made prior to routine clinical implementation , in adults but particularly also in children . the huge amount of personal medical data produced by ngs , the fact that some will be irrelevant , that some may be relevant for diseases beyond the primary reason for the test and that some may be difficult to interpret and hence unclear , make that all ethical issues raised before on genetic testing now come together in a single test because of its specific nature , certainly wes and wgs require a different kind of consent compared to the routine genetic tests . as mentioned previously , a potential benefit of these screening technologies for the patient may be the early detection of actionable diseases , the symptoms of which can occur in childhood . besides the obvious advantages for followup and prognosis , it must be taken into account that being confronted unexpectedly with the knowledge that the child may develop one or more diseases can bring about significant psychological burden for the child and the parents . while this will be so for variants associated with high risk to develop disease , -suddenly , an additional medical track needs to be established for this novel health problem- , the psychological effect may be even more pronounced when a variant is discovered with mediocre or low penetrance and hence increased uncertainty about the future of the child . therefore , it seems imperative that prior to wes or wgs for a given diagnostic question , the patient and/or his parents are informed about other diseases for which the test can reveal information and what the implications can be of each of these . the consent that is given will need to not only stipulate rigorously which diseases are being ( indirectly ) looked at , but also mention the possibility of variants of unknown significance , of which it remains uncertain what the effect may be . needless to say that this will require a much more extensive pretest counseling compared to the molecular testing that is routinely used to date as well as posttest ( psychological ) followup . to respect the right of the child not to know , it has been a standard policy not to perform presymptomatic tests in children , when the onset of the disease is in adulthood . a broad screening assay such as untargeted wes or wgs in the context of pharmacogenomics for a childhood disease will also reveal information on these specific late - onset diseases so that one can reflect on whether parents who give consent for this analysis have the right to disregard the right not to know of their child . in contrast , the situation may occur that a mutation of a late - onset disorder was inherited from a parent who does not have any symptoms yet at the moment of the test . when it concerns an actionable disease , this knowledge may improve treatment and prognosis of this individual and may play an important role in the decision - making for future pregnancies . mutation analysis in obligate or potential carriers is in most cases not performed in childhood , as there are no implications for the health of the carrier , and the child has the right to decide himself / herself whether he or she wants to know their carrier status . on the other hand , this information may be important for the parents if they would have another child and possibly other family members , particularly if the carrier frequency in the general population is considerable . these situations can be seen as a plea for targeted analysis , as the risk for such incidental findings would be minimized . though this poses few problems when the sequence data that is generated is limited to the genes of interest , but when targeted analysis is performed on a larger sequence data set in the context of targeted wes or wgs it should be considered that all sequence data on actionable diseases , although uninterpreted , is available . does the child not have the right to know whether other genetic information in his genome is present for actionable diseases , the knowledge of which may influence his health in a significant manner ? all issues mentioned previously underline that a thorough debate addressing the medical , ethical , and psychological aspects of wes and wgs with respect to the child , the parents and the rest of the family is necessary prior to the diagnostic implementation of these techniques in , for example , pharmacogenomics . such debate has begun to occur within the genetic community , and international consensus and guidelines will need to be drafted regarding late - onset disorders , carriership of recessive diseases , and actionable or non - actionable childhood diseases . however , because of the extensive impact these screening strategies can have , a more global public debate is necessary to inform the public about these novel possibilities and their challenges and to think about what people really want to learn from such a genetic test when sufficiently informed . however , as knowledge increases , more may become known about these variants ; this could imply that what was once a variant of unknown significance may turn out to be of immediate relevance to the patients health or that of his family members . knowledge of these variants , even when their meaning is initially unclear , may be useful in the followup . in this respect , the question whether the physician or diagnostic facility has the duty to recontact patients when the interpretation of their sequence data changes over the years has not been answered . legal issues that can arise around wes / wgs include the storage and access of genomic sequence data and the question of who can gain access : the individual himself , his treating physician(s ) , insurance companies , police , and so forth . the technical revolution in sequencing analysis tools has lead to new perspectives for personalized medicine in general and in pharmacogenetics / genomics specifically . next generation sequencing and its applications have increased our ability to unravel the genetic code of an individual with significant improvement of the speed of the analysis . on the other hand , the implementation of these assays brings about several considerations regarding sensitivity , data analysis , and interpretation as well as ethical aspects . of the current ngs technologies , the array - based approach seems to be the most feasible one for pharmacogenomic applications in childhood . with little doubt , the future perspective will be the application of wgs as a diagnostic tool , also in pharmacogenomics . however , many questions need to be addressed before implementing this screening technique in the clinic , including technical challenges , interpretation difficulties , and ethical considerations .

chlamydia is obligate intracellular bacteria that replicate only in the cytoplasmic inclusions of the eukaryotic host cells . they are grouped in the chlamydiales order , chlamydieceae family ; they comprise two kinds , chlamydia and chlamydophila , which in turn consist of nine species subdivided into groups depending on whether they cause pathological conditions in human beings . the group of pathogens that infect humans includes the most commonly diagnosed species chlamydia trachomatis and the least frequent chlamydophila pneumoniae ( formerly chlamydia pheumoniae ) ; the very rare species chlamydophila psittaci ( formerly chlamydia psittaci ) causes disease in birds . chlamydophila abortus ( formerly chlamydia abortus ) causes miscarriages while chlamydophila felis causes pneumonia in cats . the species that have never been found to cause medical conditions in humans include : chlamydophila caviae ( formerly chlamydia caviae ) that causes conjunctivitis in guinea pigs , chlamydophila pecorum ( formerly chlamydia pecorum ) , chlamydia suis ( formerly chlamydia trachomatis ) that causes disease in pigs , chlamydophila pecorum ( formerly chlamydia pecorum ) , and chlamydia suis ( formerly chlamydia trachomatis ) , which causes disease in mice [ 13 ] . in the past the following criteria were used to differentiate between species : the morphology of inclusions , sensitivity to sulfadiazine , ability to synthesise and accumulate glycogen in chlamydial inclusions , and estimates of the dna - dna homology . thus , chlamydia trachomatis that belongs to the chlamydia genus and chlamydophila psittaci that belongs to the chlamydophila genus were the first two species to be differentiated on the basis of differences in the morphology of inclusions , glycogen synthesis , and sensitivity to sulfadiazine . all chlamydia - like bacteria were classified as either chlamydia trachomatis or chlamydia psittaci ( chlamydophila psittaci ) of the chlamydiales order depending on their morphology and development cycle . strains of chlamydia trachomatis were identified on the basis of their ability to accumulate glycogen in inclusions and their sensitivity to sulfadiazine ; strains of chlamydia psittaci were those that lacked the ability to accumulate glycogen and could not resist sulfadiazine [ 1 , 4 ] . the chlamydophila pneumoniae species was identified as chlamydophila psittaci because it had similar phenotypic characteristics such as the density of inclusions , inability to synthesise glycogen , and resistance to sulfadiazine . however , later on chlamydophila pneumoniae was recognised as a separate species as it exhibited ultrastructural differences in the morphology of elementary bodies and dna - dna homology compared to other chlamydia . the fourth species to be recognised as a species different from chlamydophila psittaci ( chlamydia psittaci ) in its phenotypic characteristics was chlamydophila pecorum . the modern classification of bacterial species has been revised and at the moment it is based on genetic methods of estimating the dna - dna and rrna - dna homologies and comparing the results of sequencing the 16s and 23s sections of rrna , using the multipoint mapping sequencing method [ 1 , 3 , 513 ] . of all the molecular analysis methods available , comparative sequencing of rrna or ribosomal dna is most suitable for studying the phylogeny of chlamydia , which are microorganisms with similar phenotypes . when studying the phylogeny of chlamydia we primarily analyse the sequences 16s23s in rrna and 23s rrna genes . all species of chlamydia are classified as members of the chlamydiaceae family if the homology of the 16s rrna gene is more than 90% [ 12 , 14 ] . other groups of chlamydia - like organisms exhibit a homology of the 16s rrna gene with chlamydia of more than 80% . these include strain simkania z , strain hall 's coccus , and strain candidatus , that is a close relative of parachlamydia acanthamoebae , which were all obtained from amoebas and were previously interpreted as rickettsia . these bacteria were classified as members of the chlamydiales order because they are obligate intracellular microorganisms with a development cycle similar to that of chlamydia . studying the homology of the 16s rrna gene in chlamydia - like organisms may result in the discovery of new groups of organisms , or new strains of chlamydia , which will be classified as chlamydiales . there are many very known strains of chlamydophila psittaci , chlamydia trachomatis , and chlamydophila pneumoniae obtained from birds , animals , and humans , the sequencing of whose rrna is described in sundry papers and books [ 1 , 4 , 12 , 15 ] . however , nobody has ever had sequences and carried out a phylogenetic analysis of the strains chlamydia trachomatis and chlamydophila pneumoniae obtained from monkeys , which would have determined if they were related to the human strains of chlamydia . the purpose of this study is to carry out a phylogenetic analysis of the 16s23s midribosome section and the i 23s domain of rrna in the strains of chlamydia obtained from monkeys and humans suffering from pathologies caused by chlamydia . one of the strains did not have plasmids ( chlamydia trachomatis - npl ) and the other had them ( chlamydia trachomatis - pl ) ; this latter strain was obtained from the lungs of a monkey that died from pneumonia ( chlamydophila pneumoniae - pn ) ; the plasmid carrying strain chlamydia trachomatis - pl2 obtained from the cervical canal of a human being and the strain chlamydophila pneumoniae - pn2 obtained from the mouth of a human being [ 16 , 17 ] . the reference strains chlamydophila pneumoniae - b and chlamydia trachomatis - burkhan ( chlamydia trachomatis - pl3 ) were provided by the state collection of chlamydia of the d.i . the midribosome section of the 16s23s rrna genes ( include domain i ) was amplified and sequenced using purpose - built oligonulceotides : forward primer : 16sf2 5-ccg ccc gtc aca tca tgg-3 , forward primer : igsigf 5-ata ata ata gac gtt taa ga-3 , and reverse primer : 23r 5-tac taa gat gtt tca gtt c-3 . amplification was carried out in a 40 mcl mixture comprising : pcr buffer ( 10 ) : 700 mm tris - hcl , ph 8.6/25c , 166 mm ( nh4)2so4 , 25 mm mgcl2 , 0.2 mm dntps , and 2.5 u taq - polymerase , using a geneamp 2700 amplifier ( applied biosystems , us ) . the size of the products of amplification with primers 16sf2 and 23r , igsigf and 23r was 602 and 276 pairs of nucleotides , respectively . the section with the primers 16sf2 and 23r was amplified under the following conditions : 95c for3 minutes , then 40 cycles : 94c for20 seconds , 55c for 20 seconds . , 72c for 20 seconds , and 72c for 3 minutes and finally cooling to 6c with subsequent storage at 10c . v igsigf and 23r was amplified under the following conditions : 95c for 3 minutes , then 40 cycles : 94c for 15 seconds , 50c for 15 seconds , 72c for 15 seconds , and finally 72c for 2 minutes followed by cooling to 6c with subsequent storage at 10c . following the amplification 6 mcl of the sample was mixed with a 6-unit buffer for loading and then loaded into a 1.5% agarous gel ( 0.5 mkg / ml ebr ) . electrophoresis was conducted at 50 a , 100 v per 1 cm for 30 minutes . the amplification products were visualised on an ecx-20l transilluminator ( vilber lourmat , germany ) , using a gel imager electrophoresis result registration system ( helicon , russia ) . the nucleotide sequence of the fragments was determined using an abi prism 3100 genetic analyzer ( applied biosystems , us ) and a bigdye terminator v3.1 cycle sequencing kit set of sequencing reagents ( applied biosystems , us ) , as per the manufacturer 's instructions based on pynny cjsc ( post genome and nanotechnology innovations , based on the innovation centre for medical nanobio - technologies of the state research institute for physical and chemical medicine of the ministry of health of the russian federation , moscow ) . sequencing was done using both forward primers ( 16sf2 5-ccg ccc gtc aca tca tgg-3 , igsigf 5-ata ata ata gac gtt taa ga-3 ) , and reverse primers ( 23r 5-tac taa gat gtt tca gtt c-3 ) , to get more specific results . the results of the sequencing of fragments were analysed and compared with reverse primer sequencing using the vector nti advance 9.0 ( pc ) software package ( http://www.invitrogen.com/site/us/en/home.html ) . the nucleotide sequences 16s23s of the rrna genes obtained in the experiment were analysed using mega ( molecular evolutionary genetics analysis ) version 4.1 ( http://www.megasoftware.net/ ) . multiple straightening of the nucleotide sequences of the analysed strains and isolated sections of rna was done with other nucleotide sequences of the rna of different species of chlamydia available in the genbank ncbi database . genetic relation was analysed and phylogenetic trees were constructed using the mega 4.1 programme employing the neighbour - joining method , based on the p - distance model doing a bootstrap test of phylogeny ( 1000 repetitions ) and the maximum parsimony method . the degree of homology in the 16s23s sequences of the midribosome section and domain i of the 23s rrna gene of the studied strains with different species of chlamydia published in genbank ncbi were analysed using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi/ ) . phylogenetic analysis of the 16s23s midribosome section and domain i of the 23s rrna gene was carried out and tree diagrams were built in comparison with representatives of the chlamydiales order found in genbank ncbi ( http://www.ncbi.nlm.nih.gov/genbank/ ) : chlamydophila abortus eba ( u76710 ) , chlamydophila psittaci 6bc ( u68447 ) , chlamydophila psittaci nj1 ( u68419 ) , chlamydophila caviae gpic ( d85708 ) , chlamydophila felis fp baker ( u68457 ) , chlamydophila pneumoniae n16 ( u68426 ) , chlamydophila pneumoniae tw-183 ( u76711 ) , chlamydophila pecorum e58 ( u68433 ) , chlamydophila pecorum ipa ( u68434 ) , chlamydia trachomatis a / har-13 ( u68438 ) , chlamydia trachomatis b / tw-5/ot ( 68440 ) , chlamydia trachomatis d / uw-3/cx ( u68441 ) , chlamydia trachomatis l2/434/bu ( u68443 ) , chlamydia suis r22 ( u68420 ) , chlamydia suis s45 ( u73110 ) , chlamydia muridarum mopn ( u68436 ) , and chlamydia muridarum sfpd ( u68437 ) . of these chlamydia strains the following are well - documented plasmid carriers : chlamydophila psittaci 6bc pcpa1 [ 19 , 20 ] , chlamydophila pneumoniae n16pcpne1 [ 19 , 20 ] , chlamydia trachomatis b / tw-5/ot pctt1 , chlamydia trachomatis d / uw-3/cx - pchl1 , chlamydophila felis pcfe1 , chlamydophila caviae gpic - pcpgp1 , chlamydia trachomatis a / har-13-pcta , chlamydia trachomatis l2/434/bu - pl2 , chlamydia muridarum mopn - pmopn . mega 4.1 software packaged was used to estimate the evolutionary difference between the sequences and the standard estimation error(s ) for the c i of chlamydia . variation statistics methods based on calculating the arithmetic mean and standard error ( m m ) were used to mathematically process the difference in the homology of the studied strains chlamydia trachomatis ( npl , pl , pl2 , and pl3 ) and chlamydophila pneumoniae ( pn , pn2 , and b ) with the strains of chlamydia found in genbank ncbi . all mathematical processing was done in microsoft excel 2007 . our previous studies established that the strain chlamydia trachomatis - npl obtained from monkeys does not have any plasmids . this strain is less virulent than the plasmid - carrying strains chlamydia trachomatis - pl and chlamydia trachomatis - pl2 , isolated from humans and the reference strain chlamydia trachomatis - burkhan ( chlamydia trachomatis - pl3 ) [ 16 , 17 ] . plasmids contain a gene that codes for the membrane protein pgp3 in chlamydia ; this protein plays a role in the immune response of the organism to pathogens by inducing the generation of inflammatory cytokines in the macrophages and activating an inflammatory reaction . plasmids also play a dominant role in the accumulation of glycogen in chlamydia inclusions , which is explained by the presence in plasmids of sequences specific chromosome genes ( pgi , mrsa1 , glga , glgb , glgx , and glgp ) responsible for the metabolism of glycogen and performing the function of their transcription regulator . the chlamydia trachomatis strains that do not carry the critical plasmid cause asymptomatic development of the disease ( one was obtained from a patient suffering from proctocolitis and characterised as an l2 serotype l2 , another was taken from a patient suffering from asymptomatic urethritis and characterised as serovar b , a third clinical isolate , c599 was taken from the urethra of a patient with an asymptomatic condition and after being sequenced was characterised as serovar e / bour ) . 50% of the time an infecting dosage of a strain not carrying a plasmid is 400 times the dose of a plasmid - carrying strain . while the majority of the chlamydia trachomatis strains obtained from humans carry plasmids , 54% of the strains obtained from monkeys do not have plasmids . this explains the fact that monkeys suffering from chlamydia - caused conditions present with less clear symptoms than humans . the cultivation of chlamydia trachomatis strains in cells is accompanied either by the formation of multiple intracellular chlamydia inclusions or by the emergence of one big inclusion . this is first of all related to the expression of the inca gene at the level of the inca protein ( family of inc - proteins inca , incb , incc , ince , and incg ) , which is part of the composition of the membrane inclusions and is responsible for vacuolisation . if there is no expression of this gene at all , the chlamydia inclusions multiply [ 34 , 35 ] . the inca protein has a function in the formation of secondary inclusions that allow chlamydia to form intracellular recesses where they can grow and continuously infect the cells forming after the division of the mother cell . when the plasmid - free strain chlamydia trachomatis - npl genotype e cultivates multiple intracellular chlamydia inclusions ( vacuoles ) are observed inside the cell while in the case of the plasmid - carrying strains chlamydia trachomatis - pl ( obtained from monkeys ) and chlamydia trachomatis - pl2 ( obtained from humans ) , which are g genotype , one big intracellular chlamydia inclusion forms . genotyping the chlamydia trachomatis strains established that the strains obtained from monkeys were primarily genotypes e ( 42.3% ) and g ( 57.7% ) ; the plasmid carrying strain chlamydia trachomatis - pl has genotype g ; the plasmid - free strain chlamydia trachomatis - npl is genotype e. strains of five serotypes were obtained from humans ( k : 46.2% , g : 23.1% , e : 19.2% , f : 7.7% , j : 3.8% ) ; the plasmid carrying strain chlamydia trachomatis - pl2 manifests as genotype g [ 9 , 11 , 37 ] . comparative analysis of the homology was carried out and the evolutionary difference in the known sequences 15s-23s of the middle ribosome section and domain i of the 23 rrna gene of different types of chlamydia was estimated , with comparisons being made with the strains being studied ( tables 1 and 2 ) . the nucleotide sequences of the sequenced fragment of the plasmid - free strain chlamydia trachomatis - npl have a 99% homology and an evolutionary difference between the sequences of 0.672 to 0.690 ( s = 0.019 ) compared with the strains chlamydia trachomatis ( a / har-13 , b / tw-5/ot , d / uw-3/cx ) , but it exhibits the greatest homology with chlamydia trachomatis l2/434/bu ( 100% ) . in this case the difference between the sequences is 0 ( s = 0 ) , which means that the sequences of the strains npl and l2/434/bu are the same despite the fact that npl has no plasmid . this confirms the previously established similarity between strains that have no plasmid and those that have it . with regard to other species of chlamydia the difference in homology exhibited by npl was 15.92 7.04% , the difference in the sequences is 0.690 to 0.755 ( s = 0.0180.019 ) . the plasmid - carrying strains chlamydia trachomatis - pl obtained from monkeys and chlamydia trachomatis - pl2 obtained from humans are 98%99% homologous with chlamydia trachomatis ( plasmid carrying strains ) , with the difference in the sequences being 0.016 to 0.697 for the strain pl ( s = 0.0050.019 ) and from 0.034 to 0.698 for pl2 ( s = 0.0080.019 ) , with the homology differences from other types of chlamydia ranging from 13.31 5.49% to 13.77 5.97% , respectively . pl3 differs from that of chlamydia trachomatis ( a / har-13 , b / tw-5/ot , d / uw-3/cx , l2/434/bu ) by 1%2% , with the difference between the sequences ranging from 0.002 to 0.690 ( s = 0.0020.019 ) . the difference in the homology of this strain and that of other species of chlamydia of both the chlamydia and chlamydophila genus is 13.15 5.75% , with genetic distances between the sequences ranging from 0.426 to 0.691 ( s = 0.0190.021 ) . when the evolutionary difference in the nucleotide sequences of the studied strains chlamydia trachomatis between each other was studied , the following results were obtained : pl / pl2 - 0.019 ( s = 0.006 ) , pl / pl3 : 0.052 ( s = 0.009 ) , pl / npl : 0.697 ( s = 0.019 ) , pl2/pl3 : 0.033 ( s = 0.007 ) , npl / pl3 : 0.690 ( s = 0.019 ) , and npl / pl2 : 0.698 ( s = 0.019 ) . consequently the plasmid - carrying monkey strain chlamydia trachomatis - pl is the closest to the pl2 strain obtained from humans , while the plasmid - free strain npl is closer to the reference npl strain , but nevertheless this still could not be put in different clusters when the phylogenetic tree found in figures 1 and 3 was constructed . additionally , we also estimated the nucleotide sequence 16s23s of the middle ribosome section and domain i of the 23s rrna gene of the studied plasmid - carrying and plasmid - free strains chlamydia trachomatis to get a better estimate of the aforementioned evolutionary difference in the nucleotide sequences between these strains ( figures 46 ) . when comparing the monkey strains chlamydia trachomatis - pl - genotype g and chlamydia trachomatis - npl - genotype e , a difference was identified inside a section of 483 nucleotide pairs in length ( figure 4 ) . a similar tendency was found when chlamydia trachomatis - pl2-genotype g ( human ) was compared with chlamydia trachomatis - npl - genotype e ( monkey ) , where differences can be observed in a section of 197 nucleotide pairs in length ( figure 5 ) . when chlamydia trachomatis - pl2 ( human ) was compared with chlamydia trachomatis - pl ( monkey ) , isolated differences were found ( within a section 462 nucleotide pairs in length ) ; both strains are genotype g ( figure 6 ) . the studied strains of the chlamydophila pneumoniae species ( pn , pn2 and b ) exhibit a 98% and 99% homology with chlamydophila pneumoniae n16 and chlamydophila pneumoniae tw-183 , respectively . the evolutionary differences between the sequences of the strains pn2 , b , tw-183 , and n16 are between 0.066 and 0.128 ( s = 0.0100.014 ) ; between the monkey strain pn and tw-183 it makes 0.002 ( s = 0.002 ) with a homology of 99% ; between pn and n16 it makes 0.190 ( s = 0.016 ) with a homology of 98% . thus pn2 , b , and pn are close to chlamydophila pneumoniae tw-183 in terms of homology and evolutionary differences between their nucleotide sequences . the difference in homology between pn2 , b , pn , and other species is as follows : pn2 : 6.0 2.75% ; b : 13.66 3.73% ; and pn : 13.73 3.78% ; the evolutionary difference between the nucleotide sequences varies from 0.545 to 0.719 ( s = 0.0190.021 ) . estimating the evolutionary difference in the nucleotide sequences between the studied strains of chlamydophila pneumonia ( pn2 : human strain , pn : monkey strain , b : reference strain ) , we established the following : pn2/pn : 0.067 ( s = 0.010 ) ; pn2/b : 0.0 ( s = 0 ) ; and pn / b : 0.067 ( s = 0.010 ) . pn2 and the reference strain b are the closest to each other and are at the same evolutionary level ; however , the insignificance of the difference from pn means that all of the three studied strains of chlamydophila pneumonia ( human and monkey ) are very similar to each other . the homology between strain chlamydophila pneumonia : pn , isolated from monkeys , and human strains of chlamydophila pneumoniae amounted to 98%99% ; plasmid - free strain chlamydia trachomatis : npl was completely identical to chlamydia trachomatis l2/434/bu ( homology 100% ) , plasmid - carrying strain chlamydia trachomatis : pl both with human and with known chlamydia trachomatis strains from genbank ncbi had 98%99% homology , human chlamydophila pneumoniae strains ( pn2 and b ) and chlamydophila pneumoniae tw-183% : 99% ; strains chlamydia trachomatis ( pl2 , pl3 ) were homologous to chlamydia trachomatis representatives ( 98%100% ) . in order to examine the relationship between plasmid - free chlamydia trachomatis - npl and strains of chlamydia genus phylogenetically , the dendrogram was designed ( p. 1 ) , it shows that the strain is a representative of chlamydia trachomatis species and is included into one cluster with chlamydia trachomatis l2/434/bu . the phylogenetic tree of examined plasmid - carrying chlamydia trachomatis strains isolated from humans and monkeys ( pl2 and pl ) , including reference strain chlamydia trachomatis - burkhun ( pl3 ) , with strains from chlamydia genus is presented on the figure 1 . strains pl , pl2 , and pl3 are united into one combined cluster with chlamydia trachomatis ( b / tw-5/ot , d / uw-3/cx , a / har-13 ) , but into separate clusters with l2/434/bu . the phylogenetic tree of chlamydophila pneumoniae strains ( pn , pn2 , and b ) with strains from chlamydia genus ( p. 2 ) reflects the relationship between strains pn isolated from monkeys , with strain , isolated from humans ( pn2 ) , reference strain chlamydophila pneumoniae - b and strains chlamydophila pneumoniae tw-183 , chlamydophila pneumoniae n16 . strain pn is included into one subcluster with chlamydophila pneumoniae tw-183 , this fact suggests that the strain is a member of chlamydophila pneumoniae species , in one cluster with chlamydophila pneumoniae - pn2 and chlamydophila pneumoniae - b , which are in one subcluster according to evolutional discrepancy . phylogenetic analysis of strains chlamydophila pneumoniae and chlamydia trachomatis , isolated from monkeys , showed that these strains are really the members of chlamydophila genus and chlamydia genus according to general location in clusters on dendrograms with analogical human strains . considering the phylogenetic aspect of the relation between the plasmid - free strain chlamydia trachomatis - npl with strains of the chlamydia genus , we constructed a tree diagram ( figure 1 ) , which indicates that this strain is a chlamydia trachomatis and is in the same cluster as chlamydia trachomatis l2/434/bu . the phylogenetic tree of the studied plasmid - carrying strains of chlamydia trachomatis , obtained from humans and monkeys ( pl2 and pl ) , including the reference strain of chlamydia trachomatis - burkhan ( pl3 ) , with the strain of chlamydia is presented in figure 1 . strains pl , pl2 , and pl3 are grouped into the same cluster with chlamydia trachomatis ( b / tw-5/ot , d / uw-3/cx , a / har-13 ) , but they are placed in a different cluster than l2/434/bu . the phylogenetic tree of the strains of chlamydophila pneumoniae ( pn , pn2 , and b ) with strains of the chlamydophila genus ( figure 2 ) reflects the relation between the pn strain obtained from monkeys with the pn2 strain obtained from humans , the reference strain of chlamydophila pneumoniae - b and the strains of chlamydophila pneumoniae tw-183 , and chlamydophila pneumoniae n16 . it is also in the same cluster with chlamydophila pneumoniae - pn2 and chlamydophila pneumoniae - b , which are in the same subcluster in accordance with their small evolutionary differences . thus phylogenetic analysis of strains of chlamydia trachomatis and chlamydophila pneumoniae obtained from monkeys and humans has allowed us to establish their place in the phylogenetic tree of the chlamydiaceae family . it has been established that there is a close evolutionary relation between the studied original species of chlamydia and chlamydophila and similar species about which there are records in genbank . for the first time , it has been demonstrated that there are differences in the nucleotide sequence 16s23s of the middle ribosome section and domain i of the 23s rrna gene of plasmid - carrying and plasmid - free strains of chlamydia trachomatis obtained from monkeys and humans , if they have different genotypes ( group b- b , ba , d , da , e , l1 , l2 , l2a ; intermediary group- f , g , ga ) . malfunction of the expression of the chromosome gene inca that leads to a breakdown in the development and life cycle of chlamydia and their virulence may also be linked to possible changes in the nucleotide sequence of this gene .

based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16s23s nucleotide sequences of the middle ribosomal cluster and 23s rrna i domain , and based on identification of phylogenetic position for chlamydophila pneumoniae and chlamydia trichomatis strains released from monkeys , relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the genbank electronic database has been detected for the first time ever . position of these isolates in the chlamydiaceae family phylogenetic tree has been identified . the evolutional position of the investigated original chlamydia and chlamydophila strains close to analogous strains from the gen - bank electronic database has been demonstrated . differences in the 16s23s nucleotide sequence of the middle ribosomal cluster and 23s rrna i domain of plasmid and nonplasmid chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups ( group b - b , ba , d , da , e , l1 , l2 , l2a ; intermediate group - f , g , ga ) have been revealed for the first time ever . abnormality in inca chromosomal gene expression resulting in chlamydia life development cycle disorder , and decrease of chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration

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

thus , chlamydia trachomatis that belongs to the chlamydia genus and chlamydophila psittaci that belongs to the chlamydophila genus were the first two species to be differentiated on the basis of differences in the morphology of inclusions , glycogen synthesis , and sensitivity to sulfadiazine . strains of chlamydia trachomatis were identified on the basis of their ability to accumulate glycogen in inclusions and their sensitivity to sulfadiazine ; strains of chlamydia psittaci were those that lacked the ability to accumulate glycogen and could not resist sulfadiazine [ 1 , 4 ] . the modern classification of bacterial species has been revised and at the moment it is based on genetic methods of estimating the dna - dna and rrna - dna homologies and comparing the results of sequencing the 16s and 23s sections of rrna , using the multipoint mapping sequencing method [ 1 , 3 , 513 ] . all species of chlamydia are classified as members of the chlamydiaceae family if the homology of the 16s rrna gene is more than 90% [ 12 , 14 ] . studying the homology of the 16s rrna gene in chlamydia - like organisms may result in the discovery of new groups of organisms , or new strains of chlamydia , which will be classified as chlamydiales . there are many very known strains of chlamydophila psittaci , chlamydia trachomatis , and chlamydophila pneumoniae obtained from birds , animals , and humans , the sequencing of whose rrna is described in sundry papers and books [ 1 , 4 , 12 , 15 ] . however , nobody has ever had sequences and carried out a phylogenetic analysis of the strains chlamydia trachomatis and chlamydophila pneumoniae obtained from monkeys , which would have determined if they were related to the human strains of chlamydia . the purpose of this study is to carry out a phylogenetic analysis of the 16s23s midribosome section and the i 23s domain of rrna in the strains of chlamydia obtained from monkeys and humans suffering from pathologies caused by chlamydia . one of the strains did not have plasmids ( chlamydia trachomatis - npl ) and the other had them ( chlamydia trachomatis - pl ) ; this latter strain was obtained from the lungs of a monkey that died from pneumonia ( chlamydophila pneumoniae - pn ) ; the plasmid carrying strain chlamydia trachomatis - pl2 obtained from the cervical canal of a human being and the strain chlamydophila pneumoniae - pn2 obtained from the mouth of a human being [ 16 , 17 ] . the reference strains chlamydophila pneumoniae - b and chlamydia trachomatis - burkhan ( chlamydia trachomatis - pl3 ) were provided by the state collection of chlamydia of the d.i . the nucleotide sequence of the fragments was determined using an abi prism 3100 genetic analyzer ( applied biosystems , us ) and a bigdye terminator v3.1 cycle sequencing kit set of sequencing reagents ( applied biosystems , us ) , as per the manufacturer 's instructions based on pynny cjsc ( post genome and nanotechnology innovations , based on the innovation centre for medical nanobio - technologies of the state research institute for physical and chemical medicine of the ministry of health of the russian federation , moscow ) . the results of the sequencing of fragments were analysed and compared with reverse primer sequencing using the vector nti advance 9.0 ( pc ) software package ( http://www.invitrogen.com/site/us/en/home.html ) . the nucleotide sequences 16s23s of the rrna genes obtained in the experiment were analysed using mega ( molecular evolutionary genetics analysis ) version 4.1 ( http://www.megasoftware.net/ ) . multiple straightening of the nucleotide sequences of the analysed strains and isolated sections of rna was done with other nucleotide sequences of the rna of different species of chlamydia available in the genbank ncbi database . the degree of homology in the 16s23s sequences of the midribosome section and domain i of the 23s rrna gene of the studied strains with different species of chlamydia published in genbank ncbi were analysed using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi/ ) . phylogenetic analysis of the 16s23s midribosome section and domain i of the 23s rrna gene was carried out and tree diagrams were built in comparison with representatives of the chlamydiales order found in genbank ncbi ( http://www.ncbi.nlm.nih.gov/genbank/ ) : chlamydophila abortus eba ( u76710 ) , chlamydophila psittaci 6bc ( u68447 ) , chlamydophila psittaci nj1 ( u68419 ) , chlamydophila caviae gpic ( d85708 ) , chlamydophila felis fp baker ( u68457 ) , chlamydophila pneumoniae n16 ( u68426 ) , chlamydophila pneumoniae tw-183 ( u76711 ) , chlamydophila pecorum e58 ( u68433 ) , chlamydophila pecorum ipa ( u68434 ) , chlamydia trachomatis a / har-13 ( u68438 ) , chlamydia trachomatis b / tw-5/ot ( 68440 ) , chlamydia trachomatis d / uw-3/cx ( u68441 ) , chlamydia trachomatis l2/434/bu ( u68443 ) , chlamydia suis r22 ( u68420 ) , chlamydia suis s45 ( u73110 ) , chlamydia muridarum mopn ( u68436 ) , and chlamydia muridarum sfpd ( u68437 ) . of these chlamydia strains the following are well - documented plasmid carriers : chlamydophila psittaci 6bc pcpa1 [ 19 , 20 ] , chlamydophila pneumoniae n16pcpne1 [ 19 , 20 ] , chlamydia trachomatis b / tw-5/ot pctt1 , chlamydia trachomatis d / uw-3/cx - pchl1 , chlamydophila felis pcfe1 , chlamydophila caviae gpic - pcpgp1 , chlamydia trachomatis a / har-13-pcta , chlamydia trachomatis l2/434/bu - pl2 , chlamydia muridarum mopn - pmopn . variation statistics methods based on calculating the arithmetic mean and standard error ( m m ) were used to mathematically process the difference in the homology of the studied strains chlamydia trachomatis ( npl , pl , pl2 , and pl3 ) and chlamydophila pneumoniae ( pn , pn2 , and b ) with the strains of chlamydia found in genbank ncbi . this strain is less virulent than the plasmid - carrying strains chlamydia trachomatis - pl and chlamydia trachomatis - pl2 , isolated from humans and the reference strain chlamydia trachomatis - burkhan ( chlamydia trachomatis - pl3 ) [ 16 , 17 ] . plasmids contain a gene that codes for the membrane protein pgp3 in chlamydia ; this protein plays a role in the immune response of the organism to pathogens by inducing the generation of inflammatory cytokines in the macrophages and activating an inflammatory reaction . plasmids also play a dominant role in the accumulation of glycogen in chlamydia inclusions , which is explained by the presence in plasmids of sequences specific chromosome genes ( pgi , mrsa1 , glga , glgb , glgx , and glgp ) responsible for the metabolism of glycogen and performing the function of their transcription regulator . the chlamydia trachomatis strains that do not carry the critical plasmid cause asymptomatic development of the disease ( one was obtained from a patient suffering from proctocolitis and characterised as an l2 serotype l2 , another was taken from a patient suffering from asymptomatic urethritis and characterised as serovar b , a third clinical isolate , c599 was taken from the urethra of a patient with an asymptomatic condition and after being sequenced was characterised as serovar e / bour ) . while the majority of the chlamydia trachomatis strains obtained from humans carry plasmids , 54% of the strains obtained from monkeys do not have plasmids . when the plasmid - free strain chlamydia trachomatis - npl genotype e cultivates multiple intracellular chlamydia inclusions ( vacuoles ) are observed inside the cell while in the case of the plasmid - carrying strains chlamydia trachomatis - pl ( obtained from monkeys ) and chlamydia trachomatis - pl2 ( obtained from humans ) , which are g genotype , one big intracellular chlamydia inclusion forms . genotyping the chlamydia trachomatis strains established that the strains obtained from monkeys were primarily genotypes e ( 42.3% ) and g ( 57.7% ) ; the plasmid carrying strain chlamydia trachomatis - pl has genotype g ; the plasmid - free strain chlamydia trachomatis - npl is genotype e. strains of five serotypes were obtained from humans ( k : 46.2% , g : 23.1% , e : 19.2% , f : 7.7% , j : 3.8% ) ; the plasmid carrying strain chlamydia trachomatis - pl2 manifests as genotype g [ 9 , 11 , 37 ] . comparative analysis of the homology was carried out and the evolutionary difference in the known sequences 15s-23s of the middle ribosome section and domain i of the 23 rrna gene of different types of chlamydia was estimated , with comparisons being made with the strains being studied ( tables 1 and 2 ) . the nucleotide sequences of the sequenced fragment of the plasmid - free strain chlamydia trachomatis - npl have a 99% homology and an evolutionary difference between the sequences of 0.672 to 0.690 ( s = 0.019 ) compared with the strains chlamydia trachomatis ( a / har-13 , b / tw-5/ot , d / uw-3/cx ) , but it exhibits the greatest homology with chlamydia trachomatis l2/434/bu ( 100% ) . the plasmid - carrying strains chlamydia trachomatis - pl obtained from monkeys and chlamydia trachomatis - pl2 obtained from humans are 98%99% homologous with chlamydia trachomatis ( plasmid carrying strains ) , with the difference in the sequences being 0.016 to 0.697 for the strain pl ( s = 0.0050.019 ) and from 0.034 to 0.698 for pl2 ( s = 0.0080.019 ) , with the homology differences from other types of chlamydia ranging from 13.31 5.49% to 13.77 5.97% , respectively . the difference in the homology of this strain and that of other species of chlamydia of both the chlamydia and chlamydophila genus is 13.15 5.75% , with genetic distances between the sequences ranging from 0.426 to 0.691 ( s = 0.0190.021 ) . when the evolutionary difference in the nucleotide sequences of the studied strains chlamydia trachomatis between each other was studied , the following results were obtained : pl / pl2 - 0.019 ( s = 0.006 ) , pl / pl3 : 0.052 ( s = 0.009 ) , pl / npl : 0.697 ( s = 0.019 ) , pl2/pl3 : 0.033 ( s = 0.007 ) , npl / pl3 : 0.690 ( s = 0.019 ) , and npl / pl2 : 0.698 ( s = 0.019 ) . additionally , we also estimated the nucleotide sequence 16s23s of the middle ribosome section and domain i of the 23s rrna gene of the studied plasmid - carrying and plasmid - free strains chlamydia trachomatis to get a better estimate of the aforementioned evolutionary difference in the nucleotide sequences between these strains ( figures 46 ) . the evolutionary differences between the sequences of the strains pn2 , b , tw-183 , and n16 are between 0.066 and 0.128 ( s = 0.0100.014 ) ; between the monkey strain pn and tw-183 it makes 0.002 ( s = 0.002 ) with a homology of 99% ; between pn and n16 it makes 0.190 ( s = 0.016 ) with a homology of 98% . thus pn2 , b , and pn are close to chlamydophila pneumoniae tw-183 in terms of homology and evolutionary differences between their nucleotide sequences . the difference in homology between pn2 , b , pn , and other species is as follows : pn2 : 6.0 2.75% ; b : 13.66 3.73% ; and pn : 13.73 3.78% ; the evolutionary difference between the nucleotide sequences varies from 0.545 to 0.719 ( s = 0.0190.021 ) . the homology between strain chlamydophila pneumonia : pn , isolated from monkeys , and human strains of chlamydophila pneumoniae amounted to 98%99% ; plasmid - free strain chlamydia trachomatis : npl was completely identical to chlamydia trachomatis l2/434/bu ( homology 100% ) , plasmid - carrying strain chlamydia trachomatis : pl both with human and with known chlamydia trachomatis strains from genbank ncbi had 98%99% homology , human chlamydophila pneumoniae strains ( pn2 and b ) and chlamydophila pneumoniae tw-183% : 99% ; strains chlamydia trachomatis ( pl2 , pl3 ) were homologous to chlamydia trachomatis representatives ( 98%100% ) . the phylogenetic tree of examined plasmid - carrying chlamydia trachomatis strains isolated from humans and monkeys ( pl2 and pl ) , including reference strain chlamydia trachomatis - burkhun ( pl3 ) , with strains from chlamydia genus is presented on the figure 1 . strains pl , pl2 , and pl3 are united into one combined cluster with chlamydia trachomatis ( b / tw-5/ot , d / uw-3/cx , a / har-13 ) , but into separate clusters with l2/434/bu . the phylogenetic tree of chlamydophila pneumoniae strains ( pn , pn2 , and b ) with strains from chlamydia genus ( p. 2 ) reflects the relationship between strains pn isolated from monkeys , with strain , isolated from humans ( pn2 ) , reference strain chlamydophila pneumoniae - b and strains chlamydophila pneumoniae tw-183 , chlamydophila pneumoniae n16 . strain pn is included into one subcluster with chlamydophila pneumoniae tw-183 , this fact suggests that the strain is a member of chlamydophila pneumoniae species , in one cluster with chlamydophila pneumoniae - pn2 and chlamydophila pneumoniae - b , which are in one subcluster according to evolutional discrepancy . phylogenetic analysis of strains chlamydophila pneumoniae and chlamydia trachomatis , isolated from monkeys , showed that these strains are really the members of chlamydophila genus and chlamydia genus according to general location in clusters on dendrograms with analogical human strains . considering the phylogenetic aspect of the relation between the plasmid - free strain chlamydia trachomatis - npl with strains of the chlamydia genus , we constructed a tree diagram ( figure 1 ) , which indicates that this strain is a chlamydia trachomatis and is in the same cluster as chlamydia trachomatis l2/434/bu . the phylogenetic tree of the studied plasmid - carrying strains of chlamydia trachomatis , obtained from humans and monkeys ( pl2 and pl ) , including the reference strain of chlamydia trachomatis - burkhan ( pl3 ) , with the strain of chlamydia is presented in figure 1 . the phylogenetic tree of the strains of chlamydophila pneumoniae ( pn , pn2 , and b ) with strains of the chlamydophila genus ( figure 2 ) reflects the relation between the pn strain obtained from monkeys with the pn2 strain obtained from humans , the reference strain of chlamydophila pneumoniae - b and the strains of chlamydophila pneumoniae tw-183 , and chlamydophila pneumoniae n16 . it is also in the same cluster with chlamydophila pneumoniae - pn2 and chlamydophila pneumoniae - b , which are in the same subcluster in accordance with their small evolutionary differences . thus phylogenetic analysis of strains of chlamydia trachomatis and chlamydophila pneumoniae obtained from monkeys and humans has allowed us to establish their place in the phylogenetic tree of the chlamydiaceae family . it has been established that there is a close evolutionary relation between the studied original species of chlamydia and chlamydophila and similar species about which there are records in genbank . for the first time , it has been demonstrated that there are differences in the nucleotide sequence 16s23s of the middle ribosome section and domain i of the 23s rrna gene of plasmid - carrying and plasmid - free strains of chlamydia trachomatis obtained from monkeys and humans , if they have different genotypes ( group b- b , ba , d , da , e , l1 , l2 , l2a ; intermediary group- f , g , ga ) . malfunction of the expression of the chromosome gene inca that leads to a breakdown in the development and life cycle of chlamydia and their virulence may also be linked to possible changes in the nucleotide sequence of this gene .

proteins are marginally stable and rely on cooperative effects to keep them in their folded structure . a balance between charge interactions , hydrogen bonding , and hydrophobicity is responsible for the formation of stable native folds . a simple description of a hydrogen bond can be based on an electrostatic dipole the formation of a protein backbone s hydrogen bonds leads to a particular orientation of dipoles in various types of secondary structural elements . for example , it is well established that the co and nh dipoles in an -helix are electrostatically aligned and almost parallel to the axis of the helix . furthermore , studies have shown that the helical macrodipole can be exploited to stabilize or destroy a helical moiety by charge capping at the chain ends . placement of a positive amino acid at the c - terminus or negative at the n - terminus enhances helicity by a charge protein design studies have revealed that it is possible to design helix - bundle proteins and -strands considering mainly the pattern of polar and nonpolar amino acids . statistical analysis of folded structures has shown that nonpolar amino acids appear in the protein sequence , every three to four amino acids in -helical structures , and every two amino acids in solvent exposed -sheets . molecular simulations have been extremely useful in providing a molecular understanding of experimental observations . coarse - grained ( cg ) models allow us to understand which interactions are essential and which ones can be approximated . in addition , by reducing the level of resolution , cg models decrease the computational requirements compared to all - atom simulations and smooths out the free - energy landscape , facilitating the sampling from one conformation to another . simple models where residues are represented by a few beads have been very valuable in advancing our understanding of the protein folding process . but their main drawback is that the parameters are normally tuned for a particular system and are not transferable . therefore , they have to be recalibrated according to the system of interest . on the other hand , there exist intermediate resolution models where the backbone is modeled with fine resolution and the side chain coarse - grained . upon inclusion of a hydrogen bonding interaction , these models are able to fold into helical structures without the addition of biases toward a native fold but fail to stabilize -sheet structures . the inclusion of explicit dipole dipole interactions in these types of models has been shown to stabilize -sheets . however , all the above - mentioned models renormalize the role of the solvent through effective short - range , inter - residue interactions . thus , they are not appropriate for studying the effect of the environment in protein folding because explicit solvent is needed . also , the importance of the role of dipole interactions is evident from the work done by scheraga et al . his group has shown that an optimization of the electrostatic interaction , by aligning the dipole of each backbone plane to a protein s electric field , is enough to fold small peptides using monte carlo simulations without solvation . the recently developed water - explicit martini cg force field has been parametrized using water / oil and water / vacuum partitioning coefficients . by doing so , the model parameters are transferable and do not depend on a particular system . however , it fails to capture changes in secondary structure and thus it can not be used to study protein folding . in this work , we present a generic water - explicit cg model of proteins that can be used to characterize the driving forces behind protein folding without the addition of biasing potentials such as dihedral potentials or dependencies in the bending potential with secondary structure propensities . the model has roots in the martini cg force field and thus it has the potential to be extended to model membrane protein folding . in the current implementation , each amino acid is coarse - grained with two beads , backbone , and side chain . the change in orientation of the atoms underlying the backbone coarse - grained unit is captured by a flexible dipole that is created by oppositely charged dummy particles inside each backbone coarse - grained bead . these dipoles interact with each other through coulombic potentials and introduce structural polarization into the model . here we explore if just the addition of effective dipole interactions , in addition to lennard - jones interactions , is sufficient to produce secondary and supersecondary folds depending on sequence patterning . we have evaluated the performance of the model by folding protein structures on the basis of the sequence of amino acids . a molecular picture of how charge - capping stabilizes helical structures is also provided . we use the sequence patterning of helices ( -hhpphh- ) and sheets ( -hphphp- ) to fold into distinct secondary structures . we then explore the role of helix capping by terminating helices with charged residues . helix and sheet bundles are designed on the basis of the above de novo patterns with the inclusion of turning regions . the paper is organized as follows : a detailed description of the cg model is presented in the next section . the results and discussion are subdivided into two parts . in the first section , we discuss the formation of protein supersecondary structures , namely helix bundles and sheet - strands and compare the results to the model without dipoles , thus drawing attention to the relevance of the backbone dipolar interactions in determining protein structure . an amino acid is modeled by two beads , a polar backbone bead ( bb ) that embeds a dipole , and a side chain bead ( sc ) . the sc beads are broadly classified into hydrophobic ( h ) , polar ( p ) , and positively and negatively charged ( c+/c ) , as depicted in figure 1a . the protein model explores the role of backbone dipoles in driving secondary and supersecondary structure formation . the cg protein model is combined with the recently developed polarizable coarse - grained water model . the spherical backbone coarse - grained bead consists of three interaction sites , the center bead bb and two dipole particles , bbm and bbp , similar to the polarizable water model and depicted in figure 1b . the main site , the center of the bb bead , interacts with other cg beads through a pairwise lennard - jones potential . dipole particles bbm and bbp are harmonically bound to the central particle bb ( equilibrium distance ( l ) , force constant ( kl ) ) , and carry a positive and negative charge of equal magnitude ( q ) , respectively . a harmonic angle potential ( equilibrium angle ( ) and angular force constant ( k ) ) is used to control the rotation of bbm and bbp particles . because the location of the dipole particles are not fixed , the model is polarizable ; i.e. , the dipole orientation and moment of the backbone bead are dependent on the electric field of the surrounding environment . the rationale for using a polarizable backbone dipole as opposed to a fixed dipole is to make the dipoles environment sensitive , thus enabling future studies of the structural changes induced by backbone dipoles in different dielectric environments . to avoid overpolarization , a small repulsive core is added to the dipolar particles , as commonly done in polarizable all - atom force fields . because each main coarse - grained site is covalently bonded to its nearest neighbors by a harmonic bond potential , and adjacent bonds are connected by a harmonic angle potential , all the 12 and 13 nonbonded interactions are excluded from the main coarse - grained sites and the corresponding embedded dipole particles . in addition , the nonbonded interactions between dipole particles inside the backbone cg bead are excluded as well . the mass of the whole bead ( 72 amu ) is distributed equally among the three particles ( 24 amu each ) in backbone beads . c is the negatively charged residue , c+ is the positively charged residue , and p and h are the polar and hydrophobic residues , respectively . gray represents the polarizable backbone ( bb ) bead and red , blue , green , and cyan represent the side - chain ( sc ) beads . vdw radius of the bb bead encloses dummy particles , bbm ( negatively charged ) , and bbp ( positively charged ) . the five tunable parameters ( l , q , , kl , k ) are depicted . the force field consists of bonded parameters ( harmonic bonded and angular potential ; dihedral potential is not included ) and nonbonded parameters ( 12 - 6 lennard - jones ( lj ) potential and coulombic potential ) . the dipole moment distribution of the bb bead is parametrized to match that of a peptide bond ( 3.5 d ) . it is worth mentioning that with dipole moments of bb less than 3.5 d , helices are not formed . there are five parameters for the backbone cg bead that can be tuned to obtain the desired average dipole moment ( l , q , , kl , k ) . for the backbone cg bead , the following set of parameters yields an average dipole moment of 3.5 d : l = 0.14 nm , q = 0.34 , = 0 , kl = 5000 kj / mol , and k = 7.2 kj / mol . each bb bead is covalently bound to the adjacent bb bead by a bond distance of 3.85 and kl = 5000 kj / mol , distance parametrized from the average cc bond distance observed from a 12mer polyalanine atomistic simulation with gromos force field . other bonded interactions and lj interaction strengths are borrowed from marrink et al . the sc bb harmonic bond distance is chosen as 0.4 nm with kl = 5000 kj / mol on the basis of a typical two - bead residue in the martini force field . the bb bb bb harmonic angle is defined by = 105 and k = 75 kj / mol , and the sc bb harmonic angle by = 100 and k = 75 kj / mol . the interaction strength of the lj potential for the different nonbonded interactions is listed in table 1 . an effective size of = 0.47 nm is assumed for all main cg interaction pairs in the lj potential . with the interaction strengths taken from the martini force field , it has been observed that hydrophobic interactions play a dominant role in stabilizing -sheet conformations . c1 bead in the martini force field ) is not hydrophobic enough to stabilize de novo sheet formation in the current model . a 12 mer chain of backbone beads only folds into a helical structure ( results not shown ) . if hydrophobic side chains have low hydrophobicity , then the conformational ensemble is dominated by backbone properties that have an inherent preference over helical conformations . to correct for this , the between h beads and water ( c1-pol ) has been decreased from 2 to 0.2 kj / mol , and between two h beads ( c1c1 ) increased from 3.5 to 3.75 kj / mol , where sheet formation is observed . in addition , the charge sc ( c+/c ) backbone interaction strength is decreased from 5 to 4 kj / mol to observed helix capping effects , as described in the results and discussion . in parentheses all simulations were carried out using the gromacs package version 4.5.5 and visualized on vmd . all system sizes are reported in the supporting information ( table s1 ) . a nose rahman barostat ( time constant = 1 ps , isothermal compressibility = 3 10 ( kj mol nm ) ) were used to keep the temperature and pressure constant at 300 k and 1 bar . only for the construction of melting curves were nvt simulations performed instead of npt from 300 to 600 k. a time step of 5 fs was used in all the simulations , and the neighbor list was updated every 10 steps . the long - range electrostatic interactions with periodic boundary conditions ( xyz ) were calculated by the particle - mesh ewald method . a global dielectric constant of 2.5 was used . the lincs algorithm was used to constraint the bonds of the water molecules ( between the central cg site and the dipole particles ) . the starting conformation for all simulations was an extended random coil . for each system , the simulation data was collected over a 320 ns npt run , and the last 300 ns was analyzed . a speedup of 6 is observed vis - a - vis a fully atomistic system . however , because coarse - graining smooths the energy landscape , more transitions from folded to unfolded states are observed in comparison with atomistic simulations . the alignment of the backbone dipoles with respect to the protein electric field is determined from the angle i between the local electrostatic field , ei(ri ) , and the ith dipole moment ( i ) using the following equation:1where2the quantities rbbmi , rbbpi , qbbm , and qbbp represent position vectors and charge of the two dummy particles and ri is the position vector:3the electric field due to the protein molecule is computed using coulomb s law:4where the summation runs over all other charged sites in the structure , except the ones belonging to i , i 1 , and i 2 , as these interactions are excluded in the model and do not contribute to the local electric field of the considered dipole i. molecular dynamics simulation of different test systems , representing fundamental secondary and supersecondary motifs were performed . different patterns of polar and nonpolar amino acids , with and without the presence of capping charge residues were used to test the capability of our model to predict different types of motifs , and explore folding landscapes . in agreement with previous findings , a 12mer sequence of -hhpp- folds into an -helix . figure 2a depicts the potential mean force ( pmf ) of the helical system , using two reaction coordinates , the average absolute dihedral angle between backbone beads ( 50 corresponds to a perfect helix ; a comparison of dihedral angles of cg helix and sheet ensembles with those of pdb structures is shown in figure s1 , supporting information ) and the i to i + 3 distance of backbone beads , h1 ( 5.5 for a perfect helix ) . refer to figure s2 ( supporting information ) for pmf of the sheet system using two reaction coordinates , pair contacts ( q ) and end - to - end distance ( lc ) . ( a ) potential of mean force plot of a noncapped helical polymer at 300 k , as a function of the mean absolute backbone dihedral and h1 parameter . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) . ( c ) probability distribution of angles between oriented dipoles in the folded ensemble , of the capped system ( red ) and noncapped helical polymer ( blue ) . ( d ) potential of mean force plot of a capped helical protein at 300 k , as a function of mean absolute backbone dihedral and h1 parameter . ( e ) representative conformation of the capped helical peptide in the folded ensemble . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) , except for the negatively charged residue where the side chain is shown in red . ( f ) comparison of the time evolution of the second dihedral angle ( counting from the charged end ) between the capped system ( red ) and noncapped system ( blue ) . to explore charge capping effects , we introduced at one end of the 12-mer peptide a negatively charged amino acid ( c ) . the pmf plot for the capped system is shown in figure 2d and displays two distinct minima that correspond to a fully folded helical state and a partially unfolded state that has some helical content . it is evident from a comparison of figure 2a , d , that capping one end with a charge residue leads to an increase in helicity in the folded ensemble because the minima of the folded state is shifted toward h1 = 5.5 . also , upon helix capping the unfolded state becomes partially folded with a shift of h1 distances to lower values indicating more helical content . because the dipoles in the model act as pseudo hydrogen bonds , we also characterize the microdipole angles present in the folded helical structures . a dipole vector is defined from the negatively charged dipole bead of bb i ( bbm ) to the positively charged dipole bead of bb i+4 ( bbp ) , as depicted by the arrows in figure 2b , e . in figure 2c , the effect of charge capping is evident from the probability distribution of dipole angles , which shows better alignment of the dipole angles of the capped system over the noncapped system . the hydrogen bonds in helices of crystal structures have the co and nh dipole aligned , and almost parallel to the axis of the helix . therefore , the enhancement of dipole alignment with capping signals an increase in helicity . the time evolution of the second dihedral angle ( counting from the charged end ) depicted in figure 2f clearly shows that the charged end contains a helical turn , which is stable throughout the whole trajectory , even in the partially unfolded state that helical turn is seen to exist , most of the time . on the other hand , the second dihedral angle of the noncapped system displays more coil - like conformations , as evident from the large fluctuations from 50. as shown in figure 2e , the negatively charged sc bead of c - residue ( red bead ) groups the positively charged dipole particles of bonded neighboring backbone beads and creates a helical turn it is worth mentioning that similar results are obtained when a positively charged amino acid is used at one end . in that case , the dipole particles of bonded neighboring bb beads reorient pointing the negatively charged dipole particles toward the positively charged sc bead , creating a helical turn ( results not shown ) . our model mimics the fact that the n - terminus of a helix has amide groups that lack hydrogen bonds and the c - terminus has carbonyl groups that also lack hydrogen bonds ; therefore , the four residues of the ends are unable to satisfy the classical -helix hydrogen bonding interactions without proper capping , thus causing helix destabilization . our results indicate that the enhancement of helicity by charge capping is due to a charge the dipole alignment caused by charge capping promotes the formation of subsequent dipoles , thus promoting helix stabilization . this observation is in agreement with experimental data , showing that positively charged amino acids have a preference to the c terminus and , similarly , negatively charged side chains to the n terminus . however , it is important to note that our model can not distinguish n- and c - termini , as the bb beads are identical . as mentioned in the methods , the lj interaction strength between the charge side - chain and backbone beads ( qa / qd - p5 pair interaction of the martini force field ) had to be decreased , to allow for stabilization of the helical structure , as observed in figure 2d . without this change , the c sc bead was interacting strongly with the backbone beads by lj interactions through the main cg sites , which did not allow for the dipole particles of the bonded nearest neighbor bb beads to reorient toward the negative charge that creates a helical turn ( results not shown ) . to further evaluate the effect of dipolar interactions in stabilizing secondary folds , we have computed the angle i between the local electrostatic field , ei(ri ) and the ith dipole moment ( i ) using eq 1 . the folded ensembles of the 12-mer -helix and 12-mer -sheet are used for this calculation . an angle of 30.1 8.7 is observed for the -helix and 54.8 12 for the -sheet . these values agree very well with the reported values from scheraga et al . on a nonredundant set of pdb structures with atomistic resolution . the authors found that the angle made between the peptide bond dipole and the protein s local electrostatic field is approximately 35 for -helices and 50 for -sheets . furthermore , the average dipole energy ( (e) ) of the cg helix is 4.5 times higher than that of the cg sheet . this value is slightly greater than the one reported from atomistic pdb structures by scheraga et al . ( average dipole energies between helix and sheets was found to be between 2.64 and 3.24 ) . however , the same effect is observed ; helices are more stabilized by the backbone dipoles than sheets . also , helices are better aligned with the electric field than sheets . because the peptide with the repeating sequence of -hhpp- folds into a helix without any added bias , we have tested our model in predicting supersecondary structures such as helix bundles . hydrophilic loops are very common in proteins , and statistical analysis of protein structures has shown that the most common loop lengths in helix bundles are between three and six amino acids . taking all this into account , we have tested the behavior of our model in de novo folding of a 33 residue peptide with the sequence ( hhpp)14-(t)5-(pphh)14 . as shown in figure 3d , the chain folds into an -helix bundle in a hierarchical manner where initially the peptide folds into two distinct helices on either end . similar results were obtained when a turning region of three , four , or six residues was used ( results not shown ) . the initial folding of the helices is induced by the orientation of the backbone dipoles , which is a local effect . clearly , the secondary structure is sufficiently stable in the absence of tertiary interactions ; thus , folding proceeds in a hierarchical manner as observed for several proteins , such as the engrailed homeodomain . it is evident from the pmf ( figure 3a ) , using as reaction coordinates the average absolute dihedral angle between backbone beads and the distance h1 , that there is a distinct folded and unfolded state . the unfolded state is very broad due to the fact that unfolded structures have different degrees of helical content . this is in agreement with experimental observations , that for some helix bundle systems such as 3d , the thermal unfolded state is biased toward local helical content . ( a ) potential of mean force plot of a helix bundle at 300 k , as a function of the mean absolute backbone dihedral angle and the h1 parameter . ( b ) probability distribution of mean absolute dihedral angle in the folded ensemble of each of the two ( red , blue ) helices in the bundle . ( c ) folding curve of a helix bundle with dipoles ( blue ) and without dipoles ( red ) . ( d ) stages in helix bundle formation , starting from a coil - like conformation . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) . it is known that right- or left - handed helices are present in helix bundles with more proportion in the nature of right - handed helices . for the sequence pattern explored here , we observe that the helix bundles have either right- or left - handed helices ( figure 3b ) . this is because of the minimalist description of the backbone , which does not have any chirality . by adding the microdipoles of each helix ( arrows in figure 4b ) of the helix bundle , we have computed the macrodipole that exists in each helix . figure 4 depicts the probability distribution of the angle between the two macrodipoles ( big black arrows in figure 4b ) for folded conformations . the macrodipole angular distribution has a peak around 160 , which signals an almost antiparallel dipole orientation that provides a favorable electrostatic interaction energy for helix bundle formation . known helix bundles of protein structures in the literature , exhibit the same antiparallel orientation . ( a ) probability distribution of angles between macrodipole vectors of helices ( taking the helix as a whole ) in the helix bundles . ( b ) representative structure of a helix bundle , white arrows are between i and i + 4 dummy particles . to further explore the effects of dipolar interactions in the folding of helix bundles , we have computed the folded fraction as a function of temperature in figure 3c . a folded structure is considered if the average backbone dihedral of the whole protein structure is between 40 and 60 ( figure s1b ( supporting information ) and figure 3a ) . the blue curve in figure 3c is for our model , and the red one is without the inclusion of dipole particles . clearly , without backbone dipoles , the folded fraction is almost constant at around 0.1 folded fraction at all temperatures . the effect of the addition of backbone dipoles is that the folding process becomes cooperative , achieving a folding fraction of 0.6 at 300 k , as evident from the sigmoidal shape of the blue curve . furthermore , figure s3a ( supporting information ) shows that without the inclusion of dipoles , the system ( t = 300 k ) does not fold into a helix bundle , a representative of a collapsed structure is shown as an inset of that figure . clearly , the folding of helical bundles is influenced by both the effect of dipolar interactions and pairwise lj interactions ; i.e. , it is a combination of the sequence patterning and electrostatic interactions . however , we observe that even though there are two major energetic contributions , the effect of dipoles is largely significant in the systems considered . to further elaborate , we characterized these two energetic quantities at 300 k as a function of the reaction coordinate h1 . it is clear from figure 5b that the effect of dipoles has a marginal effect on the lj energetic contribution , which is lowest in the helical ensemble . it is also interesting to note that the helical ensemble is more stabilized by the coulombic contribution ( figure 5d ) , as can be seen by the larger energetic difference between folded ( h1 around 6 nm ) and unfolded conformations . also , the presence of dipoles increases cooperativity in folding the helical bundles , this is supported by both ( a ) the large drop in figure 5d , seen between folded and unfolded ensemble and ( b ) the sigmoidal behavior displayed by the melting curves of the helix bundles with dipoles ( figure 3c ) . ( a ) lj energetic contribution of sheet bundles with ( blue ) and without dipoles ( red ) , as a function of sheet contacts ( q ) . ( b ) lj energetic contribution of helix bundles with ( blue ) and without dipoles ( red ) , as a function of h1 . ( c ) coulombic energetic contribution of sheet bundles with dipoles , as a function of q. ( d ) coulombic energetic contribution of helix bundles with dipoles , as a function of h1 . all represented ensembles obtained at 300 k. finally , we tested the ability of the model in predicting -strand bundles , solely on the basis of sequence patterning . statistical analysis of -sheet structures has shown that hydrophilic short turning regions of two amino acids are very common . therefore , to model sheet bundles , we decided to use two polar residues to create turns . the sequence pattern hphphtthphphtthphph folds without any biases into a three - strand antiparallel -sheet , consisting of two hairpins , whose structure is reminiscent of the 3s protein . the folding of the three - stranded -sheet occurs through a hierarchical order of hairpin formation ( figure 6c ) . one hairpin is formed first with the second hairpin forming at the last stage of folding , in agreement with all - atom simulations of 3s . it is worth noticing the matching of hydrophobic and polar residues in the folded structure . figure 6a shows the pmf as a function of the fraction of sheet contacts ( q ) and end to end distance ( lc ) . two residues on neighboring strands within 47 are defined as a pair . as shown in the pmf ( figure 6a ) , this protein exhibits a two - state folded behavior , characterized by the folded and unfolded state . it is noteworthy that short sequences with a single turning region ( hphphtthphph ) fold into a -hairpin and four - stranded -sheets can be folded by adding a third turning region and an extra - hp- sequence ( results not shown ) . ( a ) potential of mean force plot of a sheet bundle at 300 k , as a function of the fraction of sheet contacts ( q ) and end - to - end distance ( lc ) . ( b ) folding curve of a sheet bundle with dipoles ( blue ) and without dipoles ( red ) . ( c ) stages in sheet bundle formation , starting from a coil - like conformation . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) . the melting curve of the sheet bundle exhibits a sigmoidal behavior characteristic of cooperative processes , as shown by the blue curve of figure 6b . a control run without the inclusion of dipole particles was also performed using the sequence hphphtthphphtthphph . as depicted by the red curve of figure 6b , without backbone it is also important to note that the control run at t = 300 k does not fold into a -sheet nor exhibit any secondary structure , as shown in figure s3b ( supporting information ) . to further characterize the driving forces behind the folding of sheet - bundles , we have characterized the energetic contributions ( lj and coulombic ) as a function of q at 300 k. it is evident from figure 5a that the presence of backbone dipoles has an effect on the lj contribution of the protein , which is minimum in the fully folded state . figure 5c depicts the changes in coulombic energy as a function of q. by comparing figure 5c and figure 5d ( helix bundles ) , one can say that coulombic energy plays a larger and a more cooperative role in helix bundles . therefore , in this model the secondary and supersecondary structures are driven by the presence of the dipoles and the sequence patterning ; however , the effect of the added dipoles is more significant . the explored sequences do not fold into helix or sheet bundles in the absence of backbone dipoles . in conclusion , the presented model introduces polarization into the protein backbone coarse - grained beads , which allows for the de novo folding of secondary structure and supersecondary structure assemblies based solely on the primary sequence . we have shown that our model is capable of folding helix bundles and -sheet strands . because of the minimalist description of the backbone , the helices fold with either right- or left - handedness . the chirality can be fixed by introducing ad hoc a quadratic term in the potential involving only quadruplets of successive backbone beads . we have also shown that secondary structure formation is driven by backbone dipole interactions and sequence patterning . the charge our model is currently composed of four bead types for the side chain ; however , it is possible to extend the model with more degrees of polarity and hydrophobicity ( by varying lj parameters ) , thus mimicking more amino acids . our model provides a step forward towards the development of a transferable coarse - grained force field for proteins without biases towards secondary structure . in addition , because some of the model parameters were borrowed from the martini coarse - grained force field , our new model has the potential to be extended to model membrane protein folding . also , because dipole interactions are influenced by the dielectric environment , we expect the proposed model to be sensitive to the nature of the environment ( low or high dielectric ) . future work will focus on exploring more complex folds like and protein aggregation .

we present a generic solvated coarse - grained protein model that can be used to characterize the driving forces behind protein folding . each amino acid is coarse - grained with two beads , a backbone , and a side chain . although the backbone beads are modeled as polar entities , side chains are hydrophobic , polar , or charged , thus allowing the exploration of how sequence patterning determines a protein fold . the change in orientation of the atoms of the coarse - grained unit is captured by the addition of two oppositely charged dummy particles inside the backbone coarse - grained bead . these two dummy charges represent a dipole that can fluctuate , thus introducing structural polarization into the coarse - grained model . realistic / content is achieved de novo without any biases in the force field toward a particular secondary structure . the dipoles created by the dummy particles interact with each other and drive the protein models to fold into unique structures depending on the amino acid patterning and presence of capping residues . we have also characterized the role of dipole dipole and dipole charge interactions in shaping the secondary and supersecondary structure of proteins . formation of helix bundles and -strands are also discussed .

Introduction Methods Results and Discussion Conclusions

a balance between charge interactions , hydrogen bonding , and hydrophobicity is responsible for the formation of stable native folds . a simple description of a hydrogen bond can be based on an electrostatic dipole the formation of a protein backbone s hydrogen bonds leads to a particular orientation of dipoles in various types of secondary structural elements . statistical analysis of folded structures has shown that nonpolar amino acids appear in the protein sequence , every three to four amino acids in -helical structures , and every two amino acids in solvent exposed -sheets . coarse - grained ( cg ) models allow us to understand which interactions are essential and which ones can be approximated . simple models where residues are represented by a few beads have been very valuable in advancing our understanding of the protein folding process . on the other hand , there exist intermediate resolution models where the backbone is modeled with fine resolution and the side chain coarse - grained . upon inclusion of a hydrogen bonding interaction , these models are able to fold into helical structures without the addition of biases toward a native fold but fail to stabilize -sheet structures . the inclusion of explicit dipole dipole interactions in these types of models has been shown to stabilize -sheets . however , all the above - mentioned models renormalize the role of the solvent through effective short - range , inter - residue interactions . thus , they are not appropriate for studying the effect of the environment in protein folding because explicit solvent is needed . also , the importance of the role of dipole interactions is evident from the work done by scheraga et al . his group has shown that an optimization of the electrostatic interaction , by aligning the dipole of each backbone plane to a protein s electric field , is enough to fold small peptides using monte carlo simulations without solvation . however , it fails to capture changes in secondary structure and thus it can not be used to study protein folding . in this work , we present a generic water - explicit cg model of proteins that can be used to characterize the driving forces behind protein folding without the addition of biasing potentials such as dihedral potentials or dependencies in the bending potential with secondary structure propensities . the model has roots in the martini cg force field and thus it has the potential to be extended to model membrane protein folding . in the current implementation , each amino acid is coarse - grained with two beads , backbone , and side chain . the change in orientation of the atoms underlying the backbone coarse - grained unit is captured by a flexible dipole that is created by oppositely charged dummy particles inside each backbone coarse - grained bead . these dipoles interact with each other through coulombic potentials and introduce structural polarization into the model . here we explore if just the addition of effective dipole interactions , in addition to lennard - jones interactions , is sufficient to produce secondary and supersecondary folds depending on sequence patterning . we have evaluated the performance of the model by folding protein structures on the basis of the sequence of amino acids . we use the sequence patterning of helices ( -hhpphh- ) and sheets ( -hphphp- ) to fold into distinct secondary structures . we then explore the role of helix capping by terminating helices with charged residues . helix and sheet bundles are designed on the basis of the above de novo patterns with the inclusion of turning regions . in the first section , we discuss the formation of protein supersecondary structures , namely helix bundles and sheet - strands and compare the results to the model without dipoles , thus drawing attention to the relevance of the backbone dipolar interactions in determining protein structure . an amino acid is modeled by two beads , a polar backbone bead ( bb ) that embeds a dipole , and a side chain bead ( sc ) . the sc beads are broadly classified into hydrophobic ( h ) , polar ( p ) , and positively and negatively charged ( c+/c ) , as depicted in figure 1a . the protein model explores the role of backbone dipoles in driving secondary and supersecondary structure formation . the cg protein model is combined with the recently developed polarizable coarse - grained water model . the spherical backbone coarse - grained bead consists of three interaction sites , the center bead bb and two dipole particles , bbm and bbp , similar to the polarizable water model and depicted in figure 1b . , the dipole orientation and moment of the backbone bead are dependent on the electric field of the surrounding environment . the rationale for using a polarizable backbone dipole as opposed to a fixed dipole is to make the dipoles environment sensitive , thus enabling future studies of the structural changes induced by backbone dipoles in different dielectric environments . because each main coarse - grained site is covalently bonded to its nearest neighbors by a harmonic bond potential , and adjacent bonds are connected by a harmonic angle potential , all the 12 and 13 nonbonded interactions are excluded from the main coarse - grained sites and the corresponding embedded dipole particles . in addition , the nonbonded interactions between dipole particles inside the backbone cg bead are excluded as well . the mass of the whole bead ( 72 amu ) is distributed equally among the three particles ( 24 amu each ) in backbone beads . vdw radius of the bb bead encloses dummy particles , bbm ( negatively charged ) , and bbp ( positively charged ) . the force field consists of bonded parameters ( harmonic bonded and angular potential ; dihedral potential is not included ) and nonbonded parameters ( 12 - 6 lennard - jones ( lj ) potential and coulombic potential ) . there are five parameters for the backbone cg bead that can be tuned to obtain the desired average dipole moment ( l , q , , kl , k ) . for the backbone cg bead , the following set of parameters yields an average dipole moment of 3.5 d : l = 0.14 nm , q = 0.34 , = 0 , kl = 5000 kj / mol , and k = 7.2 kj / mol . the sc bb harmonic bond distance is chosen as 0.4 nm with kl = 5000 kj / mol on the basis of a typical two - bead residue in the martini force field . c1 bead in the martini force field ) is not hydrophobic enough to stabilize de novo sheet formation in the current model . the lincs algorithm was used to constraint the bonds of the water molecules ( between the central cg site and the dipole particles ) . the alignment of the backbone dipoles with respect to the protein electric field is determined from the angle i between the local electrostatic field , ei(ri ) , and the ith dipole moment ( i ) using the following equation:1where2the quantities rbbmi , rbbpi , qbbm , and qbbp represent position vectors and charge of the two dummy particles and ri is the position vector:3the electric field due to the protein molecule is computed using coulomb s law:4where the summation runs over all other charged sites in the structure , except the ones belonging to i , i 1 , and i 2 , as these interactions are excluded in the model and do not contribute to the local electric field of the considered dipole i. molecular dynamics simulation of different test systems , representing fundamental secondary and supersecondary motifs were performed . different patterns of polar and nonpolar amino acids , with and without the presence of capping charge residues were used to test the capability of our model to predict different types of motifs , and explore folding landscapes . figure 2a depicts the potential mean force ( pmf ) of the helical system , using two reaction coordinates , the average absolute dihedral angle between backbone beads ( 50 corresponds to a perfect helix ; a comparison of dihedral angles of cg helix and sheet ensembles with those of pdb structures is shown in figure s1 , supporting information ) and the i to i + 3 distance of backbone beads , h1 ( 5.5 for a perfect helix ) . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) . ( c ) probability distribution of angles between oriented dipoles in the folded ensemble , of the capped system ( red ) and noncapped helical polymer ( blue ) . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) , except for the negatively charged residue where the side chain is shown in red . to explore charge capping effects , we introduced at one end of the 12-mer peptide a negatively charged amino acid ( c ) . because the dipoles in the model act as pseudo hydrogen bonds , we also characterize the microdipole angles present in the folded helical structures . in figure 2c , the effect of charge capping is evident from the probability distribution of dipole angles , which shows better alignment of the dipole angles of the capped system over the noncapped system . the hydrogen bonds in helices of crystal structures have the co and nh dipole aligned , and almost parallel to the axis of the helix . the time evolution of the second dihedral angle ( counting from the charged end ) depicted in figure 2f clearly shows that the charged end contains a helical turn , which is stable throughout the whole trajectory , even in the partially unfolded state that helical turn is seen to exist , most of the time . on the other hand , the second dihedral angle of the noncapped system displays more coil - like conformations , as evident from the large fluctuations from 50. as shown in figure 2e , the negatively charged sc bead of c - residue ( red bead ) groups the positively charged dipole particles of bonded neighboring backbone beads and creates a helical turn it is worth mentioning that similar results are obtained when a positively charged amino acid is used at one end . our model mimics the fact that the n - terminus of a helix has amide groups that lack hydrogen bonds and the c - terminus has carbonyl groups that also lack hydrogen bonds ; therefore , the four residues of the ends are unable to satisfy the classical -helix hydrogen bonding interactions without proper capping , thus causing helix destabilization . our results indicate that the enhancement of helicity by charge capping is due to a charge the dipole alignment caused by charge capping promotes the formation of subsequent dipoles , thus promoting helix stabilization . as mentioned in the methods , the lj interaction strength between the charge side - chain and backbone beads ( qa / qd - p5 pair interaction of the martini force field ) had to be decreased , to allow for stabilization of the helical structure , as observed in figure 2d . without this change , the c sc bead was interacting strongly with the backbone beads by lj interactions through the main cg sites , which did not allow for the dipole particles of the bonded nearest neighbor bb beads to reorient toward the negative charge that creates a helical turn ( results not shown ) . the authors found that the angle made between the peptide bond dipole and the protein s local electrostatic field is approximately 35 for -helices and 50 for -sheets . because the peptide with the repeating sequence of -hhpp- folds into a helix without any added bias , we have tested our model in predicting supersecondary structures such as helix bundles . hydrophilic loops are very common in proteins , and statistical analysis of protein structures has shown that the most common loop lengths in helix bundles are between three and six amino acids . taking all this into account , we have tested the behavior of our model in de novo folding of a 33 residue peptide with the sequence ( hhpp)14-(t)5-(pphh)14 . the initial folding of the helices is induced by the orientation of the backbone dipoles , which is a local effect . clearly , the secondary structure is sufficiently stable in the absence of tertiary interactions ; thus , folding proceeds in a hierarchical manner as observed for several proteins , such as the engrailed homeodomain . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) . it is known that right- or left - handed helices are present in helix bundles with more proportion in the nature of right - handed helices . this is because of the minimalist description of the backbone , which does not have any chirality . by adding the microdipoles of each helix ( arrows in figure 4b ) of the helix bundle , we have computed the macrodipole that exists in each helix . known helix bundles of protein structures in the literature , exhibit the same antiparallel orientation . ( a ) probability distribution of angles between macrodipole vectors of helices ( taking the helix as a whole ) in the helix bundles . ( b ) representative structure of a helix bundle , white arrows are between i and i + 4 dummy particles . to further explore the effects of dipolar interactions in the folding of helix bundles , we have computed the folded fraction as a function of temperature in figure 3c . the blue curve in figure 3c is for our model , and the red one is without the inclusion of dipole particles . the effect of the addition of backbone dipoles is that the folding process becomes cooperative , achieving a folding fraction of 0.6 at 300 k , as evident from the sigmoidal shape of the blue curve . furthermore , figure s3a ( supporting information ) shows that without the inclusion of dipoles , the system ( t = 300 k ) does not fold into a helix bundle , a representative of a collapsed structure is shown as an inset of that figure . , it is a combination of the sequence patterning and electrostatic interactions . to further elaborate , we characterized these two energetic quantities at 300 k as a function of the reaction coordinate h1 . also , the presence of dipoles increases cooperativity in folding the helical bundles , this is supported by both ( a ) the large drop in figure 5d , seen between folded and unfolded ensemble and ( b ) the sigmoidal behavior displayed by the melting curves of the helix bundles with dipoles ( figure 3c ) . ( b ) lj energetic contribution of helix bundles with ( blue ) and without dipoles ( red ) , as a function of h1 . ( d ) coulombic energetic contribution of helix bundles with dipoles , as a function of h1 . all represented ensembles obtained at 300 k. finally , we tested the ability of the model in predicting -strand bundles , solely on the basis of sequence patterning . the sequence pattern hphphtthphphtthphph folds without any biases into a three - strand antiparallel -sheet , consisting of two hairpins , whose structure is reminiscent of the 3s protein . as shown in the pmf ( figure 6a ) , this protein exhibits a two - state folded behavior , characterized by the folded and unfolded state . only backbone beads are shown ( green for polar and cyan for hydrophobic residues ) . as depicted by the red curve of figure 6b , without backbone it is also important to note that the control run at t = 300 k does not fold into a -sheet nor exhibit any secondary structure , as shown in figure s3b ( supporting information ) . to further characterize the driving forces behind the folding of sheet - bundles , we have characterized the energetic contributions ( lj and coulombic ) as a function of q at 300 k. it is evident from figure 5a that the presence of backbone dipoles has an effect on the lj contribution of the protein , which is minimum in the fully folded state . figure 5c depicts the changes in coulombic energy as a function of q. by comparing figure 5c and figure 5d ( helix bundles ) , one can say that coulombic energy plays a larger and a more cooperative role in helix bundles . therefore , in this model the secondary and supersecondary structures are driven by the presence of the dipoles and the sequence patterning ; however , the effect of the added dipoles is more significant . in conclusion , the presented model introduces polarization into the protein backbone coarse - grained beads , which allows for the de novo folding of secondary structure and supersecondary structure assemblies based solely on the primary sequence . we have shown that our model is capable of folding helix bundles and -sheet strands . because of the minimalist description of the backbone , the helices fold with either right- or left - handedness . the chirality can be fixed by introducing ad hoc a quadratic term in the potential involving only quadruplets of successive backbone beads . we have also shown that secondary structure formation is driven by backbone dipole interactions and sequence patterning . the charge our model is currently composed of four bead types for the side chain ; however , it is possible to extend the model with more degrees of polarity and hydrophobicity ( by varying lj parameters ) , thus mimicking more amino acids . our model provides a step forward towards the development of a transferable coarse - grained force field for proteins without biases towards secondary structure . in addition , because some of the model parameters were borrowed from the martini coarse - grained force field , our new model has the potential to be extended to model membrane protein folding . also , because dipole interactions are influenced by the dielectric environment , we expect the proposed model to be sensitive to the nature of the environment ( low or high dielectric ) .

the study participants were 36 990 unhospitalized residents of takayama city , gifu , japan , aged 35 years or older in september 1992 . the details of the population - based cohorts have been described previously elsewhere.15 a total of 31 552 residents ( 85.3% ) completed a baseline self - administered questionnaire which included questions on demography , anthropometric characteristics , medical history , smoking status , physical activity and diet . female participants were also asked about their reproductive characteristics , including age at menarche , age at first delivery , menopausal status , parity number and history of hormone replacement therapy . in the baseline questionnaire , the subjects were asked about their smoking habits ; never , former or current smokers . smokers were defined as people who had smoked a total of at least 20 packs of cigarettes in their life . former and current smokers were asked to choose from among five options for the number of cigarettes smoked per day ; 5 cigarettes or less , 6 to 10 cigarettes , 11 to 20 cigarettes , 21 to 30 cigarettes , or 31 cigarettes or more . although the questionnaire did not include the smoking status of each subject 's spouse , we identified married couples by the following conditions : ( i ) the pair consisted of a man and a woman with the same household number ; ( ii ) the difference between their ages was < 15 years ; ( iii ) both the man and the woman indicated their marital status as married ; and ( iv ) that he or she belonged to only one pair . the details have been described elsewhere.16 the response about smoking status elicited from the spouse 's questionnaire was used as the index of husband 's smoking . dietary data , including alcohol consumption , were assessed using a 169-item semi - quantitative food frequency questionnaire ( ffq ) . in the questionnaire , participants were asked how often on average they consumed each of the food items listed , and what the usual serving size of each item was during the past year . the questions on alcohol intake covered six different types of liquor : sake , shochu , beer , light beer , wine and hard liquor . for each item , the questions included nine categories of frequency ( never / less than once per month , once per month , 23 times per month , once per week , 23 times per week , 46 times per week , once per day , 23 times per day and 4 times per day or more ) and four categories of each serving size ( the number of cups , glasses or bottles ) . individual nutrient intake was also estimated from the frequency of ingestion and portion size using the japanese standard table of food composition ( 5th revised and enlarged edition ) , published by the science and technology agency of japan.17 the validity and reproducibility of the questionnaire were previously reported to be reliable.18 the spearman correlation coefficient between the ffq and 12-day diet records kept over a 1-year period for alcohol intake was 0.64 for women . physical activity was assessed by asking participants how much time on average they spent during the past year on activities from a list of strenuous sports , vigorous work and moderate activities . the number of hours per week spent in each activity was multiplied by the corresponding energy expenditure , expressed as a metabolic equivalent ( met ) , and the sum of the product was counted as the physical activity score ( meth / week ) . the details , including its validity , are described elsewhere.19 among 17 125 women included in the baseline survey ( 1 september 1992 ) , 543 who were diagnosed with breast cancer before the baseline and/or reported a positive history of any cancers at the baseline were excluded . 929 persons ( 5.6% ) moved out of the study area , and the date of emigration was unknown for 141 women ( 0.9% ) . the incidence of cancer was mainly confirmed through two regional population - based cancer registries in gifu . information was also collected from a local base hospital , which had played a leading role in medical care for the residents in the study area . the causes of cancer were coded according to the international classification of diseases and health related problems , 10th revision . the mortality - to - incidence ratio for breast cancer was 0.16 , and 6.0% of patients were ascertained by death certificate - only registration , indicating the very complete cancer registration of this cohort . this study was approved by the ethical board of the gifu university graduate school of medicine . after female subjects with neither information on smoking status of their own nor of their husband were excluded , our analysis finally included 15 719 women . for smoking status of the husbands , current smokers were re - categorized into one of two groups : smokers of 20 cigarettes or less per day , or smokers of 21 cigarettes or more per day . the end of follow up was determined as the date of breast cancer diagnosis , the date of emigration from the study area , the date of death or the end of the study , whichever came first . for women who moved away on a date unknown , their last confirmed date of residence in the study area was used as their censored date . relative risks and 95% confidence intervals ( ci ) for breast cancer were estimated for the groups of smoking status using the cox proportional hazards regression model . the reference group was set as the never smokers . to eliminate the impact of active smoking , the analyses for husband 's smoking status and breast cancer risk were conducted among never female smokers . to test a linear trend for breast cancer risk by smoking status of the husbands , we assigned 0 for never and former smokers , 10 for smokers of 20 cigarettes or less per day , and 21 for smokers of 21 cigarettes or more per day , and inputted them as continuous variable into the models . covariates included in the models were the following potential confounders : age ( years , continuous ) , body mass index ( quartiles ) , physical activity score ( continuous ) , alcohol consumption ( g / day ) , years of education ( 8 , 911 , 1214 , 15 years ) , age at menarche ( 12 , 1314 , 1516 , 17 years ) , age at first delivery ( non - parous , 20 , 2025 , 2630 , 31 years ) , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . these analyses were repeated among never smokers after being stratified to premenopausal and postmenopausal women at the baseline , because menopausal status was known to affect the etiology of breast cancer . to clarify whether alcohol drinking modifies the effects of smoking on breast cancer , we further examined the associations stratified by alcohol consumption ( non - drinkers or drinkers ) . all analyses were conducted using the sas computer program , version 9.3 ( sas institute ) . a p - value < 0.05 was considered statistically significant in all analyses . the study participants were 36 990 unhospitalized residents of takayama city , gifu , japan , aged 35 years or older in september 1992 . the details of the population - based cohorts have been described previously elsewhere.15 a total of 31 552 residents ( 85.3% ) completed a baseline self - administered questionnaire which included questions on demography , anthropometric characteristics , medical history , smoking status , physical activity and diet . female participants were also asked about their reproductive characteristics , including age at menarche , age at first delivery , menopausal status , parity number and history of hormone replacement therapy . in the baseline questionnaire , the subjects were asked about their smoking habits ; never , former or current smokers . smokers were defined as people who had smoked a total of at least 20 packs of cigarettes in their life . former and current smokers were asked to choose from among five options for the number of cigarettes smoked per day ; 5 cigarettes or less , 6 to 10 cigarettes , 11 to 20 cigarettes , 21 to 30 cigarettes , or 31 cigarettes or more . although the questionnaire did not include the smoking status of each subject 's spouse , we identified married couples by the following conditions : ( i ) the pair consisted of a man and a woman with the same household number ; ( ii ) the difference between their ages was < 15 years ; ( iii ) both the man and the woman indicated their marital status as married ; and ( iv ) that he or she belonged to only one pair . the details have been described elsewhere.16 the response about smoking status elicited from the spouse 's questionnaire was used as the index of husband 's smoking . dietary data , including alcohol consumption , were assessed using a 169-item semi - quantitative food frequency questionnaire ( ffq ) . in the questionnaire , participants were asked how often on average they consumed each of the food items listed , and what the usual serving size of each item was during the past year . the questions on alcohol intake covered six different types of liquor : sake , shochu , beer , light beer , wine and hard liquor . for each item , the questions included nine categories of frequency ( never / less than once per month , once per month , 23 times per month , once per week , 23 times per week , 46 times per week , once per day , 23 times per day and 4 times per day or more ) and four categories of each serving size ( the number of cups , glasses or bottles ) . individual nutrient intake was also estimated from the frequency of ingestion and portion size using the japanese standard table of food composition ( 5th revised and enlarged edition ) , published by the science and technology agency of japan.17 the validity and reproducibility of the questionnaire were previously reported to be reliable.18 the spearman correlation coefficient between the ffq and 12-day diet records kept over a 1-year period for alcohol intake was 0.64 for women . physical activity was assessed by asking participants how much time on average they spent during the past year on activities from a list of strenuous sports , vigorous work and moderate activities . the number of hours per week spent in each activity was multiplied by the corresponding energy expenditure , expressed as a metabolic equivalent ( met ) , and the sum of the product was counted as the physical activity score ( meth / week ) . the details , including its validity , are described elsewhere.19 among 17 125 women included in the baseline survey ( 1 september 1992 ) , 543 who were diagnosed with breast cancer before the baseline and/or reported a positive history of any cancers at the baseline were excluded . 929 persons ( 5.6% ) moved out of the study area , and the date of emigration was unknown for 141 women ( 0.9% ) . the incidence of cancer was mainly confirmed through two regional population - based cancer registries in gifu . information was also collected from a local base hospital , which had played a leading role in medical care for the residents in the study area . the causes of cancer were coded according to the international classification of diseases and health related problems , 10th revision . the mortality - to - incidence ratio for breast cancer was 0.16 , and 6.0% of patients were ascertained by death certificate - only registration , indicating the very complete cancer registration of this cohort . this study was approved by the ethical board of the gifu university graduate school of medicine . after female subjects with neither information on smoking status of their own nor of their husband were excluded , our analysis finally included 15 719 women . for smoking status of the husbands , current smokers were re - categorized into one of two groups : smokers of 20 cigarettes or less per day , or smokers of 21 cigarettes or more per day . the end of follow up was determined as the date of breast cancer diagnosis , the date of emigration from the study area , the date of death or the end of the study , whichever came first . for women who moved away on a date unknown , their last confirmed date of residence in the study area was used as their censored date . relative risks and 95% confidence intervals ( ci ) for breast cancer were estimated for the groups of smoking status using the cox proportional hazards regression model . the reference group was set as the never smokers . to eliminate the impact of active smoking , the analyses for husband 's smoking status and breast cancer risk were conducted among never female smokers . to test a linear trend for breast cancer risk by smoking status of the husbands , we assigned 0 for never and former smokers , 10 for smokers of 20 cigarettes or less per day , and 21 for smokers of 21 cigarettes or more per day , and inputted them as continuous variable into the models . covariates included in the models were the following potential confounders : age ( years , continuous ) , body mass index ( quartiles ) , physical activity score ( continuous ) , alcohol consumption ( g / day ) , years of education ( 8 , 911 , 1214 , 15 years ) , age at menarche ( 12 , 1314 , 1516 , 17 years ) , age at first delivery ( non - parous , 20 , 2025 , 2630 , 31 years ) , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . these analyses were repeated among never smokers after being stratified to premenopausal and postmenopausal women at the baseline , because menopausal status was known to affect the etiology of breast cancer . to clarify whether alcohol drinking modifies the effects of smoking on breast cancer , we further examined the associations stratified by alcohol consumption ( non - drinkers or drinkers ) . all analyses were conducted using the sas computer program , version 9.3 ( sas institute ) . a p - value < 0.05 was considered statistically significant in all analyses . among 15 719 subjects , 14 830 ( 94.3% ) responded to the questions about their own smoking status . it was ascertained that 10 599 participants had husbands , and 10 427 ( 98.4% ) husbands responded to the questions about smoking status . the characteristics of participants were shown as the mean ( standard deviation ) or number ( percentage ) of each category , according to the subjects ' own smoking status and each of their husbands , as shown in table1 . women who had ever smoked were likely to drink more , to have fewer experiences of childbirth , and to have histories of hormone replacement therapy . the subjects ' own smoking status was related to the smoking status of their husbands . table2 shows the associations of smoking habits of subjects and their husbands with incidence of breast cancer . active smoking by the subjects was not associated with the risk of breast cancer in the multivariate - adjusted model . when subjects were limited to never smokers , compared with women whose husbands had never smoked , the hazard ratio was 1.98 ( 95% ci : 1.033.84 ) among women whose husband was a current smoker of 21 cigarettes per day or more . the hazard ratios of breast cancer grew gradually higher along with the numbers of cigarettes husbands currently smoked per day ( p for linear trend = 0.023 ) . associations of smoking habits of subject and her husband with incidence of breast cancer from takayama study estimated hazard ratio after adjustments for age , body mass index ( quartiles ) , physical activity score , alcohol consumption , education years ( 8 , 911 , 1214 , 15 years ) , age at menarche ( 12 , 1314 , 1516 , 17 years ) , age at first delivery ( non - parous , 20 , 2125 , 2630 , 31 years ) , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . hr , hazard ratio . when the analyses were repeated among never smokers by menopausal status at the baseline , increased risks of breast cancer among women with a husband who smoked were observed among premenopausal and postmenopausal women ( table3 ) . among postmenopausal never smokers , women whose husband was a current smoker of 21 cigarettes per day or more had a relative risk of 2.18 ( 95% ci : 0.805.95 ) for breast cancer compared with women with a husband who had never smoked . associations of husband 's smoking status with incidence of breast cancer according to menopausal status and alcohol consumption at baseline among never smokers estimated hazard ratio after adjustments for age , body mass index ( quartiles ) , physical activity score , alcohol consumption , education years ( 8 , 911 , 1214y , 15 years ) , age at menarche ( 12 , 1314 , 1516 17 years ) , age at first delivery ( non - parous , 20 , 2125 , 2630 , 31 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . estimated hazard ratio after adjustments for age , body mass index , physical activity score , alcohol consumption , education years , age at menarche , age at menopause ( 49 , 50 years ) , age at first delivery , parity number and history of hormone replacement therapy . estimated hazard ratio after adjustments for age , body mass index , physical activity score , education years , age at menarche , age at first delivery , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number , and history of hormone replacement therapy . estimated hazard ratio after adjustments for age , body mass index , physical activity score , alcohol consumption , education years , age at menarche , age at first delivery , menopausal status , parity number and history of hormone replacement therapy . the analyses stratified by alcohol consumption among never smokers revealed that the increased risks of breast cancer among women having smoking husband were stronger among those who consumed no alcohol , although the interaction was not statistically significant ( p for interaction = 0.51 ) . we repeated stratified analysis using the median alcohol intake ( 1.48 g / day ) as a cut - off value . among non - drinkers and light - drinkers ( alcohol intake 1.48 g / day ) , the hazard ratios for breast cancer were 2.94 ( 95% ci : 0.8510.14 ) , 4.14 ( 95% ci : 1.2413.86 ) and 4.94 ( 95% ci : 1.4217.25 ) , respectively , for women whose husband was a former smoker , a current smoker of 20 cigarettes per day or less , and a current smoker of 21 cigarettes per day or more , compared with women whose husband had never smoked . among drinkers ( alcohol intake > 1.48 g / day ) , the corresponding values were 0.71 ( 95% ci : 0.301.67 ) , 0.95 ( 95% ci : 0.432.12 ) and 1.19 ( 95% ci : 0.522.70 ) , respectively . the p - value for interaction by alcohol drinking was 0.18 . we re - analyzed the data after excluding 12 patients who were diagnosed with breast cancer in the first 2 years of follow up to eliminate those who might have had breast cancer but had not noticed it yet at baseline . previous reports about smoking and breast cancer have mainly consisted of us and european studies , and there have been few studies on the asian population.2025 to our knowledge , there are only five prospective japanese cohorts that reported the association of active2023 and passive smoking20,2224 with breast cancer , and they showed conflicting results . only one study22 reported a positive association between active smoking and breast cancer incidence ; the other three studies reported null association.20,21,23 for passive smoking , one study revealed the increased risk for breast cancer mortality among postmenopausal nonsmoking women,20 and another showed the risk increment of incidence among premenopausal women.22 meanwhile , null23 and negative24 associations were also reported . in this study , we observed an increased risk of breast cancer among women who were exposed to cigarette smoke from their husbands , and the risk increment showed a dose - response relationship with the number of cigarettes . a stronger positive relationship between a husband 's smoking and breast cancer was observed among women who did not habitually consume alcohol . active smoking has been reported to have simultaneously opposite effects on the progress of breast cancer ; some smoke constituents , such as polycyclic aromatic hydrocarbons , aromatic amines and n - nitrosamines , potentially induce mammary carcinogenesis , while the antiestrogenic effects of smoking reduce the risk of breast cancer.7,14 although these factors might complicate a relationship between active smoking and breast cancer risk , our results regarding active smoking might have been influenced by the fact that the smoking rate of women in this generation was very low , so that it is difficult to meaningfully evaluate the effect of active smoking on breast cancer in this cohort . in addition , it is likely that japanese female smokers concealed their smoking . the reference group might be irrelevant to truly smoke - free persons ; it included never smokers who were exposed to secondhand smoke . however , the analyses of women without husbands smoking did not have enough statistical power to find any associations ( n = 1607 ) . sidestream smoke has been reported to contain chemical compounds , including some carcinogens.9 some of the carcinogens are metabolized and activated by mammary epithelial cells.26 a higher prevalence of smoking - specific dna adducts and p53 mutation was reported in smokers than in non - smokers.27,28 our results support the theory that there is a carcinogenic property of tobacco smoke for breast cancer , by demonstrating the association between husbands smoking and increased risk of breast cancer with a dose - response relationship . compared with previous us and european studies , the risks of breast cancer were estimated to be at a higher level , consistent with another japanese study by hirayama,20 showing that the relative risk for breast cancer mortality was 1.3 among women whose husband smoked 119 cigarettes daily and 2.68 among those whose husband smoked 20 cigarettes or more daily . however , we also must note that we did not detect risk increment of breast cancer among active smokers who inhale sidestream smoke through their own smoking . we additionally observed that an increased risk of breast cancer among women with a smoking husband was pronounced among those who did not habitually consume alcohol . although our finding suggests that non - drinkers were susceptible to the effect tobacco smoke has on breast cancer , the analyses were conducted with a small number of cases in subgroup analyses , showing the possibility that the results were obtained by chance . two previous case - control studies29,30 and one cohort study10 reported that alcohol consumption did not modify the association with breast cancer , which is inconsistent with our results . however , all three studies observed an overall null association between passive smoking and breast cancer , whereas we detected a significant positive association between husbands smoking and breast cancer risk . for active smoking , it was observed in a norwegian swedish cohort study that the risks from active smoking were estimated to be higher among non - drinkers than among all subjects.31 mechanisms underlying the associations between smoking , alcohol and breast cancer are unknown . because alcohol has been hypothesized to affect breast cancer by increasing estrogen levels,12 the small difference in circulating estrogen or alcohol levels might influence cigarette smoke 's carcinogenic property . slow acetylation of aromatic amines was related to an increased risk of breast cancer.5 although there might be a greater proportion of women with n - acetyltransferase 2 ( nat2 ) slow genotype among non - drinkers , the association between nat2 genotypes and drinking habits is not well known . with the increasing evidence of an association between dna methylation and breast cancer,32 smoking and drinking the modifying effects of alcohol on increased breast cancer risk from passive smoking might need to be confirmed in future studies . our cohort study was conducted with a prospective design which is expected to be less subject to bias than a case - control study because the information on smoking status was collected before the diagnosis of breast cancer . the other strengths of our study were a good participation rate , a long follow up and information obtained for several confounders . information on subjects ' or husbands ' smoking status was based on self - reports . compared with estimates based on urinary cotinine concentration , high sensitivity and specificity of self - reported smoking status were reported ( 92.1 and 98.4% for men , and 91.2 and 98.3% for women).33 however , in the present study , husbands ' smoking status was obtained only for the subjects whose spouse was ascertained from the study data ( 67.4% ) . when 3681 women who reported being single , widowed or divorced at the baseline were assigned to the group without exposure to smoke from a husband , the positive association between husband 's smoking and breast cancer was not altered . compared with the reference group of women with a husband who had never smoked or who were without a husband , the hazard ratio was 1.76 ( 95% ci : 1.112.79 ) among women whose husband was a current smoker of 21 cigarettes per day or more . passive smoking except for smoke exposure from the subjects ' husbands was not assessed ; none of the exposure to smoke from other residents at home , at the workplace or in public places was obtained . the exposure evaluation was performed only at baseline and included no information on smoking exposure during childhood . although underlying diseases or preclinical signs at the baseline may have affected lifestyles , the exclusion of those who had died during the first 2 years of follow up did not substantially change the results . finally , despite the consideration of numerous lifestyle , reproductive and dietary factors in the analyses , we might not have fully excluded some residual or unknown confounding of lifestyle or social factors . in conclusion , this prospective study of japanese women demonstrated the increased risk of breast cancer among women exposed to secondhand cigarette smoke from their husband , with a dose - response relationship . the increased risks for women with a smoking husband were stronger among those who do not habitually consume alcohol . these results suggest that exposure to smoke from husbands is a potential risk factor for breast cancer , and , considering the limited data currently available , the modifying effects of alcohol on increased breast cancer risk from passive smoking need to be confirmed in further studies .

the effects of smoking on breast cancer remain unclear . we assessed the associations of subjects ' or husbands ' smoking status with breast cancer incidence in a population - based prospective study in japan . the subjects were 15 719 women aged 35 years or older . the follow up was conducted from september 1992 to march 2008 . cancer incidence was mainly confirmed through regional population - based cancer registries . breast cancer was defined as code c50 according to the international classification of diseases and health related problems , 10th revision . lifestyle , including smoking status , was assessed with a self - administered questionnaire . alcohol consumption was assessed with a validated food - frequency questionnaire . after multivariate adjustments for age , body mass index , alcohol consumption , physical activity , education , age at menarche , age at first delivery , menopausal status , number of children and history of hormone replacement therapy , active smoking was not associated with the risk of breast cancer . compared with never smokers whose husband had never smoked , the risks of breast cancer were 1.98 ( 95% ci : 1.033.84 ) among never smokers whose husband was a current smoker of 21 cigarettes per day or more . the increased risk of breast cancer among women having a smoking husband was pronounced among those who did not habitually consume alcohol . these results suggest that exposure to smoke from husbands is a potential risk factor for breast cancer . the impact of alcohol consumption on the increased breast cancer risk from passive smoking needs to be addressed in further studies .

Materials and Methods Participants and design Statistical analyses Results Discussion Disclosure Statement

the study participants were 36 990 unhospitalized residents of takayama city , gifu , japan , aged 35 years or older in september 1992 . the details of the population - based cohorts have been described previously elsewhere.15 a total of 31 552 residents ( 85.3% ) completed a baseline self - administered questionnaire which included questions on demography , anthropometric characteristics , medical history , smoking status , physical activity and diet . female participants were also asked about their reproductive characteristics , including age at menarche , age at first delivery , menopausal status , parity number and history of hormone replacement therapy . although the questionnaire did not include the smoking status of each subject 's spouse , we identified married couples by the following conditions : ( i ) the pair consisted of a man and a woman with the same household number ; ( ii ) the difference between their ages was < 15 years ; ( iii ) both the man and the woman indicated their marital status as married ; and ( iv ) that he or she belonged to only one pair . dietary data , including alcohol consumption , were assessed using a 169-item semi - quantitative food frequency questionnaire ( ffq ) . for each item , the questions included nine categories of frequency ( never / less than once per month , once per month , 23 times per month , once per week , 23 times per week , 46 times per week , once per day , 23 times per day and 4 times per day or more ) and four categories of each serving size ( the number of cups , glasses or bottles ) . the details , including its validity , are described elsewhere.19 among 17 125 women included in the baseline survey ( 1 september 1992 ) , 543 who were diagnosed with breast cancer before the baseline and/or reported a positive history of any cancers at the baseline were excluded . the incidence of cancer was mainly confirmed through two regional population - based cancer registries in gifu . the causes of cancer were coded according to the international classification of diseases and health related problems , 10th revision . after female subjects with neither information on smoking status of their own nor of their husband were excluded , our analysis finally included 15 719 women . for smoking status of the husbands , current smokers were re - categorized into one of two groups : smokers of 20 cigarettes or less per day , or smokers of 21 cigarettes or more per day . the end of follow up was determined as the date of breast cancer diagnosis , the date of emigration from the study area , the date of death or the end of the study , whichever came first . relative risks and 95% confidence intervals ( ci ) for breast cancer were estimated for the groups of smoking status using the cox proportional hazards regression model . to eliminate the impact of active smoking , the analyses for husband 's smoking status and breast cancer risk were conducted among never female smokers . to test a linear trend for breast cancer risk by smoking status of the husbands , we assigned 0 for never and former smokers , 10 for smokers of 20 cigarettes or less per day , and 21 for smokers of 21 cigarettes or more per day , and inputted them as continuous variable into the models . covariates included in the models were the following potential confounders : age ( years , continuous ) , body mass index ( quartiles ) , physical activity score ( continuous ) , alcohol consumption ( g / day ) , years of education ( 8 , 911 , 1214 , 15 years ) , age at menarche ( 12 , 1314 , 1516 , 17 years ) , age at first delivery ( non - parous , 20 , 2025 , 2630 , 31 years ) , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . these analyses were repeated among never smokers after being stratified to premenopausal and postmenopausal women at the baseline , because menopausal status was known to affect the etiology of breast cancer . to clarify whether alcohol drinking modifies the effects of smoking on breast cancer , we further examined the associations stratified by alcohol consumption ( non - drinkers or drinkers ) . the study participants were 36 990 unhospitalized residents of takayama city , gifu , japan , aged 35 years or older in september 1992 . the details of the population - based cohorts have been described previously elsewhere.15 a total of 31 552 residents ( 85.3% ) completed a baseline self - administered questionnaire which included questions on demography , anthropometric characteristics , medical history , smoking status , physical activity and diet . female participants were also asked about their reproductive characteristics , including age at menarche , age at first delivery , menopausal status , parity number and history of hormone replacement therapy . in the baseline questionnaire , the subjects were asked about their smoking habits ; never , former or current smokers . former and current smokers were asked to choose from among five options for the number of cigarettes smoked per day ; 5 cigarettes or less , 6 to 10 cigarettes , 11 to 20 cigarettes , 21 to 30 cigarettes , or 31 cigarettes or more . although the questionnaire did not include the smoking status of each subject 's spouse , we identified married couples by the following conditions : ( i ) the pair consisted of a man and a woman with the same household number ; ( ii ) the difference between their ages was < 15 years ; ( iii ) both the man and the woman indicated their marital status as married ; and ( iv ) that he or she belonged to only one pair . dietary data , including alcohol consumption , were assessed using a 169-item semi - quantitative food frequency questionnaire ( ffq ) . for each item , the questions included nine categories of frequency ( never / less than once per month , once per month , 23 times per month , once per week , 23 times per week , 46 times per week , once per day , 23 times per day and 4 times per day or more ) and four categories of each serving size ( the number of cups , glasses or bottles ) . the details , including its validity , are described elsewhere.19 among 17 125 women included in the baseline survey ( 1 september 1992 ) , 543 who were diagnosed with breast cancer before the baseline and/or reported a positive history of any cancers at the baseline were excluded . the incidence of cancer was mainly confirmed through two regional population - based cancer registries in gifu . the causes of cancer were coded according to the international classification of diseases and health related problems , 10th revision . for smoking status of the husbands , current smokers were re - categorized into one of two groups : smokers of 20 cigarettes or less per day , or smokers of 21 cigarettes or more per day . the end of follow up was determined as the date of breast cancer diagnosis , the date of emigration from the study area , the date of death or the end of the study , whichever came first . relative risks and 95% confidence intervals ( ci ) for breast cancer were estimated for the groups of smoking status using the cox proportional hazards regression model . to eliminate the impact of active smoking , the analyses for husband 's smoking status and breast cancer risk were conducted among never female smokers . to test a linear trend for breast cancer risk by smoking status of the husbands , we assigned 0 for never and former smokers , 10 for smokers of 20 cigarettes or less per day , and 21 for smokers of 21 cigarettes or more per day , and inputted them as continuous variable into the models . covariates included in the models were the following potential confounders : age ( years , continuous ) , body mass index ( quartiles ) , physical activity score ( continuous ) , alcohol consumption ( g / day ) , years of education ( 8 , 911 , 1214 , 15 years ) , age at menarche ( 12 , 1314 , 1516 , 17 years ) , age at first delivery ( non - parous , 20 , 2025 , 2630 , 31 years ) , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . these analyses were repeated among never smokers after being stratified to premenopausal and postmenopausal women at the baseline , because menopausal status was known to affect the etiology of breast cancer . to clarify whether alcohol drinking modifies the effects of smoking on breast cancer , we further examined the associations stratified by alcohol consumption ( non - drinkers or drinkers ) . the characteristics of participants were shown as the mean ( standard deviation ) or number ( percentage ) of each category , according to the subjects ' own smoking status and each of their husbands , as shown in table1 . women who had ever smoked were likely to drink more , to have fewer experiences of childbirth , and to have histories of hormone replacement therapy . the subjects ' own smoking status was related to the smoking status of their husbands . table2 shows the associations of smoking habits of subjects and their husbands with incidence of breast cancer . active smoking by the subjects was not associated with the risk of breast cancer in the multivariate - adjusted model . when subjects were limited to never smokers , compared with women whose husbands had never smoked , the hazard ratio was 1.98 ( 95% ci : 1.033.84 ) among women whose husband was a current smoker of 21 cigarettes per day or more . the hazard ratios of breast cancer grew gradually higher along with the numbers of cigarettes husbands currently smoked per day ( p for linear trend = 0.023 ) . associations of smoking habits of subject and her husband with incidence of breast cancer from takayama study estimated hazard ratio after adjustments for age , body mass index ( quartiles ) , physical activity score , alcohol consumption , education years ( 8 , 911 , 1214 , 15 years ) , age at menarche ( 12 , 1314 , 1516 , 17 years ) , age at first delivery ( non - parous , 20 , 2125 , 2630 , 31 years ) , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . when the analyses were repeated among never smokers by menopausal status at the baseline , increased risks of breast cancer among women with a husband who smoked were observed among premenopausal and postmenopausal women ( table3 ) . among postmenopausal never smokers , women whose husband was a current smoker of 21 cigarettes per day or more had a relative risk of 2.18 ( 95% ci : 0.805.95 ) for breast cancer compared with women with a husband who had never smoked . associations of husband 's smoking status with incidence of breast cancer according to menopausal status and alcohol consumption at baseline among never smokers estimated hazard ratio after adjustments for age , body mass index ( quartiles ) , physical activity score , alcohol consumption , education years ( 8 , 911 , 1214y , 15 years ) , age at menarche ( 12 , 1314 , 1516 17 years ) , age at first delivery ( non - parous , 20 , 2125 , 2630 , 31 years ) , parity number ( 0 , 1 , 2 , 3 ) and history of hormone replacement therapy ( yes , no ) . estimated hazard ratio after adjustments for age , body mass index , physical activity score , alcohol consumption , education years , age at menarche , age at menopause ( 49 , 50 years ) , age at first delivery , parity number and history of hormone replacement therapy . estimated hazard ratio after adjustments for age , body mass index , physical activity score , education years , age at menarche , age at first delivery , menopausal status ( premenopausal , postmenopausal 49 years , postmenopausal 50 years ) , parity number , and history of hormone replacement therapy . estimated hazard ratio after adjustments for age , body mass index , physical activity score , alcohol consumption , education years , age at menarche , age at first delivery , menopausal status , parity number and history of hormone replacement therapy . the analyses stratified by alcohol consumption among never smokers revealed that the increased risks of breast cancer among women having smoking husband were stronger among those who consumed no alcohol , although the interaction was not statistically significant ( p for interaction = 0.51 ) . among non - drinkers and light - drinkers ( alcohol intake 1.48 g / day ) , the hazard ratios for breast cancer were 2.94 ( 95% ci : 0.8510.14 ) , 4.14 ( 95% ci : 1.2413.86 ) and 4.94 ( 95% ci : 1.4217.25 ) , respectively , for women whose husband was a former smoker , a current smoker of 20 cigarettes per day or less , and a current smoker of 21 cigarettes per day or more , compared with women whose husband had never smoked . among drinkers ( alcohol intake > 1.48 g / day ) , the corresponding values were 0.71 ( 95% ci : 0.301.67 ) , 0.95 ( 95% ci : 0.432.12 ) and 1.19 ( 95% ci : 0.522.70 ) , respectively . we re - analyzed the data after excluding 12 patients who were diagnosed with breast cancer in the first 2 years of follow up to eliminate those who might have had breast cancer but had not noticed it yet at baseline . previous reports about smoking and breast cancer have mainly consisted of us and european studies , and there have been few studies on the asian population.2025 to our knowledge , there are only five prospective japanese cohorts that reported the association of active2023 and passive smoking20,2224 with breast cancer , and they showed conflicting results . only one study22 reported a positive association between active smoking and breast cancer incidence ; the other three studies reported null association.20,21,23 for passive smoking , one study revealed the increased risk for breast cancer mortality among postmenopausal nonsmoking women,20 and another showed the risk increment of incidence among premenopausal women.22 meanwhile , null23 and negative24 associations were also reported . in this study , we observed an increased risk of breast cancer among women who were exposed to cigarette smoke from their husbands , and the risk increment showed a dose - response relationship with the number of cigarettes . a stronger positive relationship between a husband 's smoking and breast cancer was observed among women who did not habitually consume alcohol . active smoking has been reported to have simultaneously opposite effects on the progress of breast cancer ; some smoke constituents , such as polycyclic aromatic hydrocarbons , aromatic amines and n - nitrosamines , potentially induce mammary carcinogenesis , while the antiestrogenic effects of smoking reduce the risk of breast cancer.7,14 although these factors might complicate a relationship between active smoking and breast cancer risk , our results regarding active smoking might have been influenced by the fact that the smoking rate of women in this generation was very low , so that it is difficult to meaningfully evaluate the effect of active smoking on breast cancer in this cohort . sidestream smoke has been reported to contain chemical compounds , including some carcinogens.9 some of the carcinogens are metabolized and activated by mammary epithelial cells.26 a higher prevalence of smoking - specific dna adducts and p53 mutation was reported in smokers than in non - smokers.27,28 our results support the theory that there is a carcinogenic property of tobacco smoke for breast cancer , by demonstrating the association between husbands smoking and increased risk of breast cancer with a dose - response relationship . compared with previous us and european studies , the risks of breast cancer were estimated to be at a higher level , consistent with another japanese study by hirayama,20 showing that the relative risk for breast cancer mortality was 1.3 among women whose husband smoked 119 cigarettes daily and 2.68 among those whose husband smoked 20 cigarettes or more daily . however , we also must note that we did not detect risk increment of breast cancer among active smokers who inhale sidestream smoke through their own smoking . we additionally observed that an increased risk of breast cancer among women with a smoking husband was pronounced among those who did not habitually consume alcohol . although our finding suggests that non - drinkers were susceptible to the effect tobacco smoke has on breast cancer , the analyses were conducted with a small number of cases in subgroup analyses , showing the possibility that the results were obtained by chance . two previous case - control studies29,30 and one cohort study10 reported that alcohol consumption did not modify the association with breast cancer , which is inconsistent with our results . however , all three studies observed an overall null association between passive smoking and breast cancer , whereas we detected a significant positive association between husbands smoking and breast cancer risk . for active smoking , it was observed in a norwegian swedish cohort study that the risks from active smoking were estimated to be higher among non - drinkers than among all subjects.31 mechanisms underlying the associations between smoking , alcohol and breast cancer are unknown . slow acetylation of aromatic amines was related to an increased risk of breast cancer.5 although there might be a greater proportion of women with n - acetyltransferase 2 ( nat2 ) slow genotype among non - drinkers , the association between nat2 genotypes and drinking habits is not well known . with the increasing evidence of an association between dna methylation and breast cancer,32 smoking and drinking the modifying effects of alcohol on increased breast cancer risk from passive smoking might need to be confirmed in future studies . our cohort study was conducted with a prospective design which is expected to be less subject to bias than a case - control study because the information on smoking status was collected before the diagnosis of breast cancer . information on subjects ' or husbands ' smoking status was based on self - reports . compared with estimates based on urinary cotinine concentration , high sensitivity and specificity of self - reported smoking status were reported ( 92.1 and 98.4% for men , and 91.2 and 98.3% for women).33 however , in the present study , husbands ' smoking status was obtained only for the subjects whose spouse was ascertained from the study data ( 67.4% ) . when 3681 women who reported being single , widowed or divorced at the baseline were assigned to the group without exposure to smoke from a husband , the positive association between husband 's smoking and breast cancer was not altered . compared with the reference group of women with a husband who had never smoked or who were without a husband , the hazard ratio was 1.76 ( 95% ci : 1.112.79 ) among women whose husband was a current smoker of 21 cigarettes per day or more . passive smoking except for smoke exposure from the subjects ' husbands was not assessed ; none of the exposure to smoke from other residents at home , at the workplace or in public places was obtained . although underlying diseases or preclinical signs at the baseline may have affected lifestyles , the exclusion of those who had died during the first 2 years of follow up did not substantially change the results . in conclusion , this prospective study of japanese women demonstrated the increased risk of breast cancer among women exposed to secondhand cigarette smoke from their husband , with a dose - response relationship . the increased risks for women with a smoking husband were stronger among those who do not habitually consume alcohol . these results suggest that exposure to smoke from husbands is a potential risk factor for breast cancer , and , considering the limited data currently available , the modifying effects of alcohol on increased breast cancer risk from passive smoking need to be confirmed in further studies .

total joint arthroplasty is one of the most frequently done and successful surgeries in recent times . infection after total joint arthroplasty when occurs has devastating results . with the increasing incidence of total knee arthroplasty ( tka ) , infection at the site of a total knee arthroplasty can be classified into four basic categories : type i ( early postoperative ) , type ii ( late chronic ) , type iii ( acute hematogenous ) , and type iv ( positive intraoperative cultures but clinically unapparent infection ) . the current standard of care for late chronic infection is considered to be two - stage revision arthroplasty , including removal of the prosthesis and cement , thorough debridement , placement of an antibiotic impregnated cement spacer , a course of intravenous antibiotics , and a delayed second stage revision arthroplasty.16 the different types of spacers available are either articulating or non - articulating , pre - fabricated or custom - made , and those made on table with or without moulds . articulating spacers are better than non - articulating ones as they provide mobility to the knee besides controlling infection . we describe a new technique ( a modification of goldstein 's technique7 ) for intraoperative manufacture of a custom - made antibiotic impregnated articulating spacer , with minimal cost and good conformity . we assess the results of this technique in a larger series of patients than in the original series7 and also assess the effect of the modification that we have introduced . this is a retrospective study which conducted on twenty eight patients with thirty six infected total knee arthroplasty ( eight were bilateral ) from june 2002 to may 2007 . of the 36 knees , 26 were cases of primary osteoarthritis , while the other 10 were rheumatoid knees . the diagnosis was made on basis of clinical examination , labotary investigations and radiographic evaluation . the clinical findings included pain , warmth , redness , severe restriction of movement and frank discharging sinuses . laboratory values showed elevated total counts with neutrophilia , along with persistent elevated esr and crp . all these patients had taken conservative trial of debridemnt and antibiotics and this operation was only suggested once there was radiographic evidence of loosening of the implants . the average time between the original surgery and this surgery was 10 months 8 days ( range : 7 months to 1 year 4 months ) . the new technique is a modification of the original technique described by goldstein et al.7 all these operations were performed by the same main surgeon ( pg ) in the same institute . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . the two packets of polymethyl methacrylate high viscosity cement ( 40 gm each ) , one each for the femoral and tibial component , were used for every case . 5 ml of dextran was added for porosity as this leads to better elution of the antibiotics.8 2.4 gm of tobramycin , 2 gm of vancomycin , and 1.5 gm of cefuroxime in powder form was added because of heat resistant.9 aluminium foil of 0.05-mm thickness was autoclaved and then carefully wrapped around the freshened femur condyles so as to create all the defects and projections on the surface . the foil was then coated with a sterile lubricant ( lignocaine jelly ) in order to prevent it from sticking to the cement . a sterile trial plastic spacer or monoblock was used as a mold to give anatomical shape to the distal articulating part of the femoral spacer . the upper surface of the spacer is the surface that this component is finally going to articulate with , and this simple technique provides an exact conformation of the distal articulating surface . also , the smooth surface of the spacer provides a smooth finish to the cement , preventing the rough surfaces and the friction and wear seen with the normal hand - moulded spacers . a groove on the anterior surface of the spacer can also be made with the femoral component at the same time for better alignment of the patella , though this is not a necessary step . once the cement hardens , it is removed and the foil is taken out ; the spacer is then reapplied on the femur [ figure 1 ] . it sits well on the condyles because of exact conformity , giving it inherent stability . in all the operated knees a bone block was used for the femoral medullary canal and so we found no opening for it in any of our cases ; we did not recreate the canal as it could be a potential site of infection . ( d ) cement applied and molded with the spacer implant to prepare the femoral component . ( e ) the final prepared femoral component preoperative measurement of the tibial thickness is required for adequate soft tissue stretching . the tibial tray is prepared in a manner similar to the femoral side , with the aluminium foil and jelly covering the tibia now , using the sterile trial metallic femoral component of the same system to mould and shape the articulating surface giving a lock - and - key arrangement to the assembly . this is crucial step as it provides modularity to the spacers in terms of thickness . it is essential to maintain the needed thickness of the tibial component , which is determined preoperatively and confirmed peroperatively before this step [ figure 2 ] . ( d ) modification : k - wire being drilled into the components before setting of the cement and then embedded within . ( e ) final component articulated and placed in position fit like a lock and key nine patients , five with unilateral affection could not afford a revision surgery . three out of the five patients opted for arthrodesis . while rest two patients insisted on the spacer option the two bilateral patients also took this spacer option . to make the cement spacers last longer we reinforced the cement mantle with k - wires just before it hardens . at least one k - wire each is to be drilled , with the keel of the tibia and the intercondylar junction of the femoral component as they are the ones most prone to breakage . additional more than one k - wire can be drilled parallel or perpendicular to the first k - wire . care is to be taken to fully embed the k wire in the cement mantle . the stability and mobility are checked by flexing and extending the knee joint once both the components are placed . the wound is closed in layers and the patient kept on iv cefuroxime till reports of cultures are available . later on antibiotic was changed as guided by culture and sensitivity . immediate rehabilitation , with knee bending with partial weight bearing , was started so that maximum mobility can be attained . for the first 2 weeks the patients were allowed to walk with the help of a walker , after which they used a walking stick ; the patients were weaned off the walking aids over a period of 48 weeks . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . finally 29 patients opted for revision surgery while the rest could not afford a revision surgery . the two packets of polymethyl methacrylate high viscosity cement ( 40 gm each ) , one each for the femoral and tibial component , were used for every case . 5 ml of dextran was added for porosity as this leads to better elution of the antibiotics.8 2.4 gm of tobramycin , 2 gm of vancomycin , and 1.5 gm of cefuroxime in powder form was added because of heat resistant.9 aluminium foil of 0.05-mm thickness was autoclaved and then carefully wrapped around the freshened femur condyles so as to create all the defects and projections on the surface . the foil was then coated with a sterile lubricant ( lignocaine jelly ) in order to prevent it from sticking to the cement . a sterile trial plastic spacer or monoblock was used as a mold to give anatomical shape to the distal articulating part of the femoral spacer . the upper surface of the spacer is the surface that this component is finally going to articulate with , and this simple technique provides an exact conformation of the distal articulating surface . also , the smooth surface of the spacer provides a smooth finish to the cement , preventing the rough surfaces and the friction and wear seen with the normal hand - moulded spacers . a groove on the anterior surface of the spacer can also be made with the femoral component at the same time for better alignment of the patella , though this is not a necessary step . once the cement hardens , it is removed and the foil is taken out ; the spacer is then reapplied on the femur [ figure 1 ] . it sits well on the condyles because of exact conformity , giving it inherent stability . in all the operated knees a bone block was used for the femoral medullary canal and so we found no opening for it in any of our cases ; we did not recreate the canal as it could be a potential site of infection . ( d ) cement applied and molded with the spacer implant to prepare the femoral component . ( e ) the final prepared femoral component preoperative measurement of the tibial thickness is required for adequate soft tissue stretching . the tibial tray is prepared in a manner similar to the femoral side , with the aluminium foil and jelly covering the tibia now , using the sterile trial metallic femoral component of the same system to mould and shape the articulating surface giving a lock - and - key arrangement to the assembly . this is crucial step as it provides modularity to the spacers in terms of thickness . it is essential to maintain the needed thickness of the tibial component , which is determined preoperatively and confirmed peroperatively before this step [ figure 2 ] . ( d ) modification : k - wire being drilled into the components before setting of the cement and then embedded within . ( e ) final component articulated and placed in position fit like a lock and key nine patients , five with unilateral affection could not afford a revision surgery . three out of the five patients opted for arthrodesis . while rest two patients insisted on the spacer option the two bilateral patients also took this spacer option . to make the cement spacers last longer we reinforced the cement mantle with k - wires just before it hardens . at least one k - wire each is to be drilled , with the keel of the tibia and the intercondylar junction of the femoral component as they are the ones most prone to breakage . additional more than one k - wire can be drilled parallel or perpendicular to the first k - wire . care is to be taken to fully embed the k wire in the cement mantle . the stability and mobility are checked by flexing and extending the knee joint once both the components are placed . the wound is closed in layers and the patient kept on iv cefuroxime till reports of cultures are available . later on antibiotic was changed as guided by culture and sensitivity . immediate rehabilitation , with knee bending with partial weight bearing , was started so that maximum mobility can be attained . for the first 2 weeks the patients were allowed to walk with the help of a walker , after which they used a walking stick ; the patients were weaned off the walking aids over a period of 48 weeks . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . finally 29 patients opted for revision surgery while the rest could not afford a revision surgery . the two packets of polymethyl methacrylate high viscosity cement ( 40 gm each ) , one each for the femoral and tibial component , were used for every case . 5 ml of dextran was added for porosity as this leads to better elution of the antibiotics.8 2.4 gm of tobramycin , 2 gm of vancomycin , and 1.5 gm of cefuroxime in powder form was added because of heat resistant.9 aluminium foil of 0.05-mm thickness was autoclaved and then carefully wrapped around the freshened femur condyles so as to create all the defects and projections on the surface . the foil was then coated with a sterile lubricant ( lignocaine jelly ) in order to prevent it from sticking to the cement . a sterile trial plastic spacer or monoblock was used as a mold to give anatomical shape to the distal articulating part of the femoral spacer . the upper surface of the spacer is the surface that this component is finally going to articulate with , and this simple technique provides an exact conformation of the distal articulating surface . also , the smooth surface of the spacer provides a smooth finish to the cement , preventing the rough surfaces and the friction and wear seen with the normal hand - moulded spacers . a groove on the anterior surface of the spacer can also be made with the femoral component at the same time for better alignment of the patella , though this is not a necessary step . once the cement hardens , it is removed and the foil is taken out ; the spacer is then reapplied on the femur [ figure 1 ] . it sits well on the condyles because of exact conformity , giving it inherent stability . in all the operated knees a bone block was used for the femoral medullary canal and so we found no opening for it in any of our cases ; we did not recreate the canal as it could be a potential site of infection . ( d ) cement applied and molded with the spacer implant to prepare the femoral component . the tibial tray is prepared in a manner similar to the femoral side , with the aluminium foil and jelly covering the tibia now , using the sterile trial metallic femoral component of the same system to mould and shape the articulating surface giving a lock - and - key arrangement to the assembly . this is crucial step as it provides modularity to the spacers in terms of thickness . it is essential to maintain the needed thickness of the tibial component , which is determined preoperatively and confirmed peroperatively before this step [ figure 2 ] . ( d ) modification : k - wire being drilled into the components before setting of the cement and then embedded within . ( e ) final component articulated and placed in position fit like a lock and key nine patients , five with unilateral affection could not afford a revision surgery . three out of the five patients opted for arthrodesis . while rest two patients insisted on the spacer option the two bilateral patients also took this spacer option . to make the cement spacers last longer we reinforced the cement mantle with k - wires just before it hardens . at least one k - wire each is to be drilled , with the keel of the tibia and the intercondylar junction of the femoral component as they are the ones most prone to breakage . additional more than one k - wire can be drilled parallel or perpendicular to the first k - wire . care is to be taken to fully embed the k wire in the cement mantle . the stability and mobility are checked by flexing and extending the knee joint once both the components are placed . the wound is closed in layers and the patient kept on iv cefuroxime till reports of cultures are available . later on antibiotic immediate rehabilitation , with knee bending with partial weight bearing , was started so that maximum mobility can be attained . for the first 2 weeks the patients were allowed to walk with the help of a walker , after which they used a walking stick ; the patients were weaned off the walking aids over a period of 48 weeks . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . finally 29 patients opted for revision surgery while the rest could not afford a revision surgery . all the knees were examined by the same surgeon , who was not a part of the operating team . all the patients were administered a questionnaire before the operation and 6 weeks after the operation and the results were evaluated . clinical evidence of wound healing and infection subsidence was evident in all 36 knees within a mean period of 3.5 weeks . the 10 rheumatoid knees took slightly longer than the other knees to heal ( average : 4 weeks ) . subsidence of infection was confirmed using serial crp and esr ; these values coincided with clinical healing in all cases . the average follow - up was 5 years and 2 months [ table 1 ] . range of motion was increased by an average of 33. the womac function , pain , and stiffness scores also increased ; the average increase of the combined womac scores was 20 points . the knee society score ( clinical + functional ) increased by an average of 39 points . we specifically questioned the patients for any feeling of stickiness or any scratching or grating sound caused due to the conventional rough surface of the hand - molded spacer , or any locking episodes due to the wear particles ; none of the patients had any such symptoms . pre and postoperative range of motion , knee society score and womac score there were two cases of fracture of cement spacer in the first 15 cases that we operated without augmentation . the first case had a fracture 4 mo postoperatively while the second at 3 mo followup . in both cases fortunately , the infection in both these cases had healed by this time and we could safely remove the spacer . we added a k - wire scaffold in the last 21 knees to prevent this complication . there was one case of a minor fracture of the tibial component in one of these 21 cases but it did not restrict motion and the patient continued motion and weight bearing we also had an intraoperative fracture of the cement cast when we drilled a k - wire into the bone rather than only through the cement . the ability to change , reshape , or rebuild with just another extra packet of cement is a major advantage of this technique . we compared the cost of the standard spacers used [ table 2 ] . comparing the long - term costs ( with regard to antibiotic usage and re - operations ) , since we got eradication of infection in all our cases without any re - operations , long - term cost effectively remain same or even better than other procedures . comparison of the cost of various spacers available out of the 36 knees , 29 went for a revision surgery after an average of 1.5 years ( range : 6 months to 3.5 years ) . in all 29 cases , there was no difficulty in removing the spacer and there was minimum bone loss . one bilateral case and three unilateral cases ( five knees ) did not go for revision due to financial constraints . all of them are still walking with the spacer , without any problems till last follow up . the average follow up of these 5 cases is 4 yrs [ figures 3 and 4 ] ( range 3 years to 5.5 years ) there was one death but this was 4 years after the patient underwent the spacer operation . one patient changed her address 4.5 years after the spacer operation and she was lost to follow - up . x - rays of knee joint showing ( a ) infected tka ( anteroposterior view ) . ( c ) x ray of same patient ( lateral view ) with spacer in for 4 years now and patients walking x - rays of knee joint showing ( a ) primary tka ( anteroposterior view ) . ( c ) patient walked with this spacer for an year ( lateral view ) . ( d ) patient had a revision tka done with bone graft augmentation ( anteroposterior view ) two - stage exchange arthroplasty with an antibiotic - impregnated cement spacer remains the standard treatment for patients with an infected total knee arthroplasty.8 static spacers not only reduce the range of motion of the knee joint but also cause capsular and quadriceps scarring and shortening , with subsequent difficulty in exposure at the second stage and the possibility of additional bone loss . these had inherent problems like size discrepancy leading to early loosening , unsatisfactory antibiotic impregnation , inability to modify and redo the procedure on table and , most of all , high cost . these spacers lack modularity to increase or decrease the thickness of the spacers according to the bone loss and joint laxity . articulating spacers used in studies by hoffman10 and haddad11 and also the popular prostalac spacer have the inherent disadvantage of leaving some metallic component in the knee joint . we concur with goldstein 's thinking that any metal in the joint can act as a foreign body and as a potential surface for biofilm formation and bacterial proliferation.7 to make up for these disadvantages , intraoperative custom - made cement spacers were conceived . mcpherson et al.12 and ha et al.13 created spacers using only cement components , preparing them on the table and moulding them on the cut surfaces of the bones . the method does away with all metallic components and provides good conformation to the underlying bone . however , this approach requires technical perfection , as premature cement placement could result in interdigitation of the cement into the cancellous bone and , on the other hand , waiting until the cement is sufficiently thick may result in poor conformity of the implant and a loose fit . only with a perfect cementing technique ha used hohmann retractors and osteotomes for moulding the spacers , but we did not find this very convenient for creating good articulating surfaces . there are also cement spacers that can be made intraoperatively using moulds , which cost a little less than the prefabricated spacers , but most of the above mentioned problems still persist in these spacers.14 our technique was devised to solve some of these problems . using the aluminium foil prevents the danger of interdigitations of cement with bone . it also gives us sufficient time to mould , modify , or change the block and shape it perfectly for articulation . we think that using the cleaned prosthesis to mould the surfaces is the easiest and the most conforming way to produce good articulation . we would like to stress here that no matter what technique is used the most important step of these operations is complete and thorough debridement of the infected tissues . if this step is compromised then all techniques will fail as the primary objective of this operation is control of infection . the original technique described by goldstein is what we followed in our first few cases . but soon we had a complication in the form of fracture of the femoral component . this is an inevitable consequence if we use a cement - only spacer and advice weight - bearing to the patient . usually , this complication is not of much consequence as pointed out by the study of meek et al.,6 but the study does not state how many of their patients took to full weight - bearing . to overcome this problem we added k - wires to the spacers , drilled while the cement was setting . the k - wires provide a structural support and will go a long way in preventing fractures of the cement . even if there is a break , our k - wire inlay keeps the major fragments together , giving a satisfactory result . we advised second stage revision surgery once infection was controlled , usually 3 months after debridement and spacer . 29 did undergo revision anytime after 6 months . however , 5 which could be followed still continuing on spacer without fracture of cement spacer . in our study in contrast , there were no significant fractures in the other 21 k - wire - augmented spacers . this clearly proves the advantage of using the k - wire framework to provide longevity to the spacer . on the downside , intraoperative fracture of the cement mantle due to anchorage of k - wire in bone is a possible complication . also , drilling the k - wires in that short span of cement - setting time , along with the shaping of the spacer , is a difficult procedure and should be attempted only after the surgeon is familiar and practiced with the plain spacer . one of our major concerns , and the reason for using this technique , was the cost . infection in any arthroplasty is a huge financial burden for the patient and it is difficult to accept that the patient should pay more for an interim procedure like this than he / she did for the original procedure . the commonly used prefabricated spacer costs approximately 18 times the cost of our technique , while the local versions of the same cost around six times . this is a huge relief for the patients and an attractive option for some patients who would otherwise go for arthrodesis or amputation due to economic constraints . some patients can not ever afford a revision operation , and this system ( especially the k - wire - augmented system , with its longevity ) provides a mobile and effective solution for them . since we achieved eradication of infection in all our cases without any re - operations , the long - term cost ( which includes cost of antibiotics and re operations ) with our technique was effectively the same or even lower than with other techniques . other factors like range of motion , patient satisfaction , and infection healing were comparable with that seen with any other types of spacer and depended more on the previous state of the knees . theoretically , this technique does provide more conformation and therefore more stability to the knee , but this could not be measured objectively . overall , this is a simple technique that places only a minimal economic burden on the already burdened patient . it primarily serves to control infection , along with preservation of motion . with our modification it can last longer with lesser complications . on the strength of the excellent results achieved in this study we recommend this technique for all infected total knee cases to control infection before the final revision surgery .

background : standard treatment of chronic infected total knee arthroplasty ( tka ) is a two - stage revision , the first step being placement of an antibiotic - impregnated cement spacer . here we describe the results of a new technique ( modification of the goldstien 's technique ) for intraoperative manufacture of a customized articulating spacer at minimal cost and with relatively good conformity and longevity.materials and methods : thirty - six infected knees underwent this procedure from june 2002 to may 2007 . the technique consists of using the freshened femur and tibia interface as molds wrapped in a tin foil for manufacturing the two components of the spacer with antibiotic - impregnated methyl methycrylate cement . we used the spacer and the femoral component of the trial set of a tka system to mold them to perfect articulation . we also reinforced the spacer with a k - wire scaffold to prevent fracture of the cement mantle in the last 21 cases.results:all 36 knees showed excellent results in terms of infection control , mobility , and stability . there was significant improvement in the womac and knee society scores ( 20 and 39 points respectively ) . there were two fractures of the spacers in the initial 15 cases that did not have k - wire scaffolding but none in the last 21 that had reinforcement.conclusion:this technique provides a more conforming spacer , with good range of motion and stability . the reinforcement helps in preventing the fracture of the cement mantle and is cost effective .

I M Operative procedure Step 1 Removal of implants and debridement Step 2 Preparation of the cement Step 3 Preparation of the femoral component Step 4 Preparation of the tibial tray Step 5 Articulation and closure R D

with the increasing incidence of total knee arthroplasty ( tka ) , infection at the site of a total knee arthroplasty can be classified into four basic categories : type i ( early postoperative ) , type ii ( late chronic ) , type iii ( acute hematogenous ) , and type iv ( positive intraoperative cultures but clinically unapparent infection ) . the current standard of care for late chronic infection is considered to be two - stage revision arthroplasty , including removal of the prosthesis and cement , thorough debridement , placement of an antibiotic impregnated cement spacer , a course of intravenous antibiotics , and a delayed second stage revision arthroplasty.16 the different types of spacers available are either articulating or non - articulating , pre - fabricated or custom - made , and those made on table with or without moulds . we describe a new technique ( a modification of goldstein 's technique7 ) for intraoperative manufacture of a custom - made antibiotic impregnated articulating spacer , with minimal cost and good conformity . we assess the results of this technique in a larger series of patients than in the original series7 and also assess the effect of the modification that we have introduced . this is a retrospective study which conducted on twenty eight patients with thirty six infected total knee arthroplasty ( eight were bilateral ) from june 2002 to may 2007 . of the 36 knees , 26 were cases of primary osteoarthritis , while the other 10 were rheumatoid knees . all these patients had taken conservative trial of debridemnt and antibiotics and this operation was only suggested once there was radiographic evidence of loosening of the implants . the new technique is a modification of the original technique described by goldstein et al.7 all these operations were performed by the same main surgeon ( pg ) in the same institute . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . the two packets of polymethyl methacrylate high viscosity cement ( 40 gm each ) , one each for the femoral and tibial component , were used for every case . 5 ml of dextran was added for porosity as this leads to better elution of the antibiotics.8 2.4 gm of tobramycin , 2 gm of vancomycin , and 1.5 gm of cefuroxime in powder form was added because of heat resistant.9 aluminium foil of 0.05-mm thickness was autoclaved and then carefully wrapped around the freshened femur condyles so as to create all the defects and projections on the surface . the foil was then coated with a sterile lubricant ( lignocaine jelly ) in order to prevent it from sticking to the cement . a sterile trial plastic spacer or monoblock was used as a mold to give anatomical shape to the distal articulating part of the femoral spacer . the upper surface of the spacer is the surface that this component is finally going to articulate with , and this simple technique provides an exact conformation of the distal articulating surface . also , the smooth surface of the spacer provides a smooth finish to the cement , preventing the rough surfaces and the friction and wear seen with the normal hand - moulded spacers . a groove on the anterior surface of the spacer can also be made with the femoral component at the same time for better alignment of the patella , though this is not a necessary step . once the cement hardens , it is removed and the foil is taken out ; the spacer is then reapplied on the femur [ figure 1 ] . in all the operated knees a bone block was used for the femoral medullary canal and so we found no opening for it in any of our cases ; we did not recreate the canal as it could be a potential site of infection . ( d ) cement applied and molded with the spacer implant to prepare the femoral component . ( e ) the final prepared femoral component preoperative measurement of the tibial thickness is required for adequate soft tissue stretching . the tibial tray is prepared in a manner similar to the femoral side , with the aluminium foil and jelly covering the tibia now , using the sterile trial metallic femoral component of the same system to mould and shape the articulating surface giving a lock - and - key arrangement to the assembly . this is crucial step as it provides modularity to the spacers in terms of thickness . ( d ) modification : k - wire being drilled into the components before setting of the cement and then embedded within . three out of the five patients opted for arthrodesis . while rest two patients insisted on the spacer option the two bilateral patients also took this spacer option . to make the cement spacers last longer we reinforced the cement mantle with k - wires just before it hardens . at least one k - wire each is to be drilled , with the keel of the tibia and the intercondylar junction of the femoral component as they are the ones most prone to breakage . additional more than one k - wire can be drilled parallel or perpendicular to the first k - wire . care is to be taken to fully embed the k wire in the cement mantle . for the first 2 weeks the patients were allowed to walk with the help of a walker , after which they used a walking stick ; the patients were weaned off the walking aids over a period of 48 weeks . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . the two packets of polymethyl methacrylate high viscosity cement ( 40 gm each ) , one each for the femoral and tibial component , were used for every case . 5 ml of dextran was added for porosity as this leads to better elution of the antibiotics.8 2.4 gm of tobramycin , 2 gm of vancomycin , and 1.5 gm of cefuroxime in powder form was added because of heat resistant.9 aluminium foil of 0.05-mm thickness was autoclaved and then carefully wrapped around the freshened femur condyles so as to create all the defects and projections on the surface . the foil was then coated with a sterile lubricant ( lignocaine jelly ) in order to prevent it from sticking to the cement . a sterile trial plastic spacer or monoblock was used as a mold to give anatomical shape to the distal articulating part of the femoral spacer . the upper surface of the spacer is the surface that this component is finally going to articulate with , and this simple technique provides an exact conformation of the distal articulating surface . also , the smooth surface of the spacer provides a smooth finish to the cement , preventing the rough surfaces and the friction and wear seen with the normal hand - moulded spacers . a groove on the anterior surface of the spacer can also be made with the femoral component at the same time for better alignment of the patella , though this is not a necessary step . once the cement hardens , it is removed and the foil is taken out ; the spacer is then reapplied on the femur [ figure 1 ] . in all the operated knees a bone block was used for the femoral medullary canal and so we found no opening for it in any of our cases ; we did not recreate the canal as it could be a potential site of infection . ( d ) cement applied and molded with the spacer implant to prepare the femoral component . ( e ) the final prepared femoral component preoperative measurement of the tibial thickness is required for adequate soft tissue stretching . the tibial tray is prepared in a manner similar to the femoral side , with the aluminium foil and jelly covering the tibia now , using the sterile trial metallic femoral component of the same system to mould and shape the articulating surface giving a lock - and - key arrangement to the assembly . this is crucial step as it provides modularity to the spacers in terms of thickness . ( d ) modification : k - wire being drilled into the components before setting of the cement and then embedded within . while rest two patients insisted on the spacer option the two bilateral patients also took this spacer option . to make the cement spacers last longer we reinforced the cement mantle with k - wires just before it hardens . at least one k - wire each is to be drilled , with the keel of the tibia and the intercondylar junction of the femoral component as they are the ones most prone to breakage . additional more than one k - wire can be drilled parallel or perpendicular to the first k - wire . care is to be taken to fully embed the k wire in the cement mantle . for the first 2 weeks the patients were allowed to walk with the help of a walker , after which they used a walking stick ; the patients were weaned off the walking aids over a period of 48 weeks . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . the two packets of polymethyl methacrylate high viscosity cement ( 40 gm each ) , one each for the femoral and tibial component , were used for every case . 5 ml of dextran was added for porosity as this leads to better elution of the antibiotics.8 2.4 gm of tobramycin , 2 gm of vancomycin , and 1.5 gm of cefuroxime in powder form was added because of heat resistant.9 aluminium foil of 0.05-mm thickness was autoclaved and then carefully wrapped around the freshened femur condyles so as to create all the defects and projections on the surface . the foil was then coated with a sterile lubricant ( lignocaine jelly ) in order to prevent it from sticking to the cement . a sterile trial plastic spacer or monoblock was used as a mold to give anatomical shape to the distal articulating part of the femoral spacer . the upper surface of the spacer is the surface that this component is finally going to articulate with , and this simple technique provides an exact conformation of the distal articulating surface . also , the smooth surface of the spacer provides a smooth finish to the cement , preventing the rough surfaces and the friction and wear seen with the normal hand - moulded spacers . a groove on the anterior surface of the spacer can also be made with the femoral component at the same time for better alignment of the patella , though this is not a necessary step . once the cement hardens , it is removed and the foil is taken out ; the spacer is then reapplied on the femur [ figure 1 ] . in all the operated knees a bone block was used for the femoral medullary canal and so we found no opening for it in any of our cases ; we did not recreate the canal as it could be a potential site of infection . ( d ) cement applied and molded with the spacer implant to prepare the femoral component . the tibial tray is prepared in a manner similar to the femoral side , with the aluminium foil and jelly covering the tibia now , using the sterile trial metallic femoral component of the same system to mould and shape the articulating surface giving a lock - and - key arrangement to the assembly . this is crucial step as it provides modularity to the spacers in terms of thickness . ( d ) modification : k - wire being drilled into the components before setting of the cement and then embedded within . while rest two patients insisted on the spacer option the two bilateral patients also took this spacer option . to make the cement spacers last longer we reinforced the cement mantle with k - wires just before it hardens . at least one k - wire each is to be drilled , with the keel of the tibia and the intercondylar junction of the femoral component as they are the ones most prone to breakage . additional more than one k - wire can be drilled parallel or perpendicular to the first k - wire . care is to be taken to fully embed the k wire in the cement mantle . for the first 2 weeks the patients were allowed to walk with the help of a walker , after which they used a walking stick ; the patients were weaned off the walking aids over a period of 48 weeks . assessment was done on the basis of the following parameters : 1 ) wound healing and infection subsidence ( on the basis of clinical examination and serial esr and crp ) ; 2 ) functional results and patient satisfaction ( on the basis of range of motion , knee society score and womac score ) ; and 3 ) cost - effectiveness . all the patients were administered a questionnaire before the operation and 6 weeks after the operation and the results were evaluated . clinical evidence of wound healing and infection subsidence was evident in all 36 knees within a mean period of 3.5 weeks . range of motion was increased by an average of 33. the womac function , pain , and stiffness scores also increased ; the average increase of the combined womac scores was 20 points . the knee society score ( clinical + functional ) increased by an average of 39 points . we specifically questioned the patients for any feeling of stickiness or any scratching or grating sound caused due to the conventional rough surface of the hand - molded spacer , or any locking episodes due to the wear particles ; none of the patients had any such symptoms . pre and postoperative range of motion , knee society score and womac score there were two cases of fracture of cement spacer in the first 15 cases that we operated without augmentation . the first case had a fracture 4 mo postoperatively while the second at 3 mo followup . in both cases fortunately , the infection in both these cases had healed by this time and we could safely remove the spacer . we added a k - wire scaffold in the last 21 knees to prevent this complication . there was one case of a minor fracture of the tibial component in one of these 21 cases but it did not restrict motion and the patient continued motion and weight bearing we also had an intraoperative fracture of the cement cast when we drilled a k - wire into the bone rather than only through the cement . comparison of the cost of various spacers available out of the 36 knees , 29 went for a revision surgery after an average of 1.5 years ( range : 6 months to 3.5 years ) . in all 29 cases , there was no difficulty in removing the spacer and there was minimum bone loss . one bilateral case and three unilateral cases ( five knees ) did not go for revision due to financial constraints . all of them are still walking with the spacer , without any problems till last follow up . the average follow up of these 5 cases is 4 yrs [ figures 3 and 4 ] ( range 3 years to 5.5 years ) there was one death but this was 4 years after the patient underwent the spacer operation . ( d ) patient had a revision tka done with bone graft augmentation ( anteroposterior view ) two - stage exchange arthroplasty with an antibiotic - impregnated cement spacer remains the standard treatment for patients with an infected total knee arthroplasty.8 static spacers not only reduce the range of motion of the knee joint but also cause capsular and quadriceps scarring and shortening , with subsequent difficulty in exposure at the second stage and the possibility of additional bone loss . these spacers lack modularity to increase or decrease the thickness of the spacers according to the bone loss and joint laxity . articulating spacers used in studies by hoffman10 and haddad11 and also the popular prostalac spacer have the inherent disadvantage of leaving some metallic component in the knee joint . we concur with goldstein 's thinking that any metal in the joint can act as a foreign body and as a potential surface for biofilm formation and bacterial proliferation.7 to make up for these disadvantages , intraoperative custom - made cement spacers were conceived . however , this approach requires technical perfection , as premature cement placement could result in interdigitation of the cement into the cancellous bone and , on the other hand , waiting until the cement is sufficiently thick may result in poor conformity of the implant and a loose fit . only with a perfect cementing technique ha used hohmann retractors and osteotomes for moulding the spacers , but we did not find this very convenient for creating good articulating surfaces . we think that using the cleaned prosthesis to mould the surfaces is the easiest and the most conforming way to produce good articulation . we would like to stress here that no matter what technique is used the most important step of these operations is complete and thorough debridement of the infected tissues . but soon we had a complication in the form of fracture of the femoral component . to overcome this problem we added k - wires to the spacers , drilled while the cement was setting . the k - wires provide a structural support and will go a long way in preventing fractures of the cement . even if there is a break , our k - wire inlay keeps the major fragments together , giving a satisfactory result . however , 5 which could be followed still continuing on spacer without fracture of cement spacer . in our study in contrast , there were no significant fractures in the other 21 k - wire - augmented spacers . this clearly proves the advantage of using the k - wire framework to provide longevity to the spacer . on the downside , intraoperative fracture of the cement mantle due to anchorage of k - wire in bone is a possible complication . also , drilling the k - wires in that short span of cement - setting time , along with the shaping of the spacer , is a difficult procedure and should be attempted only after the surgeon is familiar and practiced with the plain spacer . one of our major concerns , and the reason for using this technique , was the cost . infection in any arthroplasty is a huge financial burden for the patient and it is difficult to accept that the patient should pay more for an interim procedure like this than he / she did for the original procedure . this is a huge relief for the patients and an attractive option for some patients who would otherwise go for arthrodesis or amputation due to economic constraints . some patients can not ever afford a revision operation , and this system ( especially the k - wire - augmented system , with its longevity ) provides a mobile and effective solution for them . since we achieved eradication of infection in all our cases without any re - operations , the long - term cost ( which includes cost of antibiotics and re operations ) with our technique was effectively the same or even lower than with other techniques . other factors like range of motion , patient satisfaction , and infection healing were comparable with that seen with any other types of spacer and depended more on the previous state of the knees . on the strength of the excellent results achieved in this study we recommend this technique for all infected total knee cases to control infection before the final revision surgery .

unrelieved acute pain may cause anxiety , sleep disturbances , and demoralization and may interfere with mental activity and social interactions.1,2 acute pain can also increase heart rate and blood pressure , suppress immune functioning , and reduce pulmonary function , leading to an increased risk of dangerous complications , including myocardial ischemia , deep vein thrombosis , pulmonary embolism , hypoxia , pneumonia , and stroke.2 in addition to these more severe adverse effects , uncontrolled acute pain is also associated with gastrointestinal effects , including the development of ileus , nausea , and vomiting.3 the psychologic and physiologic effects of uncontrolled acute pain can result in longer hospital stays and unscheduled readmissions following surgery.4,5 a retrospective analysis of the medical records of 20,817 patients who had undergone same - day surgery in 1999 found that pain was the most common reason that patients were hospitalized immediately after surgery or returned to the hospital within 30 days of surgery , accounting for 38% of unscheduled postoperative hospital admissions or readmissions.4 in addition , prolonged acute pain can cause sensitization of the central and peripheral nervous systems , leading to the development of chronic pain , which is often difficult and costly to treat.68 although guidelines have been developed to improve acute pain management,9,10 pain relief remains suboptimal for many patients.1116 surveys conducted among patients who had undergone ambulatory surgery indicated that 30%15 to 40%12 of patients experience moderate to severe pain following discharge . among hospitalized patients who were receiving treatment in various medical wards14 or who had undergone surgery,11 the percentages were even higher , with 52%14 to 80%11 of patients experiencing acute pain ; of these patients , as many as 86% experienced moderate to extreme pain.11 the undertreatment of acute pain may result from several contributing factors related to patient care , such as infrequent pain assessments , underestimation of the severity of pain , concerns about side effects associated with analgesic treatment , and delays or dose reductions in the administration of analgesics by health care providers.13,14,1720 in addition , patients may underreport acute pain because of low expectations of pain relief and concerns about the adverse effects of analgesic treatment.19,21 a 2004 survey of patients undergoing major abdominal surgery found that many patients were willing to sacrifice pain relief for a reduction in the severity of side effects.22 current treatment options for the management of acute pain include opioid analgesics ( eg , morphine , hydromorphone , and oxycodone ) and nonopioid analgesics ( eg , acetaminophen , acetylsalicylic acid , and nonsteroidal anti - inflammatory drugs [ nsaids]).23 most nsaids are limited by a therapeutic ceiling and are appropriate for the relief of only mild to moderate pain.23 in addition , nsaids are contraindicated in patients with peptic ulcer disease , renal impairment , and any tendency for bleeding.24 cyclooxygenase-2 ( cox-2)-specific inhibitors do not impair platelet function and have an improved gastrointestinal tolerability profile relative to other nsaids25 ; however , certain cox-2specific inhibitors have been linked to an increase in the risk of cardiovascular side effects , including myocardial infarction.26 opioids are typically used for the management of moderate to severe acute pain,27 but opioid use is limited by the occurrence of a range of side effects.28 opioids exert their analgesic effects primarily through agonistic interactions with -opioid receptors in neurons in the pain pathway , which lead to a reduction in neurotransmitter release and associated pain.29 however , the agonistic interactions responsible for opioid activity are not limited to neurons of the pain pathway.23 opioid receptors are present throughout the nervous system , and the interactions of opioids with nonanalgesic receptors are responsible for many of the side effects associated with opioid treatment.23 for example , opioids may induce nausea and vomiting by direct stimulation of receptors at the chemoreceptor trigger zone and vestibular apparatus.30 a systematic review31 of randomized controlled trials of opioids in postoperative patients found that the most common side effects occurring in these patients were gastrointestinal side effects , central nervous system ( cns ) side effects , pruritus , and urinary retention . pruritus occurred in 18.3% of patients who were treated with opioids following surgery and was most common with epidural administration of opioids.31 somnolence and sedation were the most commonly reported cns side effects ; the incidence of somnolence ranged from less than 2% to more than 90% , depending on the route of administration and type of opioid used.31 gastrointestinal side effects , including nausea , vomiting , and constipation , were the most common side effects associated with opioid analgesia and were reported by 31.0% of patients.31 opioid - induced postoperative nausea and vomiting ( ponv ) is associated with negative effects on patient outcomes and quality of life,32 which may lead to increases in recovery time , duration of hospitalization , and cost of medical care.33,34 the underuse of opioid analgesics by health care providers to relieve acute pain may be related to attempts to balance analgesia against concerns about opioid - induced side effects and subsequent deleterious repercussions for patient outcomes.20 there is a continuing need for a potent analgesic agent that will provide adequate relief of acute pain , but with a reduction in side effects . tapentadol is a novel , centrally acting analgesic that offers analgesic efficacy that is similar to that provided by a pure -opioid agonist comparator , but with an improved side effect profile , which may represent a significant advancement in the management of moderate to severe acute pain . tapentadol ( figure 1 ) is a centrally acting analgesic with 2 complementary mechanisms of action , -opioid receptor agonism and norepinephrine reuptake inhibition.35,36 in opioid receptor binding studies , tapentadol was found to have only a modest affinity ( dissociation constant [ ki ] = 0.096 m [ rat])35 for the -opioid receptor relative to pure -opioid receptor agonists such as oxycodone ( ki = 0.018 m [ rat ] ) or morphine ( ki = 0.002 m [ rat]).37 a similar binding affinity to that observed in native rat receptors was demonstrated for tapentadol at the human recombinant -opioid receptor ( ki = 0.16 m [ human]).35 despite the approximately 50-fold difference in binding affinity for the -opioid receptor relative to morphine , tapentadol demonstrated only a 2- to 3-fold reduction in analgesic potency in a series of acute and persistent animal pain models.36 this disparity in potency and binding affinity for the -opioid receptor may be related to the contribution of the second mechanism of action , inhibition of norepinephrine reuptake , to the analgesic effects of tapentadol . in rat synaptosomal reuptake assays , tapentadol inhibited the norepinephrine reuptake transporter with a ki of 0.48 0.11 m and , when administered in intraperitoneal doses of 4.64 to 10 mg / kg , produced a dose - dependent increase in extracellular levels of norepinephrine in the ventral hippocampus of freely moving rats to a maximum of 450% above baseline with 10 mg / kg , as measured by microdialysis.35 in contrast to tapentadol , morphine administered to rats in intraperitoneal doses of 1 to 10 mg / kg produced a small , nonsignificant decrease in extracellular norepinephrine levels.35 the contributions of both -opioid receptor agonism and norepinephrine reuptake inhibition to the analgesic effects of tapentadol were further elucidated by examining the extent to which analgesia was blocked by the selective -opioid receptor antagonist naloxone and the norepinephrine 2-receptor antagonist yohimbine.35 in an animal model35 of acute ( writhing ) pain , it was observed that intravenous tapentadol and morphine induced dose - dependent inhibition of writhing ( ed50 for tapentadol , 0.7 mg / kg ; ed50 for morphine , 0.4 mg / kg ) . when combined with naloxone , the antinociceptive effect of morphine ( 0.681 mg / kg ) was more potently reduced than that of tapentadol ( 3.16 mg / kg ) ; the ed50 for naloxone antagonism was 0.007 mg / kg when combined with morphine and 0.099 mg / kg when combined with tapentadol ( p < 0.001 for tapentadol vs morphine ) . in a spinal nerve ligation model of mononeuropathic pain,35 coadministration of intraperitoneal naloxone ( 0.3 mg / kg ) with equianalgesic intravenous doses of tapentadol ( 10 mg / kg ) or morphine ( 6.81 mg / kg ) reduced the anti - allodynic effect of tapentadol from 72% to 42% of the maximal possible effect ( mpe ) ; the anti - allodynic effect of morphine was reduced from 83% to 25% of the mpe . in contrast , when yohimbine ( 2.15 mg / kg ) was administered intraperitoneally in combination with intravenous doses of morphine ( 6.81 mg / kg ) or tapentadol ( 10 mg / kg ) , the anti - allodynic effect of tapentadol was reduced from 81% to 19% of the mpe , whereas the anti - allodynic effect of morphine was only minimally reduced ( from 80% to 54% of the mpe).35 these results indicate that both -opioid receptor agonist and norepinephrine reuptake inhibitor mechanisms are involved in the analgesic effect of tapentadol and that , in contrast to morphine , norepinephrine reuptake inhibition plays a prominent role in tapentadol - induced analgesia . in addition to contributing to the analgesic activity of tapentadol , the opioid - sparing effect of norepinephrine reuptake inhibition may also contribute to a reduction of adverse effects associated with pure -opioid agonists.36 such an opioid - sparing effect has been achieved by combining other analgesics , such as nsaids or cox-2specific inhibitors , with opioids to control acute pain.38 this type of multimodal strategy achieves an additive analgesic effect by combining 2 different mechanisms of analgesic activity , but reduces the consumption of opioid analgesics and , thereby , the incidence of adverse effects associated with -opioid agonist activity.38 for example , in a study of 200 patients undergoing outpatient anterior cruciate ligament surgery , patients who received perioperative doses of the cox-2specific inhibitor celecoxib in addition to oxycodone experienced less pain ( p < 0.01 ) in the recovery room , had lower postoperative opioid consumption ( p < 0.001 ) , and reported a lower incidence of ponv ( p < 0.05 ) than patients taking oxycodone alone.39 by combining a second mechanism of analgesic activity with -opioid receptor agonism , tapentadol may offer the benefits of multimodal analgesia within a single molecule . the analgesic effects of tapentadol are independent of metabolic activation , and tapentadol has no active metabolites.40 orally administered tapentadol is principally cleared by hepatic glucuronidation via the uridine 5-diphospho - glucuronosyl transferases ( ugts ) ugt1a9 and ugt2b7 , which are responsible for approximately 55% of tapentadol metabolism in humans.41 the major metabolite of tapentadol , tapentadol - o - glucuronide , has no activity at opioid receptors , synaptosomal reuptake systems , or other binding sites.35 morphine is likewise primarily metabolized by hepatic glucuronidation via ugt2b7 to morphine-3-glucuronide ( m3 g ) and morphine-6-glucuronide ( m6g).42 morphine-3-glucuronide has no analgesic activity , but m6 g has an affinity for the -opioid receptor and contributes significantly to the analgesic effect of morphine.43 in patients with renal insufficiency , m6 g accumulates and may contribute to the higher incidence of morphine toxicity observed in these patients.44,45 many other opioids , including oxycodone , codeine , dihydrocodeine , hydrocodone , and tramadol , are primarily metabolized by the cytochrome p450 ( cyp ) enzymes cyp2d6 or cyp3a4.46 mutations in the cyp2d6 gene , which occur in approximately 1% to 7% of the caucasian population , can either decrease or increase enzyme activity , leading to alterations in opioid analgesia.47 the analgesic effects of codeine are highly dependent on conversion of codeine to morphine by cyp2d6 ; a poor cyp2d6 metabolic phenotype can suppress codeine analgesia , and an ultra - rapid cyp2d6 metabolic phenotype can lead to increased opioid effects , such as euphoria , dizziness , and visual disturbances.47 in addition to the potential for varied individual responses to cytochrome p450-metabolized opioids due to genetic mutations , opioids metabolized by the cytochrome p450 pathway are associated with an increased risk for drug drug interactions . more than half of all drugs are metabolized by cyp3a4 , and opioids metabolized by this isozyme ( including fentanyl , buprenorphine , and methadone ) are prone to drug drug interactions with antiretroviral agents and antidepressants.48 opioids metabolized by cyp2d6 , including codeine , dihydrocodeine , and oxycodone , are also associated with a number of drug drug interactions . substrates of cyp2d6 include antiarrhythmic agents , antidepressants , antipsychotics , antiparasitic agents , and tamoxifen.48 the analgesic activity of codeine is particularly impaired by inhibition of cyp2d6 because the analgesic effects of codeine result from the formation of metabolites , including morphine and , possibly , codeine-6-glucuronide.48 in vitro studies41 were used to evaluate the inhibitory or inducing effects of tapentadol on the 7 major cyp isoforms involved in drug metabolism ( cyp2d6 , cyp3a4 , cyp1a2 , cyp2a6 , cyp2c9 , cyp2c19 , and cyp2e1 ) . tapentadol did not undergo significant metabolism by cyp enzymes and did not inhibit or induce the activity of any of the cyp isoforms tested , with the exception of cyp2d6.41 limited inhibition of cyp2d6 was observed with tapentadol , with competitive inhibition occurring with a ki of 181 m and noncompetitive inhibition with a ki of 1,410 m.41 the ki values for both competitive and noncompetitive inhibition are much higher than the expected tapentadol plasma concentrations of 0.5 to 1 m ( following therapeutic dosing ) , indicating that cyp2d6 inhibition by tapentadol is unlikely to be clinically relevant.41 two randomized , open - label studies were performed to evaluate the potential for drug drug interactions between tapentadol and 3 common analgesics that , like tapentadol , are metabolized by ugt pathways ( acetaminophen , naproxen , and acetylsalicylic acid).49 mean serum concentrations of tapentadol and tapentadol - o - glucuronide were similar after administration of tapentadol ir alone and after coadministration with acetaminophen and acetylsalicylic acid . a slight increase in the serum concentration of tapentadol and a slight decrease in the serum concentration of tapentadol - o - glucuronide were observed after coadministration with naproxen , but these changes were not considered clinically relevant , due to the relatively small magnitude of change . thus , no dosing adjustments are needed for administration of tapentadol with any of these commonly coadministered analgesics . the efficacy of tapentadol ir for the relief of moderate to severe pain has been evaluated in 3 randomized , double - blind , phase 3 studies in patients with postoperative ( bunionectomy ) pain50,51 and pain related to end - stage degenerative joint disease52 and as a secondary measure in a phase 3 randomized , double - blind , 90-day safety study.53 in one of the postoperative pain studies,51 patients received tapentadol ir ( 50 , 75 , or 100 mg ) , oxycodone hcl ir ( 15 mg ) , or placebo every 4 to 6 hours for 72 hours following bunionectomy ; in the other postoperative pain study,50 patients received tapentadol ir ( 50 or 75 mg ) , oxycodone hcl ir ( 10 mg ) , or placebo every 4 to 6 hours for 72 hours following bunionectomy . in the end - stage joint disease study,52 patients received tapentadol ir ( 50 or 75 mg ) , oxycodone hcl ir ( 10 mg ) , or placebo every 4 to 6 hours for 10 days . in the 90-day safety study,53 patients received flexible doses of tapentadol ir 50 or 100 mg ( up to 600 mg / day ) or oxycodone hcl 10 mg or 15 mg ( up to 90 mg / day ) every 4 to 6 hours as needed for up to 90 days . in all 4 phase 3 studies5053 of tapentadol ir for acute pain , improvements in pain intensity were observed with tapentadol ir treatment ( 50 , 75 , or 100 mg every 4 to 6 hours ) that were similar to those observed with oxycodone hcl ir treatment ( 10 or 15 mg every 4 to 6 hours ) based on pain intensity measurements on an 11-point numerical rating scale ( nrs ; 0 = no pain to 10 = worst pain imaginable ) . for example , in the postoperative pain study50 in which patients received tapentadol ir ( 50 or 75 mg ) for 72 hours following bunionectomy , 901 patients were randomized to treatment . in that study,50 efficacy was evaluated based on the following measures : the sum of the pain intensity difference ( spid ) over the first 12 , 24 , 48 ( primary efficacy endpoint ) , and 72 hours of treatment ; responder rates at 48 hours ; and the patient global impression of change ( pgic ) . based on increases in the mean ( sd ) spid48 , significantly greater reductions in pain intensity from baseline were observed with tapentadol ir 50 mg ( 122.2 [ 98.66 ] ) , tapentadol ir 75 mg ( 143.7 [ 96.52 ] ) , and oxycodone hcl ir 10 mg ( 140.3 [ 99.52 ] ) than with placebo ( 54.1 [ 105.74 ] ; p < 0.001 for all comparisons ; figure 2 ) . additionally , the efficacy of both doses of tapentadol ir was noninferior to the efficacy of oxycodone hcl ir 10 mg , based on the lower bounds of the 2-sided 97.5% confidence intervals for tapentadol ir 50 mg ( 36.05 ) and 75 mg ( 12.91 ) , which were within the prespecified noninferiority margin of 48 ( 10% of the total possible value ) . secondary efficacy measurements supported the results of the primary efficacy endpoint in this phase 3 postoperative pain study.50 compared with placebo , significant reductions in pain intensity ( based on the spid ) were observed for all active treatment groups at 12 , 24 , and 72 hours ( p < 0.001 for all comparisons ; figure 2 ) . the distribution of responder rates was also significantly different between both tapentadol ir dose groups and placebo ( p < 0.001 ) and oxycodone hcl ir 10 mg and placebo ( p < 0.001 ; figure 3 ) . compared with placebo , a significantly greater percentage of patients in all active treatment groups reported reductions in pain intensity at 48 hours of at least 30% ( tapentadol ir 50 mg , 77.5% ; tapentadol ir 75 mg , 76.3% ; oxycodone hcl ir 10 mg , 75.2% ; placebo , 58.0% ; p 0.004 for all comparisons ) and at least 50% ( tapentadol ir 50 mg , 64.7% ; tapentadol ir 75 mg , 64.0% ; oxycodone hcl ir 10 mg , 64.4% ; placebo , 47.8% ; p 0.012 for all comparisons ) . for the pgic , patients rated their overall status since beginning study medication on a 7-point scale ( 1 = very much improved to 7 = very much worse ) . at the end of the study or early discontinuation , a rating of much improved or very much improved on the pgic was reported by 83% of patients in the tapentadol ir 50-mg group , 88% of patients in the tapentadol ir 75-mg group , 86% of patients in the oxycodone hcl ir 10-mg group , and 65% of patients in the placebo group . the overall distribution of pgic scores was significantly more favorable for all active treatment groups compared with placebo ( p < 0.001 for all comparisons ) . in all 4 studies,5053 the most commonly reported treatment emergent adverse events ( teaes ) for patients who received any dosage of tapentadol ir were typical of drugs with -opioid agonist activity , and there were no major teaes suggestive of hyper - adrenergism . in the phase 3 study50 of tapentadol ir 50 and 75 mg in patients with postoperative pain following bunionectomy , the most common teaes ( reported by 10% of patients in any treatment group ) were nausea , vomiting , dizziness , headache , somnolence , pruritus , and constipation . tapentadol ir 50 mg was associated with a lower incidence of all of these teaes than oxycodone hcl ir 10 mg , and tapentadol ir 75 mg was associated with a lower incidence of nausea , headache , and constipation than oxycodone hcl ir 10 mg ( figure 4 ) . a significantly lower percentage of patients reported nausea and/or vomiting in the tapentadol ir 50-mg group ( 35% ) than in the oxycodone hcl ir 10-mg group ( 59% ; p < 0.001 ) . thus , at a dose ( 50 mg ) that provided efficacy that was noninferior to that provided by oxycodone hcl ir 10 mg , tapentadol ir was associated with a significantly lower incidence of gastrointestinal adverse events than oxycodone ir . a lower percentage of patients in the tapentadol ir 75-mg ( 51% ) than in the oxycodone hcl ir 10-mg group ( 59% ) reported nausea and/or vomiting , but the difference did not reach statistical significance ( p = 0.057 ) . a low percentage ( < 3% in any treatment group ) of patients reported teaes that led to study discontinuation in the tapentadol ir 50-mg group ( 1.1% ) , the tapentadol ir 75-mg group ( 2.9% ) , the oxycodone hcl ir 10-mg group ( 1.8% ) , and the placebo group ( 1.5% ) . the 90-day safety study53 permitted an evaluation of the long - term safety of flexible doses of tapentadol ir ( 50 or 100 mg every 4 to 6 hours ) in comparison with flexible doses of oxycodone hcl ir ( 10 or 15 mg every 4 to 6 hours ) in patients with acute osteoarthritis hip or knee pain or low back pain . similar to the phase 3 postoperative pain study,50 the most common teaes ( occurring in 10% of patients in either treatment group ) included gastrointestinal teaes ( nausea , vomiting , and constipation ) , nervous system teaes ( dizziness , headache , and somnolence ) , and pruritus . a lower percentage of patients in the tapentadol ir group than in the oxycodone ir group reported nausea ( 18.4% vs 29.4% ) , vomiting ( 16.9% vs 30.0% ) , constipation ( 12.8% vs 27.1% ) , and pruritus ( 4.3% vs 11.8% ) . odds ratios demonstrated that patients treated with tapentadol ir were significantly less likely than patients treated with oxycodone ir to report nausea ( 0.542 ) , vomiting ( 0.476 ) , the composite of nausea and/or vomiting ( 0.458 ) , or constipation ( 0.396 ; p < 0.001 for all comparisons ) . in addition , a lower percentage of patients discontinued from the study because of aes in the tapentadol ir group ( 20.8% ) than in the oxycodone ir group ( 30.6% ) , and patients in the oxycodone ir group discontinued significantly earlier than those in the tapentadol ir group ( nominal p < 0.05 ; figure 5 ) . the percentage of patients who discontinued from the study due to gastrointestinal aes was lower in the tapentadol ir group ( 8.8% ) than in the oxycodone ir group ( 21.1% ) . compared with placebo , tapentadol ir ( 50 to 100 mg every 4 to 6 hours ) and oxycodone hcl ir ( 10 or 15 mg every 4 to 6 hours ) were associated with a higher incidence of teaes at all doses studied , and the majority of teaes increased with increasing dose.5052 however , tapentadol ir was associated with a reduction in teaes , particularly gastrointestinal teaes and pruritus , relative to oxycodone ir in all 4 phase 3 studies.5053 the lower incidence of gastrointestinal teaes observed with tapentadol ir relative to oxycodone ir5053 was associated with a lower percentage of patients who discontinued treatment because of teaes . nausea and vomiting are often associated with the initiation of opioid therapy27 and are considered to be among the most undesirable adverse effects associated with analgesic therapy.54 these opioid - induced gastrointestinal teaes may be dose - limiting and are often severe enough to cause patients to discontinue therapy , leading to disruption of pain relief.55,56 tapentadol is a novel , centrally acting analgesic with 2 mechanisms of action , -opioid receptor agonism and norepinephrine reuptake inhibition,35,36 which may contribute to an improved gastrointestinal tolerability profile . in patients with moderate to severe acute pain of various etiologies , tapentadol ir has been shown to offer comparable analgesia to that provided by oxycodone ir , with lower incidences of gastrointestinal adverse effects and pruritus and lower rates of discontinuation due to adverse effects.5053 this combination of potent analgesia and tolerability may represent a substantial improvement over current acute pain relief strategies .

the undertreatment of acute pain is common in many health care settings . insufficient management of acute pain may lead to poor patient outcomes and potentially life - threatening complications . opioids provide relief of moderate to severe acute pain ; however , therapy with pure -opioid agonists is often limited by the prevalence of side effects , particularly opioid - induced nausea and vomiting . tapentadol is a novel , centrally acting analgesic with 2 mechanisms of action , -opioid receptor agonism and norepinephrine reuptake inhibition . the analgesic effects of tapentadol are independent of metabolic activation and tapentadol has no active metabolites ; therefore , in theory , tapentadol may be associated with a low potential for interindividual efficacy variations and drug drug interactions . previous phase 3 trials in patients with various types of moderate to severe acute pain have shown that tapentadol immediate release ( ir ; 50 to 100 mg every 4 to 6 hours ) provides analgesia comparable to that provided by the pure -opioid agonist comparator , oxycodone hcl ir ( 10 or 15 mg every 4 to 6 hours ) , with a lower incidence of nausea , vomiting , and constipation . findings suggest tapentadol may represent an improved treatment option for acute pain .

Introduction Pharmacology and pharmacokinetics of tapentadol Tapentadol IR for moderate to severe pain Safety and tolerability of tapentadol IR Conclusions

unrelieved acute pain may cause anxiety , sleep disturbances , and demoralization and may interfere with mental activity and social interactions.1,2 acute pain can also increase heart rate and blood pressure , suppress immune functioning , and reduce pulmonary function , leading to an increased risk of dangerous complications , including myocardial ischemia , deep vein thrombosis , pulmonary embolism , hypoxia , pneumonia , and stroke.2 in addition to these more severe adverse effects , uncontrolled acute pain is also associated with gastrointestinal effects , including the development of ileus , nausea , and vomiting.3 the psychologic and physiologic effects of uncontrolled acute pain can result in longer hospital stays and unscheduled readmissions following surgery.4,5 a retrospective analysis of the medical records of 20,817 patients who had undergone same - day surgery in 1999 found that pain was the most common reason that patients were hospitalized immediately after surgery or returned to the hospital within 30 days of surgery , accounting for 38% of unscheduled postoperative hospital admissions or readmissions.4 in addition , prolonged acute pain can cause sensitization of the central and peripheral nervous systems , leading to the development of chronic pain , which is often difficult and costly to treat.68 although guidelines have been developed to improve acute pain management,9,10 pain relief remains suboptimal for many patients.1116 surveys conducted among patients who had undergone ambulatory surgery indicated that 30%15 to 40%12 of patients experience moderate to severe pain following discharge . among hospitalized patients who were receiving treatment in various medical wards14 or who had undergone surgery,11 the percentages were even higher , with 52%14 to 80%11 of patients experiencing acute pain ; of these patients , as many as 86% experienced moderate to extreme pain.11 the undertreatment of acute pain may result from several contributing factors related to patient care , such as infrequent pain assessments , underestimation of the severity of pain , concerns about side effects associated with analgesic treatment , and delays or dose reductions in the administration of analgesics by health care providers.13,14,1720 in addition , patients may underreport acute pain because of low expectations of pain relief and concerns about the adverse effects of analgesic treatment.19,21 a 2004 survey of patients undergoing major abdominal surgery found that many patients were willing to sacrifice pain relief for a reduction in the severity of side effects.22 current treatment options for the management of acute pain include opioid analgesics ( eg , morphine , hydromorphone , and oxycodone ) and nonopioid analgesics ( eg , acetaminophen , acetylsalicylic acid , and nonsteroidal anti - inflammatory drugs [ nsaids]).23 most nsaids are limited by a therapeutic ceiling and are appropriate for the relief of only mild to moderate pain.23 in addition , nsaids are contraindicated in patients with peptic ulcer disease , renal impairment , and any tendency for bleeding.24 cyclooxygenase-2 ( cox-2)-specific inhibitors do not impair platelet function and have an improved gastrointestinal tolerability profile relative to other nsaids25 ; however , certain cox-2specific inhibitors have been linked to an increase in the risk of cardiovascular side effects , including myocardial infarction.26 opioids are typically used for the management of moderate to severe acute pain,27 but opioid use is limited by the occurrence of a range of side effects.28 opioids exert their analgesic effects primarily through agonistic interactions with -opioid receptors in neurons in the pain pathway , which lead to a reduction in neurotransmitter release and associated pain.29 however , the agonistic interactions responsible for opioid activity are not limited to neurons of the pain pathway.23 opioid receptors are present throughout the nervous system , and the interactions of opioids with nonanalgesic receptors are responsible for many of the side effects associated with opioid treatment.23 for example , opioids may induce nausea and vomiting by direct stimulation of receptors at the chemoreceptor trigger zone and vestibular apparatus.30 a systematic review31 of randomized controlled trials of opioids in postoperative patients found that the most common side effects occurring in these patients were gastrointestinal side effects , central nervous system ( cns ) side effects , pruritus , and urinary retention . pruritus occurred in 18.3% of patients who were treated with opioids following surgery and was most common with epidural administration of opioids.31 somnolence and sedation were the most commonly reported cns side effects ; the incidence of somnolence ranged from less than 2% to more than 90% , depending on the route of administration and type of opioid used.31 gastrointestinal side effects , including nausea , vomiting , and constipation , were the most common side effects associated with opioid analgesia and were reported by 31.0% of patients.31 opioid - induced postoperative nausea and vomiting ( ponv ) is associated with negative effects on patient outcomes and quality of life,32 which may lead to increases in recovery time , duration of hospitalization , and cost of medical care.33,34 the underuse of opioid analgesics by health care providers to relieve acute pain may be related to attempts to balance analgesia against concerns about opioid - induced side effects and subsequent deleterious repercussions for patient outcomes.20 there is a continuing need for a potent analgesic agent that will provide adequate relief of acute pain , but with a reduction in side effects . tapentadol is a novel , centrally acting analgesic that offers analgesic efficacy that is similar to that provided by a pure -opioid agonist comparator , but with an improved side effect profile , which may represent a significant advancement in the management of moderate to severe acute pain . tapentadol ( figure 1 ) is a centrally acting analgesic with 2 complementary mechanisms of action , -opioid receptor agonism and norepinephrine reuptake inhibition.35,36 in opioid receptor binding studies , tapentadol was found to have only a modest affinity ( dissociation constant [ ki ] = 0.096 m [ rat])35 for the -opioid receptor relative to pure -opioid receptor agonists such as oxycodone ( ki = 0.018 m [ rat ] ) or morphine ( ki = 0.002 m [ rat]).37 a similar binding affinity to that observed in native rat receptors was demonstrated for tapentadol at the human recombinant -opioid receptor ( ki = 0.16 m [ human]).35 despite the approximately 50-fold difference in binding affinity for the -opioid receptor relative to morphine , tapentadol demonstrated only a 2- to 3-fold reduction in analgesic potency in a series of acute and persistent animal pain models.36 this disparity in potency and binding affinity for the -opioid receptor may be related to the contribution of the second mechanism of action , inhibition of norepinephrine reuptake , to the analgesic effects of tapentadol . in rat synaptosomal reuptake assays , tapentadol inhibited the norepinephrine reuptake transporter with a ki of 0.48 0.11 m and , when administered in intraperitoneal doses of 4.64 to 10 mg / kg , produced a dose - dependent increase in extracellular levels of norepinephrine in the ventral hippocampus of freely moving rats to a maximum of 450% above baseline with 10 mg / kg , as measured by microdialysis.35 in contrast to tapentadol , morphine administered to rats in intraperitoneal doses of 1 to 10 mg / kg produced a small , nonsignificant decrease in extracellular norepinephrine levels.35 the contributions of both -opioid receptor agonism and norepinephrine reuptake inhibition to the analgesic effects of tapentadol were further elucidated by examining the extent to which analgesia was blocked by the selective -opioid receptor antagonist naloxone and the norepinephrine 2-receptor antagonist yohimbine.35 in an animal model35 of acute ( writhing ) pain , it was observed that intravenous tapentadol and morphine induced dose - dependent inhibition of writhing ( ed50 for tapentadol , 0.7 mg / kg ; ed50 for morphine , 0.4 mg / kg ) . in contrast , when yohimbine ( 2.15 mg / kg ) was administered intraperitoneally in combination with intravenous doses of morphine ( 6.81 mg / kg ) or tapentadol ( 10 mg / kg ) , the anti - allodynic effect of tapentadol was reduced from 81% to 19% of the mpe , whereas the anti - allodynic effect of morphine was only minimally reduced ( from 80% to 54% of the mpe).35 these results indicate that both -opioid receptor agonist and norepinephrine reuptake inhibitor mechanisms are involved in the analgesic effect of tapentadol and that , in contrast to morphine , norepinephrine reuptake inhibition plays a prominent role in tapentadol - induced analgesia . in addition to contributing to the analgesic activity of tapentadol , the opioid - sparing effect of norepinephrine reuptake inhibition may also contribute to a reduction of adverse effects associated with pure -opioid agonists.36 such an opioid - sparing effect has been achieved by combining other analgesics , such as nsaids or cox-2specific inhibitors , with opioids to control acute pain.38 this type of multimodal strategy achieves an additive analgesic effect by combining 2 different mechanisms of analgesic activity , but reduces the consumption of opioid analgesics and , thereby , the incidence of adverse effects associated with -opioid agonist activity.38 for example , in a study of 200 patients undergoing outpatient anterior cruciate ligament surgery , patients who received perioperative doses of the cox-2specific inhibitor celecoxib in addition to oxycodone experienced less pain ( p < 0.01 ) in the recovery room , had lower postoperative opioid consumption ( p < 0.001 ) , and reported a lower incidence of ponv ( p < 0.05 ) than patients taking oxycodone alone.39 by combining a second mechanism of analgesic activity with -opioid receptor agonism , tapentadol may offer the benefits of multimodal analgesia within a single molecule . the analgesic effects of tapentadol are independent of metabolic activation , and tapentadol has no active metabolites.40 orally administered tapentadol is principally cleared by hepatic glucuronidation via the uridine 5-diphospho - glucuronosyl transferases ( ugts ) ugt1a9 and ugt2b7 , which are responsible for approximately 55% of tapentadol metabolism in humans.41 the major metabolite of tapentadol , tapentadol - o - glucuronide , has no activity at opioid receptors , synaptosomal reuptake systems , or other binding sites.35 morphine is likewise primarily metabolized by hepatic glucuronidation via ugt2b7 to morphine-3-glucuronide ( m3 g ) and morphine-6-glucuronide ( m6g).42 morphine-3-glucuronide has no analgesic activity , but m6 g has an affinity for the -opioid receptor and contributes significantly to the analgesic effect of morphine.43 in patients with renal insufficiency , m6 g accumulates and may contribute to the higher incidence of morphine toxicity observed in these patients.44,45 many other opioids , including oxycodone , codeine , dihydrocodeine , hydrocodone , and tramadol , are primarily metabolized by the cytochrome p450 ( cyp ) enzymes cyp2d6 or cyp3a4.46 mutations in the cyp2d6 gene , which occur in approximately 1% to 7% of the caucasian population , can either decrease or increase enzyme activity , leading to alterations in opioid analgesia.47 the analgesic effects of codeine are highly dependent on conversion of codeine to morphine by cyp2d6 ; a poor cyp2d6 metabolic phenotype can suppress codeine analgesia , and an ultra - rapid cyp2d6 metabolic phenotype can lead to increased opioid effects , such as euphoria , dizziness , and visual disturbances.47 in addition to the potential for varied individual responses to cytochrome p450-metabolized opioids due to genetic mutations , opioids metabolized by the cytochrome p450 pathway are associated with an increased risk for drug drug interactions . more than half of all drugs are metabolized by cyp3a4 , and opioids metabolized by this isozyme ( including fentanyl , buprenorphine , and methadone ) are prone to drug drug interactions with antiretroviral agents and antidepressants.48 opioids metabolized by cyp2d6 , including codeine , dihydrocodeine , and oxycodone , are also associated with a number of drug drug interactions . tapentadol did not undergo significant metabolism by cyp enzymes and did not inhibit or induce the activity of any of the cyp isoforms tested , with the exception of cyp2d6.41 limited inhibition of cyp2d6 was observed with tapentadol , with competitive inhibition occurring with a ki of 181 m and noncompetitive inhibition with a ki of 1,410 m.41 the ki values for both competitive and noncompetitive inhibition are much higher than the expected tapentadol plasma concentrations of 0.5 to 1 m ( following therapeutic dosing ) , indicating that cyp2d6 inhibition by tapentadol is unlikely to be clinically relevant.41 two randomized , open - label studies were performed to evaluate the potential for drug drug interactions between tapentadol and 3 common analgesics that , like tapentadol , are metabolized by ugt pathways ( acetaminophen , naproxen , and acetylsalicylic acid).49 mean serum concentrations of tapentadol and tapentadol - o - glucuronide were similar after administration of tapentadol ir alone and after coadministration with acetaminophen and acetylsalicylic acid . the efficacy of tapentadol ir for the relief of moderate to severe pain has been evaluated in 3 randomized , double - blind , phase 3 studies in patients with postoperative ( bunionectomy ) pain50,51 and pain related to end - stage degenerative joint disease52 and as a secondary measure in a phase 3 randomized , double - blind , 90-day safety study.53 in one of the postoperative pain studies,51 patients received tapentadol ir ( 50 , 75 , or 100 mg ) , oxycodone hcl ir ( 15 mg ) , or placebo every 4 to 6 hours for 72 hours following bunionectomy ; in the other postoperative pain study,50 patients received tapentadol ir ( 50 or 75 mg ) , oxycodone hcl ir ( 10 mg ) , or placebo every 4 to 6 hours for 72 hours following bunionectomy . in the end - stage joint disease study,52 patients received tapentadol ir ( 50 or 75 mg ) , oxycodone hcl ir ( 10 mg ) , or placebo every 4 to 6 hours for 10 days . in the 90-day safety study,53 patients received flexible doses of tapentadol ir 50 or 100 mg ( up to 600 mg / day ) or oxycodone hcl 10 mg or 15 mg ( up to 90 mg / day ) every 4 to 6 hours as needed for up to 90 days . in all 4 phase 3 studies5053 of tapentadol ir for acute pain , improvements in pain intensity were observed with tapentadol ir treatment ( 50 , 75 , or 100 mg every 4 to 6 hours ) that were similar to those observed with oxycodone hcl ir treatment ( 10 or 15 mg every 4 to 6 hours ) based on pain intensity measurements on an 11-point numerical rating scale ( nrs ; 0 = no pain to 10 = worst pain imaginable ) . based on increases in the mean ( sd ) spid48 , significantly greater reductions in pain intensity from baseline were observed with tapentadol ir 50 mg ( 122.2 [ 98.66 ] ) , tapentadol ir 75 mg ( 143.7 [ 96.52 ] ) , and oxycodone hcl ir 10 mg ( 140.3 [ 99.52 ] ) than with placebo ( 54.1 [ 105.74 ] ; p < 0.001 for all comparisons ; figure 2 ) . in the phase 3 study50 of tapentadol ir 50 and 75 mg in patients with postoperative pain following bunionectomy , the most common teaes ( reported by 10% of patients in any treatment group ) were nausea , vomiting , dizziness , headache , somnolence , pruritus , and constipation . tapentadol ir 50 mg was associated with a lower incidence of all of these teaes than oxycodone hcl ir 10 mg , and tapentadol ir 75 mg was associated with a lower incidence of nausea , headache , and constipation than oxycodone hcl ir 10 mg ( figure 4 ) . thus , at a dose ( 50 mg ) that provided efficacy that was noninferior to that provided by oxycodone hcl ir 10 mg , tapentadol ir was associated with a significantly lower incidence of gastrointestinal adverse events than oxycodone ir . the 90-day safety study53 permitted an evaluation of the long - term safety of flexible doses of tapentadol ir ( 50 or 100 mg every 4 to 6 hours ) in comparison with flexible doses of oxycodone hcl ir ( 10 or 15 mg every 4 to 6 hours ) in patients with acute osteoarthritis hip or knee pain or low back pain . similar to the phase 3 postoperative pain study,50 the most common teaes ( occurring in 10% of patients in either treatment group ) included gastrointestinal teaes ( nausea , vomiting , and constipation ) , nervous system teaes ( dizziness , headache , and somnolence ) , and pruritus . compared with placebo , tapentadol ir ( 50 to 100 mg every 4 to 6 hours ) and oxycodone hcl ir ( 10 or 15 mg every 4 to 6 hours ) were associated with a higher incidence of teaes at all doses studied , and the majority of teaes increased with increasing dose.5052 however , tapentadol ir was associated with a reduction in teaes , particularly gastrointestinal teaes and pruritus , relative to oxycodone ir in all 4 phase 3 studies.5053 the lower incidence of gastrointestinal teaes observed with tapentadol ir relative to oxycodone ir5053 was associated with a lower percentage of patients who discontinued treatment because of teaes . nausea and vomiting are often associated with the initiation of opioid therapy27 and are considered to be among the most undesirable adverse effects associated with analgesic therapy.54 these opioid - induced gastrointestinal teaes may be dose - limiting and are often severe enough to cause patients to discontinue therapy , leading to disruption of pain relief.55,56 tapentadol is a novel , centrally acting analgesic with 2 mechanisms of action , -opioid receptor agonism and norepinephrine reuptake inhibition,35,36 which may contribute to an improved gastrointestinal tolerability profile . in patients with moderate to severe acute pain of various etiologies , tapentadol ir has been shown to offer comparable analgesia to that provided by oxycodone ir , with lower incidences of gastrointestinal adverse effects and pruritus and lower rates of discontinuation due to adverse effects.5053 this combination of potent analgesia and tolerability may represent a substantial improvement over current acute pain relief strategies .

the mechanism of action for most cancer vaccines is mainly mediated through cytotoxic t lymphocytes ( ctls ) . we are now gaining a clear understanding of the cellular events leading to an effective ctl - mediated antitumor immunity . the antigen - presenting cells ( apcs ) most suitable for cancer vaccines are dendritic cells ( dcs ) , which can be distinguished from b cells and macrophages by their abundant expression of costimulatory molecules and abilities to initiate a strong primary immune response [ 1 , 2 ] . dcs are specialized to capture and process tumor - associated antigens ( taas ) , converting the proteins to peptides that are presented on major histocompatibility complex ( mhc ) class i and class ii molecules . after taas uptake and inflammatory stimulation , immature dcs in peripheral tissues undergo a maturation process characterized by the upregulation of costimulatory molecules [ 2 , 3 ] . during this process , mature dcs migrate to t - cell areas of secondary lymphoid organs , where they present antigenic peptides to cd8 + and cd4 + t cells through mhc class i and class ii pathways , respectively , and become competent to present antigens to t cells , thus initiating antigen - specific ctl responses . antigen - specific ctls in turn can attack tumor cells that express cognate antigenic determinants or can provide help for b - cell responses that produce antibodies , which can also lead to tumor cell death in some cases . thus , the mechanism of action for cancer vaccines , based on harnessing host immune cells to infiltrate tumors and to exert ctl responses , is quite different from that of a traditional cytotoxic chemotherapy . a major area of investigation in induction of antitumor immunity involves the design of dc - based cancer vaccines . dcs derive their potency from constitutive and inducible expression of essential costimulatory molecules including b7 , icam-1 , lfa-1 , lfa-3 , and cd40 on the cell surface [ 1 , 8 , 9 ] . these proteins function in concert to generate a network of secondary signals essential for reinforcing the primary antigen - specific signals in t - cell activation . therefore , many strategies have been developed to load taas onto dcs and used as cancer vaccines . for example , dcs are pulsed with synthetic peptides derived from the known antigens , tumor lysates , tumor rna [ 12 , 13 ] , and dying tumor cells to induce antigen - specific antitumor immunity . although the production of dc - based cancer vaccines for individual patients with cancer has currently been addressed in clinical trials , a major drawback of these strategies comes from the limited number of known antigenic peptides available in many hla contexts . moreover , the results of clinical trials using dcs pulsed with antigen - specific peptides show that clinical responses have been found in a small number of patients [ 15 , 16 ] . to overcome this limitation , we have proposed the fusions of dcs and whole tumor cell ( dc / tumor ) to generate cell hybrids with the characteristics of apcs able to process endogenously provided whole taas . the whole tumor cells may be postulated to serve as the best source of antigens [ 1721 ] . the fusion of dc and tumor cell through chemical , physical , or biological means creates a heterokaryon which combines dc - derived costimulatory molecules , efficient antigen - processing and -presentation machinery , and an abundance of tumor - derived antigens including those yet to be unidentified ( figure 1 ) . thus , the dc / tumor fusion cells combine the essential elements for presenting tumor antigens to host immune cells and for inducing effective antitumor responses . now , it is becoming clear that the tumor antigens are processed along the endogenous pathway , through the antigen processing machinery of human dc . thus , it is conceivable that tumor antigens synthesized de novo in the heterokaryon are processed and presented through the endogenous pathway . the advantage of dc / tumor fusion vaccines over pulsing dc with whole tumor lysates is that endogenously synthesized antigens have better access to mhc class i pathway [ 1821 ] . indeed , it has been demonstrated that dc / tumor fusion vaccines are superior to those involving other methods of dc loaded with antigenic proteins , peptides , tumor cell lysates , or irradiated tumor cells in murine models [ 1821 ] . the efficacy of antitumor immunity induced by dc / tumor fusion vaccines has been demonstrated in murine models using melanoma [ 2432 ] , colorectal [ 17 , 30 , 31 , 3341 ] , breast [ 4247 ] , esophageal , pancreatic [ 49 , 50 ] , hepatocellular [ 5155 ] , lung [ 22 , 41 , 5659 ] , renal cell carcinoma , sarcoma [ 6166 ] , myeloma [ 6774 ] , mastocytoma , lymphoma , and neuroblastoma . the fusion cells generated with human dc and tumor cell also have the ability to present multiple tumor antigens , thus increasing the frequency of responding t cells and maximizing antitumor immunity capable of killing tumor targets such as colon [ 7884 ] , gastric [ 85 , 86 ] , pancreatic , breast [ 47 , 8893 ] , laryngeal , ovarian [ 9597 ] , lung [ 85 , 98 ] , prostate [ 99 , 100 ] , renal cell [ 101 , 102 ] , hepatocellular [ 103105 ] carcinoma , leukemia [ 106111 ] , myeloma [ 112 , 113 ] , sarcoma [ 114 , 115 ] , melanoma [ 116119 ] , glioma , and plasmacytoma . despite the strong preclinical evidences supporting the use of dc / tumor fusions for cancer vaccination , the results of clinical trials so far reported are conflicting [ 1821 ] . one of the reasons is the evidence for fusion cell formation used as clinical trials is not definitive . the levels of fusion efficiency , which can be quantified by determining the percentage of cells that coexpress tumor and dc antigens , are closely correlated with ctl induction in vitro [ 82 , 83 ] . another reason is immunosuppressive substances such as tgf- derived from tumor cells used for fusion cell preparations [ 35 , 47 ] . although tumor - derived tgf- reduces the efficacy of dc / tumor fusion vaccines via an in vivo mechanism , the reduction of tgf- derived from the fusions inhibits the generation of tregs and enhances antitumor immunity . moreover , the therapeutic effects in patients vaccinated by dc / tumor fusions are correlated with the characteristics of the dcs used as the fusion vaccines [ 82 , 83 ] . indeed , patient - derived fusions show inferior levels of mhc class ii and costimulatory molecules and produce decreased levels of il-12 and increased levels of il-10 , as compared with those obtained from fusions of tumor cell and dc from healthy donors [ 87 , 103 ] . therefore , it is important to assess the phenotype and function of dc / tumor fusion cell preparations used in each vaccination . the delayed - type hypersensitivity ( dth ) is an inflammatory reaction mainly mediated by cd4 + effector memory t cells that infiltrate the site of injection of an antigen against which the immune system has been primed by cancer vaccines . actually , soluble proteins , peptides , or antigens pulsed dcs have been injected intradermally , and the diameter of erythema or induration after 4872 h is measured . cd4 + effector memory t cells that recognize the antigens presented on local apcs mediate the immune responses by releasing cytokines , resulting in an increased vascular permeability and the recruitment of monocytes and other inflammatory cells in the site . cd8 + it has been reported that antigen - specific t cells can be readily detected in skin biopsies from dth sites , much less in abdominal lymph nodes , and not in peripheral blood and tumor site . moreover , there is a significant correlation between favorable clinical outcome and the presence of vaccine - related antigen - specific t cells in biopsies from dth sites . indeed , the increased dth reactivity against tumor antigens has been observed in clinical responders by dc / tumor fusion vaccines . in almost patients with cancer , t cells from lymph nodes and . therefore , functional assessment of antigen - specific t cells from such dth sites may serve as an additional strategy to evaluate antigen - specific antitumor immune responses [ 122 , 126 , 127 ] . the mechanism of cancer vaccines , based on inducing ctls , infiltrating tumors , and exerting t - cell - mediated cytotoxic effects , is quite different from that of cytotoxic chemotherapy . as cancer vaccines do not work as quickly as chemotherapy which has a direct cytotoxic effect , the response evaluation criteria in solid tumors ( recist ) and who criteria [ 128 , 129 ] , designed to detect early effects of cytotoxic chemotherapy , can not appropriately evaluate the response patterns observed with cancer vaccines . they presume that linear measures are an adequate substitute for 2-dimentional methods and register four response categories : cr ( complete response ) , pr ( partial response ) , sd ( stable disease ) , and pd ( progressive disease ) . however , in the solid tumors , there exist not only antigen - specific ctls but also immune suppressive cells such as myeloid - derived suppressor cells ( mdscs ) , immunosuppressive tumor - associated macrophages ( tams ) , and cancer associated fibroblasts ( cafs ) ( figure 2 ) . after vaccination , the solid tumors may become heavily infiltrated by immune - related cells resulting in an apparent increase in size of lesions , which is , at least in part , due to the infiltration of ctls induced by cancer vaccines . therefore , the development of new response criteria , including immunologic cellular monitoring , is of great importance in the development of cancer vaccines . in clinical trials , . the currently used methods of assessing t - cells from patients treated with cancer vaccines are t - cell proliferation , cytokine profile , cytotoxic t lymphocyte assays ( ctl assays ) , ctl - associated molecules ( cd107 , perforin , granzyme b , and cd154 ) , multimer analysis , t - cell receptor ( tcr ) gene usage , and immune suppression assays ( table 1 ) . while these assays can be also used for monitoring cellular immune responses induced by dc / tumor fusion vaccines , none has been standardized . as dc / tumor fusion vaccines can induce defined and undefined antigen - specific antitumor activities , immunologic cellular monitoring for the fusion vaccines is much more complex . furthermore , as immune responses induced by dc / tumor fusion vaccines are a balanced mosaic of both immune stimulatory and suppressive responses , multiple monitoring assays for the clinical efficacy parameters may be needed to evaluate the antitumor immune responses . t - cell proliferation assay assesses the number and function at the level of the entire t - cell population in the culture . therefore , the ability to detect t - cell responses is based on the proliferative potential of the cells in response to antigens . the most commonly used in vitro method for measuring antigen - specific t - cell proliferation is the assessment of t - cell clonal expansion following incubation of t - cells with antigens in the presence of a radio - labeled nucleotide ( e.g. , [ h ] thymidine ) in vitro . cfse ( 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester ) staining can be also used to directly detect proliferative responses of t - cells . because cfse is partitioned equally during cell division , this technique can monitor t - cell division and determine the relationship between t - cell division and differentiation in vitro and in vivo . the extensive t - cell proliferation can be demonstrated by the few undivided t - cells left and from proper accumulation of activated t cells , as shown by the increase in t - cell counts correlating with the decrease in cfse label for each division . the cfse - based assays are equivalent to traditional measures of antigen - specific t - cell responsiveness and have significant advantages for the ability to gate on a specific population of t - cells and the concomitant measurement of t - cell phenotype . after vaccination , dc / tumor fusion cells can migrate to the t - cell area in the regional lymph nodes and form clusters with cd8 + and cd4 + t cells . simultaneous recognition of cognate peptides presented by mhc class i and class ii molecules on dc / tumor fusion cell is essential in the induction of efficient ctls . therefore , measuring antigen - specific cd8 + and cd4 + t - cell proliferation is essential to evaluate the induction of vaccine - specific immune responses . although t - cell proliferation assay is usefulness to detect immune responses in vitro , the results are strongly influenced by the in vitro stimulation procedures . stimulation of naive t cells from healthy donors with dc / tumor fusions in vitro results in potent proliferation of cd4 + and cd8 + t cells [ 34 , 80 ] . therefore , to assess dc / tumor fusion vaccines , antigen - specific cd4 + and cd8 + t cells need to be expanded by stimulation with autologous tumor lysates . in addition , the frozen peripheral blood mononuclear cells ( pbmcs ) obtained before and after vaccination must be processed in the same set of experiments [ 103 , 135 , 136 ] . as t - cell proliferation assay is biologically irrelevant and imprecise for the reasons stated above , this assay may not be emphasized in future studies . there is a currently great interest in the assay of polyfunctional t cells , secreting multiple cytokines ( e.g. , secreting ifn- and tnf- rather than either alone ) , or expressing multiple surface markers . as the release of th1 cytokines such as ifn- and tnf- is important to determine long - lasting antitumor immunity , a shift to th1 response by cancer vaccines is essential for therapeutic potential in murine models [ 36 , 37 , 67 , 77 , 137 , 138 ] therefore , it is important to test whether cancer vaccines can induce a th1 response in the tumor - specific t cells , and what impact might this have on the clinical responses . cytokine production by t cells in response to antigens can be detected in individual t cells by enzyme - linked immunospot ( elispot ) assay [ 1821 , 139 ] . moreover , production of ifn- captured by antibodies bound to t - cell surface can be detected by flow cytometry analysis [ 96 , 140 ] . the actual state of antigen specific t - cell reactivity directly from peripheral blood t cells can be quantified by ifn- elispot assay and flow cytometry analysis [ 1821 , 141 ] . as the ifn- elispot assay shows highly reproducible results among different laboratories , the elispot may be an ideal candidate for robust monitoring of t - cell activity [ 1821 , 142 ] . coculture of cd4 + and cd8 + t cells from healthy donors with dc / tumor fusions results in high levels of ifn- production and low levels of il-10 production [ 50 , 54 , 80 , 143 ] . therefore , to assess dc / tumor fusion vaccines precisely , t cells obtained before and after vaccination might be directly quantified with stimulation of autologous tumor lysates in vitro . in effective clinical responders , comparable levels of ifn- production in response to tumor lysates may be detected in pbmcs obtained before vaccination . a correlation between ifn- elispot outcome and effective clinical responses ( period free of relapse or survival ) has been found in patients treated with cancer vaccines including dc / tumor fusions [ 103 , 135 , 136 , 144 ] . for immune monitoring of cancer vaccines , t - cell - mediated ctl assays are appealing because measurement of the ability of ctl to kill tumor targets is thought to be a relevant marker for antitumor activity . it has been assumed that the cytotoxicity has been measured in cr - release assays in vitro . instead of cr release assays , flow cytometry - based methods have been developed to assess ctl activity [ 145 , 146 ] . flow cytometry ctl assays can be predicated on measurement of ctl - induced caspase-3 or annexin - v activation in target cells through fluorescence detection , which are more sensitive to conventional cr release assays [ 145147 ] . these assays show increased sensitivity at early time points after target / effector cell mixing and allow for analysis of target cells in real time at the single - cell level . however , it is unusual to detect antigen - specific killing by t cells directly isolated from the patients vaccinated with dc / tumor fusions even with the use of flow cytometry - based ctl assays [ 103 , 148 ] . therefore , there is a need to stimulate and expand the antigen - specific t cells in vitro for several days . these stimulations may distort the phenotype and function of the t - cell populations from tumor state . moreover , it is difficult to obtain sufficient numbers of viable tumor cells from primary lesion due to the length of culture time and potential contamination of bacteria and fungus . thus , semiallogeneic targets with shared taas and mhc class i molecules are necessary instead of autologous targets . importantly , a majority of the antigen - specific cd8 + ctls in peripheral blood may not be tumor reactive due to various mechanisms such as downmodulation of mhc class i molecules on tumors and presence of tregs at the tumor site . indeed , cytotoxic activity against autologous targets has been observed in peripheral blood t cells from patients vaccinated with dc / tumor fusions by ctl assays [ 103 , 148 ] , but the clinical responses from early clinical trails in patients with melanoma , glioma , gastric , breast , and renal cancer are muted [ 103 , 130 , 134 , 135 , 142 , 143 , 148154 ] . the defects of the clinical responses may be caused by the immunosuppressive influences derived from the local tumor microenvironment . in addition , therapeutic antitumor immunity depends on highly migratory ctls capable of trafficking between lymphoid and tumor sites . therefore , localization of antigen - specific ctls demonstrated by analysis of biopsy samples from tumor sites may be directly associated with clinical responses . the population of cd8 + ctls can destroy tumor cells through effector molecules ( e.g. , perforin and granzyme b ) . degranulation of cd107a and b is a requisite process of perforin / granzyme b - dependent lytic fashions mediated by responding antigen - specific ctls . these perforin / granzyme b - dependent lytic fashions require degranulation of cd107a and b in cd8 + ctls . therefore , measurement of cd107a and b , perforin , or granzyme b expression by flow cytometric analysis can be combined with intracellular ifn- staining to more completely assess the functionality of cd8 + ctls [ 83 , 87 ] . moreover , autologous tumor - induced de novo cd154 expression in cd4 + t cells is highly sensitive for tumor - specific th cells . the coupling of cd154 expression with multiplexed measurements of ifn- production provides a greater level of detail for the study of tumor - specific cd4 + t - cell responses . although dc / tumor fusion vaccines have abilities to induce cd107 + ifn-+ cd8 + t cells and cd154 + ifn-+ cd4 + t cells upon autologous tumor encounter in vitro [ 83 , 87 ] , it has now been unclear the correlation of the assay with clinical outcome . now , it has become possible to analyze antigen - specific cd8 + and cd4 + t cells by flow cytometric analysis using multimeric mhc - peptide complexes , measuring the affinity of the tcr to a given epitope . although dc / tumor fusion vaccines can induce defined and undefined antigens - specific cd8 + and cd4 + t cells , the multimer analysis can only be used to detect immune responses against defined antigenic epitopes expressing in tumor cells . mhc - peptide multimers stably bind to the tcr exhibiting a certain minimal avidity . hence , there are principal limitations of the multimeric analysis including the suitability and specificity of multimers and the lack of information about the functionality of multimer - positive t cells . the specific role of the multimer - positive t cells for cancer vaccine efficacy has not yet been well established in the setting of clinical trials . recent studies suggest that effective cancer vaccines not only stimulate ctl activity , but also sustain long - term memory t cells capable of mounting strong proliferative and functional responses to secondary tumor antigen challenge . therefore , it is more important to assess whether multimer - positive t cells are effector or effector - memory cells . moreover , the combined use of multimers and functional assays such as ifn- analysis may have provided some insight into the functional activity of these cells . it has been demonstrated that cryopreserved pbmcs from melanoma patients vaccinated with gp100 peptide show that the majority of multimer - positive cd8 + t cells had either a long - term effector ( cd45ra+ ccr7 ) or an effector - memory ( cd45ra ccr7 ) phenotype . interestingly , after vaccination , the resected melanoma patients can mount a significant antigen - specific cd8 + t cell immune response with a production of ifn- and high proliferation potential . to date , no studies have evaluated the functional activity of multimer - positive t cells in the blood after vaccination with dc / tumor fusions . only t cells having a tcr specific for a given antigen are triggered by interaction with cancer vaccines . this activation results in the clonal expansion of antigen - specific t cells that can be followed by tcr v gene usage . recently , the availability of a large panel of monoclonal antibodies against tcrs , mainly v epitopes , allows one to study the tcr repertoire by flow cytometry . pcr techniques can also be used to detect a restricted tcr repertoire from small amounts of t cells without biases caused by ex vivo expansions . although dc / tumor fusion vaccines have resulted in selection and expansion of t - cell clones , the generation of antitumor immunity by ctls has not correlated with clinical responses . immunogenic tolerance to a particular set of antigens is the absence of an immune response to those antigens , which can be achieved by processes that result in t - cell anergy ( antigen - specific unresponsiveness ) , t - cell unresponsiveness ( generalized dysfunction ) , and t - cell deletion ( apoptosis ) . future fusion vaccine studies should be designed to determine whether t - cell dysfunction correlated with clinical outcome . although antigen - specific ctls can be generated and detected in the circulation of vaccinated patients , these do not usually act against the tumor . it has been documented that immune suppressive cells can counteract antitumor immune responses . in tumor microenvironment , there are not only ctls but also many immune suppressive cells such as cd4 + cd25high+ foxp3 + tregs [ 103 , 164 ] , mdscs , tams , and cafs ( figure 2 ) . moreover , tumor cells produce immunosuppressive substances such as transforming growth factor ( tgf- ) vascular endothelial growth factor ( vegf ) , il-6 , il-10 , soluble fas ligand ( fas - l ) , programmed death-1 ligand ( pd - l1 ) , indolamine-2,3-dioxygenase ( ido ) , and microvesicles . type 1 regulatory t cells ( tr1 ) expressing cd39 may mediate suppression by il-10 , tgf- , and adenosine secretion , and whereby accumulation strongly correlates with the cancer progression . the mechanisms that suppress the immune system provide a fundamental reason why cancer vaccines fail to induce consistently robust antitumor immune responses . in dc / tumor fusion vaccines , cd4 + cd25high+ foxp3 + tregs were promoted in the presence of the local tumor - related factors in vitro . moreover , increased cd4 + cd25high+ foxp3 + tregs impaired the effector function of ctls induced by dc / tumor fusion vaccines . therefore , monitoring of immune suppressive cells in cancer patients vaccinated with dc / tumor fusions is also essential . the development of assays for detecting immune responses associated with clinical outcome has been limited . a variety of assays had been introduced to provide monitoring tools necessary for following changes in the frequency of antigen - specific ctls and to assess the impact of cancer vaccines on the immune system . as the mechanisms of immune response that cause tumor regression are not simple , the currently available assays may not actually measure a function with direct relevance to how tumors are actually attacked immunologically in cancer patients . a high reproducibility of results among different laboratories leads to the conclusion that cytokine flow cytometry or elispot may be an ideal candidate for robust and reproducible monitoring of t - cell activity in vivo . however , the widely used elispot assay often does not correlate the best with clinical outcome . therefore , it may be important to use a functional assay like cytokine flow cytometry or elispot in combination with a quantitative assay like multimers for immune monitoring . furthermore , it is necessary to understand the immune responses seen in peripheral blood versus the responses at the tumor site . monitoring of antigen - specific ctls at the tumor site may be directly associated with clinical responses . however , t cells from lymph nodes and the tumor site itself are not readily available for monitoring purposes in almost all patients . therefore , the ability to assess the function of antigen - specific t cells from dth site may serve as an additional strategy to evaluate cancer vaccines [ 122 , 126 , 127 ] . in our opinion , monitoring of multimer - positive cd8 + ( effector or effector memory ) t cells from the dth sites or pbmcs with ifn- production by flow cytometry may be sensitive markers particularly associated with overall survival . in addition , the dc / tumor fusion vaccine studies should be designed to determine whether t cell dysfunction in the tumor microenvironment correlated with clinical outcome . this informations may help us more fully understand the mechanisms of cancer vaccines and its potency to hasten the progress of efficient cancer vaccine strategies into the clinic .

although dendritic cell ( dc)- based cancer vaccines induce effective antitumor activities in murine models , only limited therapeutic results have been obtained in clinical trials . as cancer vaccines induce antitumor activities by eliciting or modifying immune responses in patients with cancer , the response evaluation criteria in solid tumors ( recist ) and who criteria , designed to detect early effects of cytotoxic chemotherapy in solid tumors , may not provide a complete assessment of cancer vaccines . the problem may , in part , be resolved by carrying out immunologic cellular monitoring , which is one prerequisite for rational development of cancer vaccines . in this review , we will discuss immunologic monitoring of cellular responses for the evaluation of cancer vaccines including fusions of dc and whole tumor cell .

1. Introduction 2. DC-Based Cancer Vaccines 3. DC/Tumor Fusion Vaccines 4. Monitoring of DC/Tumor Fusion Cell Preparations 5. In Vivo Monitoring 6. T-Cell Monitoring In Vitro 7. Conclusion

the mechanism of action for most cancer vaccines is mainly mediated through cytotoxic t lymphocytes ( ctls ) . the antigen - presenting cells ( apcs ) most suitable for cancer vaccines are dendritic cells ( dcs ) , which can be distinguished from b cells and macrophages by their abundant expression of costimulatory molecules and abilities to initiate a strong primary immune response [ 1 , 2 ] . dcs are specialized to capture and process tumor - associated antigens ( taas ) , converting the proteins to peptides that are presented on major histocompatibility complex ( mhc ) class i and class ii molecules . during this process , mature dcs migrate to t - cell areas of secondary lymphoid organs , where they present antigenic peptides to cd8 + and cd4 + t cells through mhc class i and class ii pathways , respectively , and become competent to present antigens to t cells , thus initiating antigen - specific ctl responses . antigen - specific ctls in turn can attack tumor cells that express cognate antigenic determinants or can provide help for b - cell responses that produce antibodies , which can also lead to tumor cell death in some cases . thus , the mechanism of action for cancer vaccines , based on harnessing host immune cells to infiltrate tumors and to exert ctl responses , is quite different from that of a traditional cytotoxic chemotherapy . a major area of investigation in induction of antitumor immunity involves the design of dc - based cancer vaccines . dcs derive their potency from constitutive and inducible expression of essential costimulatory molecules including b7 , icam-1 , lfa-1 , lfa-3 , and cd40 on the cell surface [ 1 , 8 , 9 ] . therefore , many strategies have been developed to load taas onto dcs and used as cancer vaccines . for example , dcs are pulsed with synthetic peptides derived from the known antigens , tumor lysates , tumor rna [ 12 , 13 ] , and dying tumor cells to induce antigen - specific antitumor immunity . although the production of dc - based cancer vaccines for individual patients with cancer has currently been addressed in clinical trials , a major drawback of these strategies comes from the limited number of known antigenic peptides available in many hla contexts . moreover , the results of clinical trials using dcs pulsed with antigen - specific peptides show that clinical responses have been found in a small number of patients [ 15 , 16 ] . to overcome this limitation , we have proposed the fusions of dcs and whole tumor cell ( dc / tumor ) to generate cell hybrids with the characteristics of apcs able to process endogenously provided whole taas . the whole tumor cells may be postulated to serve as the best source of antigens [ 1721 ] . the fusion of dc and tumor cell through chemical , physical , or biological means creates a heterokaryon which combines dc - derived costimulatory molecules , efficient antigen - processing and -presentation machinery , and an abundance of tumor - derived antigens including those yet to be unidentified ( figure 1 ) . thus , the dc / tumor fusion cells combine the essential elements for presenting tumor antigens to host immune cells and for inducing effective antitumor responses . thus , it is conceivable that tumor antigens synthesized de novo in the heterokaryon are processed and presented through the endogenous pathway . the advantage of dc / tumor fusion vaccines over pulsing dc with whole tumor lysates is that endogenously synthesized antigens have better access to mhc class i pathway [ 1821 ] . indeed , it has been demonstrated that dc / tumor fusion vaccines are superior to those involving other methods of dc loaded with antigenic proteins , peptides , tumor cell lysates , or irradiated tumor cells in murine models [ 1821 ] . the efficacy of antitumor immunity induced by dc / tumor fusion vaccines has been demonstrated in murine models using melanoma [ 2432 ] , colorectal [ 17 , 30 , 31 , 3341 ] , breast [ 4247 ] , esophageal , pancreatic [ 49 , 50 ] , hepatocellular [ 5155 ] , lung [ 22 , 41 , 5659 ] , renal cell carcinoma , sarcoma [ 6166 ] , myeloma [ 6774 ] , mastocytoma , lymphoma , and neuroblastoma . the fusion cells generated with human dc and tumor cell also have the ability to present multiple tumor antigens , thus increasing the frequency of responding t cells and maximizing antitumor immunity capable of killing tumor targets such as colon [ 7884 ] , gastric [ 85 , 86 ] , pancreatic , breast [ 47 , 8893 ] , laryngeal , ovarian [ 9597 ] , lung [ 85 , 98 ] , prostate [ 99 , 100 ] , renal cell [ 101 , 102 ] , hepatocellular [ 103105 ] carcinoma , leukemia [ 106111 ] , myeloma [ 112 , 113 ] , sarcoma [ 114 , 115 ] , melanoma [ 116119 ] , glioma , and plasmacytoma . despite the strong preclinical evidences supporting the use of dc / tumor fusions for cancer vaccination , the results of clinical trials so far reported are conflicting [ 1821 ] . one of the reasons is the evidence for fusion cell formation used as clinical trials is not definitive . the levels of fusion efficiency , which can be quantified by determining the percentage of cells that coexpress tumor and dc antigens , are closely correlated with ctl induction in vitro [ 82 , 83 ] . although tumor - derived tgf- reduces the efficacy of dc / tumor fusion vaccines via an in vivo mechanism , the reduction of tgf- derived from the fusions inhibits the generation of tregs and enhances antitumor immunity . moreover , the therapeutic effects in patients vaccinated by dc / tumor fusions are correlated with the characteristics of the dcs used as the fusion vaccines [ 82 , 83 ] . indeed , patient - derived fusions show inferior levels of mhc class ii and costimulatory molecules and produce decreased levels of il-12 and increased levels of il-10 , as compared with those obtained from fusions of tumor cell and dc from healthy donors [ 87 , 103 ] . therefore , it is important to assess the phenotype and function of dc / tumor fusion cell preparations used in each vaccination . the delayed - type hypersensitivity ( dth ) is an inflammatory reaction mainly mediated by cd4 + effector memory t cells that infiltrate the site of injection of an antigen against which the immune system has been primed by cancer vaccines . actually , soluble proteins , peptides , or antigens pulsed dcs have been injected intradermally , and the diameter of erythema or induration after 4872 h is measured . cd4 + effector memory t cells that recognize the antigens presented on local apcs mediate the immune responses by releasing cytokines , resulting in an increased vascular permeability and the recruitment of monocytes and other inflammatory cells in the site . indeed , the increased dth reactivity against tumor antigens has been observed in clinical responders by dc / tumor fusion vaccines . in almost patients with cancer , t cells from lymph nodes and . therefore , functional assessment of antigen - specific t cells from such dth sites may serve as an additional strategy to evaluate antigen - specific antitumor immune responses [ 122 , 126 , 127 ] . the mechanism of cancer vaccines , based on inducing ctls , infiltrating tumors , and exerting t - cell - mediated cytotoxic effects , is quite different from that of cytotoxic chemotherapy . as cancer vaccines do not work as quickly as chemotherapy which has a direct cytotoxic effect , the response evaluation criteria in solid tumors ( recist ) and who criteria [ 128 , 129 ] , designed to detect early effects of cytotoxic chemotherapy , can not appropriately evaluate the response patterns observed with cancer vaccines . they presume that linear measures are an adequate substitute for 2-dimentional methods and register four response categories : cr ( complete response ) , pr ( partial response ) , sd ( stable disease ) , and pd ( progressive disease ) . however , in the solid tumors , there exist not only antigen - specific ctls but also immune suppressive cells such as myeloid - derived suppressor cells ( mdscs ) , immunosuppressive tumor - associated macrophages ( tams ) , and cancer associated fibroblasts ( cafs ) ( figure 2 ) . after vaccination , the solid tumors may become heavily infiltrated by immune - related cells resulting in an apparent increase in size of lesions , which is , at least in part , due to the infiltration of ctls induced by cancer vaccines . therefore , the development of new response criteria , including immunologic cellular monitoring , is of great importance in the development of cancer vaccines . in clinical trials , . the currently used methods of assessing t - cells from patients treated with cancer vaccines are t - cell proliferation , cytokine profile , cytotoxic t lymphocyte assays ( ctl assays ) , ctl - associated molecules ( cd107 , perforin , granzyme b , and cd154 ) , multimer analysis , t - cell receptor ( tcr ) gene usage , and immune suppression assays ( table 1 ) . while these assays can be also used for monitoring cellular immune responses induced by dc / tumor fusion vaccines , none has been standardized . as dc / tumor fusion vaccines can induce defined and undefined antigen - specific antitumor activities , immunologic cellular monitoring for the fusion vaccines is much more complex . furthermore , as immune responses induced by dc / tumor fusion vaccines are a balanced mosaic of both immune stimulatory and suppressive responses , multiple monitoring assays for the clinical efficacy parameters may be needed to evaluate the antitumor immune responses . t - cell proliferation assay assesses the number and function at the level of the entire t - cell population in the culture . therefore , the ability to detect t - cell responses is based on the proliferative potential of the cells in response to antigens . the most commonly used in vitro method for measuring antigen - specific t - cell proliferation is the assessment of t - cell clonal expansion following incubation of t - cells with antigens in the presence of a radio - labeled nucleotide ( e.g. cfse ( 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester ) staining can be also used to directly detect proliferative responses of t - cells . the cfse - based assays are equivalent to traditional measures of antigen - specific t - cell responsiveness and have significant advantages for the ability to gate on a specific population of t - cells and the concomitant measurement of t - cell phenotype . therefore , measuring antigen - specific cd8 + and cd4 + t - cell proliferation is essential to evaluate the induction of vaccine - specific immune responses . although t - cell proliferation assay is usefulness to detect immune responses in vitro , the results are strongly influenced by the in vitro stimulation procedures . in addition , the frozen peripheral blood mononuclear cells ( pbmcs ) obtained before and after vaccination must be processed in the same set of experiments [ 103 , 135 , 136 ] . as t - cell proliferation assay is biologically irrelevant and imprecise for the reasons stated above , this assay may not be emphasized in future studies . there is a currently great interest in the assay of polyfunctional t cells , secreting multiple cytokines ( e.g. as the release of th1 cytokines such as ifn- and tnf- is important to determine long - lasting antitumor immunity , a shift to th1 response by cancer vaccines is essential for therapeutic potential in murine models [ 36 , 37 , 67 , 77 , 137 , 138 ] therefore , it is important to test whether cancer vaccines can induce a th1 response in the tumor - specific t cells , and what impact might this have on the clinical responses . as the ifn- elispot assay shows highly reproducible results among different laboratories , the elispot may be an ideal candidate for robust monitoring of t - cell activity [ 1821 , 142 ] . coculture of cd4 + and cd8 + t cells from healthy donors with dc / tumor fusions results in high levels of ifn- production and low levels of il-10 production [ 50 , 54 , 80 , 143 ] . therefore , to assess dc / tumor fusion vaccines precisely , t cells obtained before and after vaccination might be directly quantified with stimulation of autologous tumor lysates in vitro . in effective clinical responders , comparable levels of ifn- production in response to tumor lysates may be detected in pbmcs obtained before vaccination . a correlation between ifn- elispot outcome and effective clinical responses ( period free of relapse or survival ) has been found in patients treated with cancer vaccines including dc / tumor fusions [ 103 , 135 , 136 , 144 ] . for immune monitoring of cancer vaccines , t - cell - mediated ctl assays are appealing because measurement of the ability of ctl to kill tumor targets is thought to be a relevant marker for antitumor activity . it has been assumed that the cytotoxicity has been measured in cr - release assays in vitro . instead of cr release assays , flow cytometry - based methods have been developed to assess ctl activity [ 145 , 146 ] . flow cytometry ctl assays can be predicated on measurement of ctl - induced caspase-3 or annexin - v activation in target cells through fluorescence detection , which are more sensitive to conventional cr release assays [ 145147 ] . however , it is unusual to detect antigen - specific killing by t cells directly isolated from the patients vaccinated with dc / tumor fusions even with the use of flow cytometry - based ctl assays [ 103 , 148 ] . these stimulations may distort the phenotype and function of the t - cell populations from tumor state . importantly , a majority of the antigen - specific cd8 + ctls in peripheral blood may not be tumor reactive due to various mechanisms such as downmodulation of mhc class i molecules on tumors and presence of tregs at the tumor site . indeed , cytotoxic activity against autologous targets has been observed in peripheral blood t cells from patients vaccinated with dc / tumor fusions by ctl assays [ 103 , 148 ] , but the clinical responses from early clinical trails in patients with melanoma , glioma , gastric , breast , and renal cancer are muted [ 103 , 130 , 134 , 135 , 142 , 143 , 148154 ] . therefore , localization of antigen - specific ctls demonstrated by analysis of biopsy samples from tumor sites may be directly associated with clinical responses . , perforin and granzyme b ) . these perforin / granzyme b - dependent lytic fashions require degranulation of cd107a and b in cd8 + ctls . the coupling of cd154 expression with multiplexed measurements of ifn- production provides a greater level of detail for the study of tumor - specific cd4 + t - cell responses . although dc / tumor fusion vaccines have abilities to induce cd107 + ifn-+ cd8 + t cells and cd154 + ifn-+ cd4 + t cells upon autologous tumor encounter in vitro [ 83 , 87 ] , it has now been unclear the correlation of the assay with clinical outcome . although dc / tumor fusion vaccines can induce defined and undefined antigens - specific cd8 + and cd4 + t cells , the multimer analysis can only be used to detect immune responses against defined antigenic epitopes expressing in tumor cells . the specific role of the multimer - positive t cells for cancer vaccine efficacy has not yet been well established in the setting of clinical trials . recent studies suggest that effective cancer vaccines not only stimulate ctl activity , but also sustain long - term memory t cells capable of mounting strong proliferative and functional responses to secondary tumor antigen challenge . moreover , the combined use of multimers and functional assays such as ifn- analysis may have provided some insight into the functional activity of these cells . interestingly , after vaccination , the resected melanoma patients can mount a significant antigen - specific cd8 + t cell immune response with a production of ifn- and high proliferation potential . only t cells having a tcr specific for a given antigen are triggered by interaction with cancer vaccines . this activation results in the clonal expansion of antigen - specific t cells that can be followed by tcr v gene usage . recently , the availability of a large panel of monoclonal antibodies against tcrs , mainly v epitopes , allows one to study the tcr repertoire by flow cytometry . pcr techniques can also be used to detect a restricted tcr repertoire from small amounts of t cells without biases caused by ex vivo expansions . although dc / tumor fusion vaccines have resulted in selection and expansion of t - cell clones , the generation of antitumor immunity by ctls has not correlated with clinical responses . immunogenic tolerance to a particular set of antigens is the absence of an immune response to those antigens , which can be achieved by processes that result in t - cell anergy ( antigen - specific unresponsiveness ) , t - cell unresponsiveness ( generalized dysfunction ) , and t - cell deletion ( apoptosis ) . future fusion vaccine studies should be designed to determine whether t - cell dysfunction correlated with clinical outcome . it has been documented that immune suppressive cells can counteract antitumor immune responses . the mechanisms that suppress the immune system provide a fundamental reason why cancer vaccines fail to induce consistently robust antitumor immune responses . in dc / tumor fusion vaccines , cd4 + cd25high+ foxp3 + tregs were promoted in the presence of the local tumor - related factors in vitro . therefore , monitoring of immune suppressive cells in cancer patients vaccinated with dc / tumor fusions is also essential . the development of assays for detecting immune responses associated with clinical outcome has been limited . a variety of assays had been introduced to provide monitoring tools necessary for following changes in the frequency of antigen - specific ctls and to assess the impact of cancer vaccines on the immune system . as the mechanisms of immune response that cause tumor regression are not simple , the currently available assays may not actually measure a function with direct relevance to how tumors are actually attacked immunologically in cancer patients . a high reproducibility of results among different laboratories leads to the conclusion that cytokine flow cytometry or elispot may be an ideal candidate for robust and reproducible monitoring of t - cell activity in vivo . however , the widely used elispot assay often does not correlate the best with clinical outcome . furthermore , it is necessary to understand the immune responses seen in peripheral blood versus the responses at the tumor site . monitoring of antigen - specific ctls at the tumor site may be directly associated with clinical responses . therefore , the ability to assess the function of antigen - specific t cells from dth site may serve as an additional strategy to evaluate cancer vaccines [ 122 , 126 , 127 ] . in our opinion , monitoring of multimer - positive cd8 + ( effector or effector memory ) t cells from the dth sites or pbmcs with ifn- production by flow cytometry may be sensitive markers particularly associated with overall survival . in addition , the dc / tumor fusion vaccine studies should be designed to determine whether t cell dysfunction in the tumor microenvironment correlated with clinical outcome . this informations may help us more fully understand the mechanisms of cancer vaccines and its potency to hasten the progress of efficient cancer vaccine strategies into the clinic .

gap formation at cavity wall - restorative material interface is always of great importance , because of its significance to microleakage , postoperative sensitivity and secondary caries formation . bond strength and polymerization shrinkage of restorative materials are directly related to their adaptation to cavity walls through their flow , modulus of elasticity and wettability . cavity factors such as number of walls , access to the walls and wall quality , along with operator factors such as material type , material handling and method of insertion are also very important to wall adaptation . if these factors are not properly handled , a gap of variable size will be formed between cavity walls and filling material , during or following the hardening of the material . light , scanning electron , laser and confocal microscopy , dyes , tracers , air - pressure , electrical conductance and optical coherence tomography have all been used in the investigation of this gap . most of the above methods measure representative two - dimensional sections of cavities , interpolating the results for three - dimensional estimations . although , efforts in measuring the three - dimensional space in small cavities ( 2 mm in diameter ) were made , in minimally invasive interventions for caries , the question of how well a material can be adapted to the walls of cavities with smaller diameters ( 1.0 - 1.5 mm ) , still remains . opdam et al . found that voids and wall gaps were greater for thicker in consistency materials than medium or thin materials . in another study , olmez et al . found that internal voids correlated with marginal microleakage in class ii composite restorations . since material voids are equally important to gap space , and both are depended on the manipulation of the material , it is important to see if voids are also affected by the size of the cavity , during clinicians effort to insert the material to the bottom of the cavity . in the study of lioumi , it was noted that the width of material - cavity interface was not uniformly distributed over the entire cavity walls but it was different in different parts of the cavity , due to certain parameters . the widest gap formed at the internal line angles of the cavity when margins were beveled , etched and bonded , while a significant amount of voids appeared within the material close to or in contact with the interface . investigating the effect of configuration factor on gap formation in composites found that high c - factor values generate large gap formation . x - ray computed micro - tomography ( micro xct ) is a method that uses x - rays to create two - dimensional density images of the specimen 's cross sections and then reconstruct the specimen in a three - dimensional model . for this reason , it is very useful in many areas in dentistry including the investigation of material adaptation to cavity wall and margins , with relative accuracy , visualizing the different in density entities of the specimens . until now , a number of studies have been published on the adaptation of several different composite materials on cavity walls . however , most of these studies were focused on polymerization shrinkage in cavities of normal dimensions and the attention was given to a rather laboratory technique in order to facilitate measurements . for these reasons , a method that could permit the investigation of gaps in a more clinically relevant setting of the entire cavity preparation , and in dimensions closer to a very conservative cavity would be of significant importance to the study of gap formation and location in such restorations . the purpose of this study was to validate a microtomographic procedure for the comparison of voids ( densities ) in different ultraconservative cavities and to test the hypotheses that : the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface , voids and gaps are not related to cavity dimension , andlocation of voids is not dependent to a specific restorative material . the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface , voids and gaps are not related to cavity dimension , and location of voids is not dependent to a specific restorative material . for the purpose of this study , 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials . the required sample size at a = 0.05 , a power of 0.85 and an effect size of 0.6 , was a priori computed using g power v.3.1.5 ( franz ful , universitat kiel , germany ) . visually intact teeth were carefully selected from recently extracted teeth , without the presence of hypomineralized or hypolplastic areas , caries or fracture lines on their buccal or lingual surfaces . teeth were kept continually in tap water until cavity preparations . in order to prepare cavities , as much as possible on the same horizontal tooth plane , the roots of all teeth , 2 - 3 mm below their cemento - enamel junction , were cut and removed . two buccal ans two linguall cavities of 2.5 mm in depth were prepared in each tooth under air - water spray , with a diamond bur rotating in a high - speed hand piece . two of the cavities were opened with a 0.8 mm in diameter bur ( fgd0 , strauss and co ) and two with a 1.0 mm diameter ( fgd1 , strauss and co ) . cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [ figure 1 ] . no special attention was given for opening the cavities with an exact width or depth , in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids . only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces . after preparation of the cavities , all teeth were stored in water of 37c until their first microtomographic examination by skyscan 1072 microtomographer ( micro xct , model 1072 , skyscan aartselaar , belgium ) . the cavities were examined before insertion of the filling material , to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [ figure 2 ] . operating parameters used for skyscan 1072 microtomographer upper left : microtomographic section of a tooth showing the prepared cavities . lower : a screen image showing the binarization settings with tview software , before calculation starts . the restorative materials used to fill the experimental cavities are shown in table 2 , and the procedure followed for each material is described below . the assignment of restorative material and bur diameter on tooth surfaces was based on a latin square design , in order to have teeth , with all the designed treatment variables , in a systematic rather than a random fashion . all cavities were acid etched with 37% ortho - phosphoric acid for 30 s , rinsed with water for 2 - 3 s and dried with air for 4 - 5 s and 1 - 2 absorbent paper points . cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions ( prime and bond nt , dentsply int inc , milford , usa ) and polymerized for 20 s , using a curing unit with a light intensity of 550 - 600 mw / cm ( demetron lc / kerr gmbh , rastatt , germany ) . it must be mentioned that adhesive material was used with caution since excess of adhesive remain undetected by x - rays and even a small excess could be measured as gap . materials used in the study aeliteflo / bisco inc , chicago , usa ( af ) was placed in the cavity with a small diameter tip on material syringes . the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity . the material was polymerized for 30 s , and the excess , was finished and polished using medium , fine and superfine sof - lex discs ( 3 m espe , dental products , st . permaflo / ultradent products inc , stlouis / usa(pf ) was placed in the cavity exactly as af . aelite aesthetic / bisco inc , chicago , usa ( aa ) was placed in the cavity in small parts and condensed with the use of a dentin excavator , modified to a small diameter condenser ( 0.8 mm ) , in order to enter the cavities . after condensation and polymerization , the same finishing and polishing procedure as in af was followed . fuji ix / gc corporation , tokyo , japan ( fu ) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions , by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser , as before . immediately after finishing and polishing of restorations , teeth were placed in distilled water of 37c and retrieved after 24 h to receive 500 thermal cycles ( 5 - 55c , 3 min each ) . the second x - ray microtomographic examination was made under the same operating parameters with the first examination , immediately after thermocycling procedure . the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections , using the tview v.1.3 software ( skyscan n.v . the software calculates on binary images of sample 's tomographic sections , the different in density areas , within a predefined region of interest ( roi ) . running all relative sections , the total volume ( vt ) and the volume fraction percent ( vf % ) of the interested densities ( called active pixels ) in the roi can be calculated . a specific process had to be followed , to ensure that all calculations were based exactly on the same roi . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the sections showing all four cavities at their largest width were located and copied on a photoshop cs file . a circular area over the cavity was drawn on a transparent layer with the marquee tool . then , the circular marquee line was copied in new transparent layers , moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single .bmp file . this process was repeated for all tooth cavities [ figure 2 ] and the saved .bmp files were placed as the last tomographic image , in order to remain available , but outside the range of the selected sections . the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas . the upper and lower sections of each cavity were inserted , and the color palette was set to binary colors ( 247 in level-1 and 255 in level-2 ) , for best discrimination of empty spaces from tooth / material substance [ figure 2 ] . the three - dimensional analysis was used , in order to collect information from the set of predefined horizontal sections on the variables described below . this three - dimensional analysis was repeated three times for each cavity and for all teeth . data collected concerned the following morphometric values : vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . the location of voids and gaps was evaluated using the tview software and its no1 color palette . all relative to each cavity .bmp images ( 1024 1024 pixels ) were viewed by two examiners for the location of voids if present , on a 19 computer screen . for their classification three locations were recorded : the material itself , the side walls of the cavity andthe bottom of the cavity . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials , in respect to the morphometric parameters . mann - whitney test was also used to search for possible differences between low and high viscosity materials , for each morphometric parameter . the degree of correlation of cavity dimensions with all morphometric parameters was analyzed by spearman 's test . all statistical tests performed with medcalc v.10.0 ( medcalc software , mariakerke , belgium ) at a 0.05 level of significance . for the purpose of this study , 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials . the required sample size at a = 0.05 , a power of 0.85 and an effect size of 0.6 , was a priori computed using g power v.3.1.5 ( franz ful , universitat kiel , germany ) . visually intact teeth were carefully selected from recently extracted teeth , without the presence of hypomineralized or hypolplastic areas , caries or fracture lines on their buccal or lingual surfaces . in order to prepare cavities , as much as possible on the same horizontal tooth plane , the roots of all teeth , 2 - 3 mm below their cemento - enamel junction , were cut and removed . two buccal ans two linguall cavities of 2.5 mm in depth were prepared in each tooth under air - water spray , with a diamond bur rotating in a high - speed hand piece . two of the cavities were opened with a 0.8 mm in diameter bur ( fgd0 , strauss and co ) and two with a 1.0 mm diameter ( fgd1 , strauss and co ) . cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [ figure 1 ] . no special attention was given for opening the cavities with an exact width or depth , in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids . only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces . after preparation of the cavities , all teeth were stored in water of 37c until their first microtomographic examination by skyscan 1072 microtomographer ( micro xct , model 1072 , skyscan aartselaar , belgium ) . the cavities were examined before insertion of the filling material , to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [ figure 2 ] . operating parameters used for skyscan 1072 microtomographer upper left : microtomographic section of a tooth showing the prepared cavities . lower : a screen image showing the binarization settings with tview software , before calculation starts . the restorative materials used to fill the experimental cavities are shown in table 2 , and the procedure followed for each material is described below . the assignment of restorative material and bur diameter on tooth surfaces was based on a latin square design , in order to have teeth , with all the designed treatment variables , in a systematic rather than a random fashion . all cavities were acid etched with 37% ortho - phosphoric acid for 30 s , rinsed with water for 2 - 3 s and dried with air for 4 - 5 s and 1 - 2 absorbent paper points . cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions ( prime and bond nt , dentsply int inc , milford , usa ) and polymerized for 20 s , using a curing unit with a light intensity of 550 - 600 mw / cm ( demetron lc / kerr gmbh , rastatt , germany ) . it must be mentioned that adhesive material was used with caution since excess of adhesive remain undetected by x - rays and even a small excess could be measured as gap . materials used in the study aeliteflo / bisco inc , chicago , usa ( af ) was placed in the cavity with a small diameter tip on material syringes . the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity . the material was polymerized for 30 s , and the excess , was finished and polished using medium , fine and superfine sof - lex discs ( 3 m espe , dental products , st . permaflo / ultradent products inc , stlouis / usa(pf ) was placed in the cavity exactly as af . aelite aesthetic / bisco inc , chicago , usa ( aa ) was placed in the cavity in small parts and condensed with the use of a dentin excavator , modified to a small diameter condenser ( 0.8 mm ) , in order to enter the cavities . after condensation and polymerization , the same finishing and polishing procedure as in af was followed . fuji ix / gc corporation , tokyo , japan ( fu ) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions , by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser , as before . immediately after finishing and polishing of restorations , teeth were placed in distilled water of 37c and retrieved after 24 h to receive 500 thermal cycles ( 5 - 55c , 3 min each ) . the second x - ray microtomographic examination was made under the same operating parameters with the first examination , immediately after thermocycling procedure . the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections , using the tview v.1.3 software ( skyscan n.v . the software calculates on binary images of sample 's tomographic sections , the different in density areas , within a predefined region of interest ( roi ) . running all relative sections , the total volume ( vt ) and the volume fraction percent ( vf % ) of the interested densities ( called active pixels ) in the roi can be calculated . a specific process had to be followed , to ensure that all calculations were based exactly on the same roi . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the sections showing all four cavities at their largest width were located and copied on a photoshop cs file . a circular area over the cavity was drawn on a transparent layer with the marquee tool . then , the circular marquee line was copied in new transparent layers , moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single .bmp file . this process was repeated for all tooth cavities [ figure 2 ] and the saved .bmp files were placed as the last tomographic image , in order to remain available , but outside the range of the selected sections . the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas . the upper and lower sections of each cavity were inserted , and the color palette was set to binary colors ( 247 in level-1 and 255 in level-2 ) , for best discrimination of empty spaces from tooth / material substance [ figure 2 ] . the three - dimensional analysis was used , in order to collect information from the set of predefined horizontal sections on the variables described below . this three - dimensional analysis was repeated three times for each cavity and for all teeth . data collected concerned the following morphometric values : vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the sections showing all four cavities at their largest width were located and copied on a photoshop cs file . a circular area over the cavity was drawn on a transparent layer with the marquee tool . then , the circular marquee line was copied in new transparent layers , moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single .bmp file . this process was repeated for all tooth cavities [ figure 2 ] and the saved .bmp files were placed as the last tomographic image , in order to remain available , but outside the range of the selected sections . the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas . the upper and lower sections of each cavity were inserted , and the color palette was set to binary colors ( 247 in level-1 and 255 in level-2 ) , for best discrimination of empty spaces from tooth / material substance [ figure 2 ] . the three - dimensional analysis was used , in order to collect information from the set of predefined horizontal sections on the variables described below . this three - dimensional analysis was repeated three times for each cavity and for all teeth . data collected concerned the following morphometric values : vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . the location of voids and gaps was evaluated using the tview software and its no1 color palette . all relative to each cavity .bmp images ( 1024 1024 pixels ) were viewed by two examiners for the location of voids if present , on a 19 computer screen . for their classification three locations were recorded : the material itself , the side walls of the cavity andthe bottom of the cavity . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials , in respect to the morphometric parameters . mann - whitney test was also used to search for possible differences between low and high viscosity materials , for each morphometric parameter . the degree of correlation of cavity dimensions with all morphometric parameters was analyzed by spearman 's test . all statistical tests performed with medcalc v.10.0 ( medcalc software , mariakerke , belgium ) at a 0.05 level of significance . unfilled cavity dimensions were measured and found to have a mean length of 2.6 0.4 mm and a mean width 1.30 0.19 mm . the mean values of all morphometric elements ( vt , vf and st ) , estimated by three - dimensional analysis for the cavities filled with four restorative materials are shown in table 3 . morphometric parameters of voids and gaps upper left : a tooth section showing two of the filled cavities with voids within fuji ix ( fu ) material . lower right : voids at the bottom of an fu and in the middle of an aa restoration . statistical analysis by kruskal - wallis nonparametric analysis of variance indicated significant differences among filled cavity groups , for all three parameters ( kwvt = 38.0 , kwvf = 38.29 , kwst = 29.28 , p < 0.0001 ) . post - hoc analysis based on pairwise comparisons showed significantly higher values of vf % in cavities filled with fu than those filled with other materials ( p < 0.05 ) [ table 3 ] . restorations with af and pf materials showed the lowest vf % values of all ( p < 0.05 ) . mann - whitney test also indicated a significant difference between cavities filled with high and low viscosity materials ( u = 565.5 , p < 0.0001 ) . cavity length , width and volume of high and low viscosity materials as well as in most individual materials correlated poorly and not significantly with vf % . however , in cavities filled with aa , a significant correlation was found between vf % and depth of the cavity . spearman 's rho coefficients of vf % with cavity dimensions the proportion of cavities with voids at different locations ( within the material , bottom and side walls of the cavity ) is shown in table 5 . in 50 - 75% of the restorations , voids were present within the material , without difference in their proportion among the different materials . in 90 - 100% of the cavities filled with aa and fu , gaps were present at the bottom of the cavity , and significantly higher than those filled with af or pf . all cavities filled with aa showed also gaps at the side walls of the cavity , significantly higher than all other materials . lower and upper limits of the 95% ci for the proportion of cavities with voids within the material , and gaps at the bottom or side walls of the cavity the results showed that the procedure followed to compare voids fraction among different cavities on the same sample , is capable of revealing significant differences . the study rejected all three hypotheses : for no differences between materials in vf % of voids ( and gaps),for no association of cavity dimensions with vf % of voids ( and gaps ) , andfor no association of voids location with a specific restorative material . for no differences between materials in vf % of voids ( and gaps ) , for no association of cavity dimensions with vf % of voids ( and gaps ) , and for no association of voids location with a specific restorative material . the procedure introduced in this study for the selection of exactly the same roi on all cavities was not a simple one , but since differences between repeated measurements ranged only from 0.002 to 0.005 vf % , this indicates that the method was able to reveal small differences in the volume of voids in very conservative cavities . however , the need to add extra digital sections to a number of cavities smaller in depth than others , or the inclusion in the experiment of materials with high variability in the volume of voids , like fu material , may introduce an error to the measurements . in our study , the low viscous materials were the best among different materials in filling the ultraconservative cavities without gap and this is in agreement with the results of other studies for normal cavities . found that thinner composites had fewer problems with voids and wall adaptation than thicker materials . peutzfeldt and asmussen , who also studied gap formation , found a positive correlation of high viscosity and polymerization shrinkage with gap formation . moreira da silva et al . found a correlation of gap and voids formation with high viscous flow and low flexural modulus of composite materials . these results can be explained by the high thixotropic effect of low viscous composite materials , and their low polymerization shrinkage due to their low modulus of elasticity . opdam et al . showed that injection technique , like the one we used with low viscous composites , leads to reduced voids formation within the material and a smaller interfacial gaps . hence , in cavities of very small diameter ( 1.0 - 1.5 mm ) where the insertion of the restorative material and its adaptation to the cavity walls is difficult , low viscous material should be syringed deep into the cavity instead of using higher viscosity composites . thicker composite materials even if they are managed to be inserted in such cavities can potentially form internal voids between layers , and for this reason their use should be done with caution . glass - ionomer material was inserted with difficulty in small cavities , due to its adherence to the instruments and the cavity walls during insertion . as a result of this this type of material should not be used in such small cavities unless perhaps a special small in diameter tip designed to allow proper insertion of the material deep into the cavity before its hardening . our study also showed a significant correlation of initial cavity volume with vf of spaces around and within aa filling material only . this positive correlation of deeper or wider cavities with the presence of voids is probably a result of higher polymerization contraction in larger restorations , as he et al . their study showed that in larger cavities with a high c - factor ( class i and v ) , the bonding was riskier than in small cavities . therefore , larger cavities filled with high viscous composite material may present a higher amount of voids and gaps . our study showed that the voids within the restorative material were not associated with a specific type of material . however , gaps at the bottom of the cavity were more frequently associated with the more viscous aa and fu materials , as other studies indicated . they presented a higher polymerization contraction than af and pf materials , and need a higher force and more attention to make a closer contact with the bottom of the cavity . the reason is probably that polymerization contraction , which is evident with all cavity walls in a nonbonded restoration , creates a space at the sides of the cavity , more evidently than in other materials . fu , for instance , has the ability of creating a bond with the side walls of the cavity , but also seems to create larger gaps at the bottom of the cavity , which helps to accommodate a significant part of its contraction during hardening . it must be noticed that pixel size in microtomographic images was 14 in this study , although system 's detection limit was 1.8 . for this reason , smaller in size voids could not be recorded or located and voids of such dimensions may exist in all types of materials and locations . this is probably the limitation of the suggested method and the reason why low viscous materials show a low proportion of voids or gaps . however , newer ct technology ( x - ray computed nanotomography ) will be able to estimate gaps and voids more accurately . voids are present in the same frequency within the mass of all restorative materials , but gaps are present more frequently in restorative materials with high viscosity , at the bottom and the side cavity walls . for these reasons , more research is needed to refine the available techniques for placing correctly and precisely a restorative material within a very conservative cavity , the way of using adhesives in cavities of a very small opening , the type of material that can actually minimize the hardening shrinkage of the material and of course the measurement of voids of very small dimensions . there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities.low viscous composite resins were the best in filling cavities without voids or gaps.high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps.cavity depth , width and volume do correlate with the amount of voids and gap spaces , but only for the high viscous composite material.voids are located in the same frequency within all materials , but gaps are more frequently located within high viscous composites , both at the bottom and the side cavity walls . there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities . high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps . cavity depth , width and volume do correlate with the amount of voids and gap spaces , but only for the high viscous composite material . voids are located in the same frequency within all materials , but gaps are more frequently located within high viscous composites , both at the bottom and the side cavity walls . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .

background : volume fraction ( vf ) and location of internal voids and gaps in relation to material type and cavity dimensions in ultraconservative restorations were investigated in this study.materials and methods : forty - eight round cavities of 1.3 mm mean diameter and 2.6 mm mean depth were made on buccal and lingual surfaces of recently extracted human teeth . these were filled and thermocycled with two low viscosity composites ( aeliteflo lv [ af ] , permaflo [ pf ] ) , one high viscosity composite ( aelite aesthetic enamel [ aa ] ) and one glass - ionomer ( gcfuji ix gp ) . x - ray microtomography , following a specific procedure , was applied to all cavities before and after their restoration , using skyscan-1072 microtomographer . vf percent ( vf% ) and location of voids and gaps were recorded and analysed statistically at a = 0.05 . kruskal - wallis nonparametric analysis of variance , post - hoc analysis , mann - whitney test , spearman 's correlation analysis were used to analyze data.results:cavities filled with af and pf showed significantly lower vf % of voids and gaps than all other restorations ( p < 0.05 ) . only for the cavities filled with aa , cavity width and depth was significantly correlated with vf % ( p < 0.05 ) . 50 - 75% of the filled cavities contained internal voids regardless of the restorative material ( p > 0.05 ) . the proportion of cavities with gaps at the bottom and side walls was lower in those filled with af and pf ( p < 0.05).conclusion : cavities filled with low viscosity composites presented the lowest amount of internal voids and gaps . glass - ionomer and high viscosity composite restorative materials showed the highest amount of interfacial gaps . only in the high viscosity composite restorations the amount of voids and gaps correlated with the cavity depth , width and volume .

INTRODUCTION MATERIALS AND METHODS Sampling Cavity preparations First microtomographic examination Cavity filling Second microtomographic examination Selection of measuring areas Measuring process Voids and gaps location Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

although , efforts in measuring the three - dimensional space in small cavities ( 2 mm in diameter ) were made , in minimally invasive interventions for caries , the question of how well a material can be adapted to the walls of cavities with smaller diameters ( 1.0 - 1.5 mm ) , still remains . found that internal voids correlated with marginal microleakage in class ii composite restorations . since material voids are equally important to gap space , and both are depended on the manipulation of the material , it is important to see if voids are also affected by the size of the cavity , during clinicians effort to insert the material to the bottom of the cavity . in the study of lioumi , it was noted that the width of material - cavity interface was not uniformly distributed over the entire cavity walls but it was different in different parts of the cavity , due to certain parameters . the widest gap formed at the internal line angles of the cavity when margins were beveled , etched and bonded , while a significant amount of voids appeared within the material close to or in contact with the interface . x - ray computed micro - tomography ( micro xct ) is a method that uses x - rays to create two - dimensional density images of the specimen 's cross sections and then reconstruct the specimen in a three - dimensional model . for these reasons , a method that could permit the investigation of gaps in a more clinically relevant setting of the entire cavity preparation , and in dimensions closer to a very conservative cavity would be of significant importance to the study of gap formation and location in such restorations . the purpose of this study was to validate a microtomographic procedure for the comparison of voids ( densities ) in different ultraconservative cavities and to test the hypotheses that : the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface , voids and gaps are not related to cavity dimension , andlocation of voids is not dependent to a specific restorative material . the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface , voids and gaps are not related to cavity dimension , and location of voids is not dependent to a specific restorative material . for the purpose of this study , 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials . the required sample size at a = 0.05 , a power of 0.85 and an effect size of 0.6 , was a priori computed using g power v.3.1.5 ( franz ful , universitat kiel , germany ) . two of the cavities were opened with a 0.8 mm in diameter bur ( fgd0 , strauss and co ) and two with a 1.0 mm diameter ( fgd1 , strauss and co ) . cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [ figure 1 ] . no special attention was given for opening the cavities with an exact width or depth , in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids . only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces . the cavities were examined before insertion of the filling material , to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [ figure 2 ] . the restorative materials used to fill the experimental cavities are shown in table 2 , and the procedure followed for each material is described below . cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions ( prime and bond nt , dentsply int inc , milford , usa ) and polymerized for 20 s , using a curing unit with a light intensity of 550 - 600 mw / cm ( demetron lc / kerr gmbh , rastatt , germany ) . the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity . aelite aesthetic / bisco inc , chicago , usa ( aa ) was placed in the cavity in small parts and condensed with the use of a dentin excavator , modified to a small diameter condenser ( 0.8 mm ) , in order to enter the cavities . fuji ix / gc corporation , tokyo , japan ( fu ) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions , by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser , as before . the second x - ray microtomographic examination was made under the same operating parameters with the first examination , immediately after thermocycling procedure . the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections , using the tview v.1.3 software ( skyscan n.v . running all relative sections , the total volume ( vt ) and the volume fraction percent ( vf % ) of the interested densities ( called active pixels ) in the roi can be calculated . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . the location of voids and gaps was evaluated using the tview software and its no1 color palette . all relative to each cavity .bmp images ( 1024 1024 pixels ) were viewed by two examiners for the location of voids if present , on a 19 computer screen . for their classification three locations were recorded : the material itself , the side walls of the cavity andthe bottom of the cavity . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials , in respect to the morphometric parameters . mann - whitney test was also used to search for possible differences between low and high viscosity materials , for each morphometric parameter . for the purpose of this study , 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials . the required sample size at a = 0.05 , a power of 0.85 and an effect size of 0.6 , was a priori computed using g power v.3.1.5 ( franz ful , universitat kiel , germany ) . two of the cavities were opened with a 0.8 mm in diameter bur ( fgd0 , strauss and co ) and two with a 1.0 mm diameter ( fgd1 , strauss and co ) . cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [ figure 1 ] . no special attention was given for opening the cavities with an exact width or depth , in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids . only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces . the cavities were examined before insertion of the filling material , to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [ figure 2 ] . the restorative materials used to fill the experimental cavities are shown in table 2 , and the procedure followed for each material is described below . the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity . permaflo / ultradent products inc , stlouis / usa(pf ) was placed in the cavity exactly as af . aelite aesthetic / bisco inc , chicago , usa ( aa ) was placed in the cavity in small parts and condensed with the use of a dentin excavator , modified to a small diameter condenser ( 0.8 mm ) , in order to enter the cavities . fuji ix / gc corporation , tokyo , japan ( fu ) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions , by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser , as before . the second x - ray microtomographic examination was made under the same operating parameters with the first examination , immediately after thermocycling procedure . the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections , using the tview v.1.3 software ( skyscan n.v . running all relative sections , the total volume ( vt ) and the volume fraction percent ( vf % ) of the interested densities ( called active pixels ) in the roi can be calculated . a circular area over the cavity was drawn on a transparent layer with the marquee tool . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . the location of voids and gaps was evaluated using the tview software and its no1 color palette . all relative to each cavity .bmp images ( 1024 1024 pixels ) were viewed by two examiners for the location of voids if present , on a 19 computer screen . for their classification three locations were recorded : the material itself , the side walls of the cavity andthe bottom of the cavity . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials , in respect to the morphometric parameters . mann - whitney test was also used to search for possible differences between low and high viscosity materials , for each morphometric parameter . the mean values of all morphometric elements ( vt , vf and st ) , estimated by three - dimensional analysis for the cavities filled with four restorative materials are shown in table 3 . morphometric parameters of voids and gaps upper left : a tooth section showing two of the filled cavities with voids within fuji ix ( fu ) material . lower right : voids at the bottom of an fu and in the middle of an aa restoration . statistical analysis by kruskal - wallis nonparametric analysis of variance indicated significant differences among filled cavity groups , for all three parameters ( kwvt = 38.0 , kwvf = 38.29 , kwst = 29.28 , p < 0.0001 ) . post - hoc analysis based on pairwise comparisons showed significantly higher values of vf % in cavities filled with fu than those filled with other materials ( p < 0.05 ) [ table 3 ] . restorations with af and pf materials showed the lowest vf % values of all ( p < 0.05 ) . mann - whitney test also indicated a significant difference between cavities filled with high and low viscosity materials ( u = 565.5 , p < 0.0001 ) . cavity length , width and volume of high and low viscosity materials as well as in most individual materials correlated poorly and not significantly with vf % . however , in cavities filled with aa , a significant correlation was found between vf % and depth of the cavity . spearman 's rho coefficients of vf % with cavity dimensions the proportion of cavities with voids at different locations ( within the material , bottom and side walls of the cavity ) is shown in table 5 . in 50 - 75% of the restorations , voids were present within the material , without difference in their proportion among the different materials . in 90 - 100% of the cavities filled with aa and fu , gaps were present at the bottom of the cavity , and significantly higher than those filled with af or pf . all cavities filled with aa showed also gaps at the side walls of the cavity , significantly higher than all other materials . lower and upper limits of the 95% ci for the proportion of cavities with voids within the material , and gaps at the bottom or side walls of the cavity the results showed that the procedure followed to compare voids fraction among different cavities on the same sample , is capable of revealing significant differences . the study rejected all three hypotheses : for no differences between materials in vf % of voids ( and gaps),for no association of cavity dimensions with vf % of voids ( and gaps ) , andfor no association of voids location with a specific restorative material . for no differences between materials in vf % of voids ( and gaps ) , for no association of cavity dimensions with vf % of voids ( and gaps ) , and for no association of voids location with a specific restorative material . the procedure introduced in this study for the selection of exactly the same roi on all cavities was not a simple one , but since differences between repeated measurements ranged only from 0.002 to 0.005 vf % , this indicates that the method was able to reveal small differences in the volume of voids in very conservative cavities . however , the need to add extra digital sections to a number of cavities smaller in depth than others , or the inclusion in the experiment of materials with high variability in the volume of voids , like fu material , may introduce an error to the measurements . showed that injection technique , like the one we used with low viscous composites , leads to reduced voids formation within the material and a smaller interfacial gaps . hence , in cavities of very small diameter ( 1.0 - 1.5 mm ) where the insertion of the restorative material and its adaptation to the cavity walls is difficult , low viscous material should be syringed deep into the cavity instead of using higher viscosity composites . glass - ionomer material was inserted with difficulty in small cavities , due to its adherence to the instruments and the cavity walls during insertion . as a result of this this type of material should not be used in such small cavities unless perhaps a special small in diameter tip designed to allow proper insertion of the material deep into the cavity before its hardening . this positive correlation of deeper or wider cavities with the presence of voids is probably a result of higher polymerization contraction in larger restorations , as he et al . their study showed that in larger cavities with a high c - factor ( class i and v ) , the bonding was riskier than in small cavities . therefore , larger cavities filled with high viscous composite material may present a higher amount of voids and gaps . our study showed that the voids within the restorative material were not associated with a specific type of material . however , gaps at the bottom of the cavity were more frequently associated with the more viscous aa and fu materials , as other studies indicated . they presented a higher polymerization contraction than af and pf materials , and need a higher force and more attention to make a closer contact with the bottom of the cavity . the reason is probably that polymerization contraction , which is evident with all cavity walls in a nonbonded restoration , creates a space at the sides of the cavity , more evidently than in other materials . fu , for instance , has the ability of creating a bond with the side walls of the cavity , but also seems to create larger gaps at the bottom of the cavity , which helps to accommodate a significant part of its contraction during hardening . it must be noticed that pixel size in microtomographic images was 14 in this study , although system 's detection limit was 1.8 . this is probably the limitation of the suggested method and the reason why low viscous materials show a low proportion of voids or gaps . however , newer ct technology ( x - ray computed nanotomography ) will be able to estimate gaps and voids more accurately . voids are present in the same frequency within the mass of all restorative materials , but gaps are present more frequently in restorative materials with high viscosity , at the bottom and the side cavity walls . for these reasons , more research is needed to refine the available techniques for placing correctly and precisely a restorative material within a very conservative cavity , the way of using adhesives in cavities of a very small opening , the type of material that can actually minimize the hardening shrinkage of the material and of course the measurement of voids of very small dimensions . there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities.low viscous composite resins were the best in filling cavities without voids or gaps.high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps.cavity depth , width and volume do correlate with the amount of voids and gap spaces , but only for the high viscous composite material.voids are located in the same frequency within all materials , but gaps are more frequently located within high viscous composites , both at the bottom and the side cavity walls . there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities . high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps . cavity depth , width and volume do correlate with the amount of voids and gap spaces , but only for the high viscous composite material . voids are located in the same frequency within all materials , but gaps are more frequently located within high viscous composites , both at the bottom and the side cavity walls .

cutaneous t cell lymphoma ( ctcl ) is a rare group of lymphoproliferative disorders characterized by localization of t lymphocytes to the skin with an overall incidence of 10.2 cases per million person - years . it is becoming increasingly recognized that ctcl is heterogeneous , with multiple variants that have unique clinical presentations , histologic features , and therapeutic considerations . the most common subtype of ctcl is mycosis fungoides ( mf ) , which typically presents with pruritic patches and plaques in areas of skin not commonly exposed to sun , and may evolve to cutaneous tumors or erythroderma . demographic features including african - american race , male gender , and age have been found to be risk factors for ctcl , while extent of skin involvement , overall disease stage , and age are prognostic indicators [ 2 , 3 ] . the prognostic significance of lymphocytic cd30 positivity in ctcl prognosis remains controversial : some have reported it as a favorable prognostic factor , while others have shown it to be associated with advanced disease and independently associated with poorer survival . total skin electron irradiation ( tsei ) has been shown to be an effective palliative treatment for ctcl , even following failure with previous treatment modalities . this has been demonstrated in multiple retrospective reviews showing that conventional dose ( 30 gy ) tsei achieves high clinical response rates [ 3 , 611 ] typically using large - field / modified stanford technique ( discontinuous irradiation ) . however , more recent clinical series using dual - field rotational tsei ( rtsei ) technique , where a patient is rotated at a constant speed about the vertical axis while being irradiated with continuous dual - field irradiation ( providing a theoretical advantage in dose homogeneity compared to its large - field / modified stanford counterpart ) , showed promising results [ 12 , 13 ] . given that there have been no previous studies that have examined the response of cd30 + ctcl to rtsei , we investigated the effect of cd30 status on treatment response and survival in our cohort of patients with ctcl treated with conventional dose rtsei . after institutional review board ( irb ) approval was obtained from emory university for this study , we retrospectively reviewed medical records of patients treated with rtsei identified from billing records . patients were treated between 2000 and 2013 at emory university , atlanta , ga , usa . patient electronic medical records and a previous irb - approved dermatology database were used to ascertain information regarding demographics , diagnosis , histology , staging , treatment regimens , rtsei treatment , clinical response , recurrence , and overall survival ( os ) . eligibility criteria for the study included : histologically confirmed ctcl and a completed first course of conventional dose ( 30 gy ) rtsei . all cd30 + patients slides were reviewed by a board - certified hematopathologist ( k.b . ) . pathologists typically perform cd30 immunohistochemistry when there is concern for transformation with increased numbers of large lymphoid cells . cd30-not - tested cases lacked histological evidence of transformation and thus were grouped for statistical purposes with those that are known to be cd30 negative per pathology report . rtsei was administered using a 21ex varian linear accelerator ( linac ; varian , palo alto , ca , usa ) equipped with 6 mev high - dose total skin electron ( hdtse ) mode at a dose rate of 888 mu / min . rtsei used dual angles field at gantry angles of 241 and 299 to cover the upper and the lower halves of the patient body . in the 6 mev hdtse mode , our linac calibration was 2.99 cgy / mu at a maximum source to skin distance ( ssd ) of 100 cm . the patient is placed on a rotating platform at an extended ssd ( 315 cm ) from the gantry . the platform rotates at a constant speed of four revolutions per minute to ensure adequate surface dose buildup . all patients were treated using a standard method : 36 gy delivered at 1.5 gy per fraction , administered three times weekly , typically on a monday wednesday friday schedule ( 4.5 gy / week ) . a one - week mid - treatment break was allowed after delivery of 18 gy . optically stimulated luminescent dosimeter ( osld ) measurements at typically under - dosed areas including but not limited to scalp , palms , inner thighs , and bottom of feet were taken prior to the mid - treatment break . approximately 84% of patients received additional boost to these under - dosed regions based on clinical assessment of disease response at the end of rtsei . in total , the total rtsei regimen required 9 weeks followed by a 23-week boost portion . the overall study cohort was compared across the following covariates : sex , age at diagnosis , age at rtsei , histology , t stage at start of rtsei , pre - rtsei ctcl treatments ( therapies received prior to primary rtsei course including topical , systemic antineoplastic , systemic dermatologic , and phototherapies ) , maintenance therapy ( systemic or topical therapy started within 36 months after rtsei ) , time from diagnosis to rtsei , and recurrence . primary outcome measures were complete clinical response ( ccr ) rate , relapse - free survival ( rfs ) , and os . ccr rate was defined as the proportion of follow - up patients with at least 90% reduction in cutaneous tumor burden . patients considered non - complete responders ( non - cr ) included those with partial response ( > 50% reduction but less than 90% ) , stable disease , recurrence , progression , or death at each of three time points : ( 1 ) end of rtsei treatment , ( 2 ) 6 weeks post - rtsei , and ( 3 ) 6 months post - rtsei . patients with no previous record of recurrence , those who did not follow - up or had no recorded clinical response at one of the time points were not included in the ccr proportion . response rates were compared across cd30 status and by t stage using chi - square or fisher s exact test , as appropriate . rfs was calculated as the number of days between start of rtsei and the date of recurrence . in the case of patients who did not have recurrence , rfs was censored at the date of last follow - up or date of death . os was the number of days between diagnosis and the date of death or last known follow - up . both rfs and os were compared across race , t stage , and previously mentioned covariates , using log - rank tests and cox proportional hazards models . multivariate cox models were fit , adjusting for t stage pre - tsei , and the proportional hazards assumption was checked for all models . the cutoff for statistical significance for all analyses was set at the two - sided alpha error of 0.05 . all analysis was done in sas 9.3 ( sas institute inc . , cary , nc , usa ) . after institutional review board ( irb ) approval was obtained from emory university for this study , we retrospectively reviewed medical records of patients treated with rtsei identified from billing records . patients were treated between 2000 and 2013 at emory university , atlanta , ga , usa . patient electronic medical records and a previous irb - approved dermatology database were used to ascertain information regarding demographics , diagnosis , histology , staging , treatment regimens , rtsei treatment , clinical response , recurrence , and overall survival ( os ) . eligibility criteria for the study included : histologically confirmed ctcl and a completed first course of conventional dose ( 30 gy ) rtsei . all cd30 + patients slides were reviewed by a board - certified hematopathologist ( k.b . ) . pathologists typically perform cd30 immunohistochemistry when there is concern for transformation with increased numbers of large lymphoid cells . cd30-not - tested cases lacked histological evidence of transformation and thus were grouped for statistical purposes with those that are known to be cd30 negative per pathology report . rtsei was administered using a 21ex varian linear accelerator ( linac ; varian , palo alto , ca , usa ) equipped with 6 mev high - dose total skin electron ( hdtse ) mode at a dose rate of 888 mu / min . rtsei used dual angles field at gantry angles of 241 and 299 to cover the upper and the lower halves of the patient body . in the 6 mev hdtse mode , our linac calibration was 2.99 cgy / mu at a maximum source to skin distance ( ssd ) of 100 cm . the patient is placed on a rotating platform at an extended ssd ( 315 cm ) from the gantry . the platform rotates at a constant speed of four revolutions per minute to ensure adequate surface dose buildup . all patients were treated using a standard method : 36 gy delivered at 1.5 gy per fraction , administered three times weekly , typically on a monday wednesday friday schedule ( 4.5 gy / week ) . a one - week mid - treatment break was allowed after delivery of 18 gy . optically stimulated luminescent dosimeter ( osld ) measurements at typically under - dosed areas including but not limited to scalp , palms , inner thighs , and bottom of feet were taken prior to the mid - treatment break . approximately 84% of patients received additional boost to these under - dosed regions based on clinical assessment of disease response at the end of rtsei . in total , the total rtsei regimen required 9 weeks followed by a 23-week boost portion . the overall study cohort was compared across the following covariates : sex , age at diagnosis , age at rtsei , histology , t stage at start of rtsei , pre - rtsei ctcl treatments ( therapies received prior to primary rtsei course including topical , systemic antineoplastic , systemic dermatologic , and phototherapies ) , maintenance therapy ( systemic or topical therapy started within 36 months after rtsei ) , time from diagnosis to rtsei , and recurrence . primary outcome measures were complete clinical response ( ccr ) rate , relapse - free survival ( rfs ) , and os . ccr rate was defined as the proportion of follow - up patients with at least 90% reduction in cutaneous tumor burden . patients considered non - complete responders ( non - cr ) included those with partial response ( > 50% reduction but less than 90% ) , stable disease , recurrence , progression , or death at each of three time points : ( 1 ) end of rtsei treatment , ( 2 ) 6 weeks post - rtsei , and ( 3 ) 6 months post - rtsei . patients with no previous record of recurrence , those who did not follow - up or had no recorded clinical response at one of the time points were not included in the ccr proportion . response rates were compared across cd30 status and by t stage using chi - square or fisher s exact test , as appropriate . rfs was calculated as the number of days between start of rtsei and the date of recurrence . in the case of patients who did not have recurrence , rfs was censored at the date of last follow - up or date of death . os was the number of days between diagnosis and the date of death or last known follow - up . both rfs and os were compared across race , t stage , and previously mentioned covariates , using log - rank tests and cox proportional hazards models . multivariate cox models were fit , adjusting for t stage pre - tsei , and the proportional hazards assumption was checked for all models . the cutoff for statistical significance for all analyses was set at the two - sided alpha error of 0.05 . all analysis was done in sas 9.3 ( sas institute inc . , cary , nc , usa ) . medical records of 110 patients treated with rtsei identified from billing records were retrospectively reviewed . after excluding 42 patients who either had a primary diagnosis of leukemia cutis or did not complete conventional dose rtsei , 68 eligible patients remained . patient characteristics of the study cohort , stratified by cd30 status , are summarized in table 2 . sixty - eight patients were treated with conventional dose rtsei ( range 3036 gy ) . median follow - up time for these patients was 80.2 months.table 1summary of cd30 + casescaseage at rtsei ( years)sexracectcl histologyt stage pre - rtseicd30 + lymphocytetransformation statustime from dx to rtsei ( months)response at end of rtseiresponse at 6 weeksresponse at 6 months153femaleaamft410%equivocal35crcrcr241femaleaamft35%no40crcrnon - cr354femalewhitemft3<5%no30crcrnon - cr461femalewhitemft310%no27crcrnon - cr567malewhitelypt3diffuse*unknown14crcrnon - cr671maleaamft4<5%no18crcrcr786maleaamft4cd30+*unknown18crcrnon - cr849femaleaamft45%no3crcrnon - cr954maleaamft430%yes2non - crnon - crnon - cr1051femaleaamft35%no4crcrcr1172malewhitepcptcl nost35%no93crcrnon - cr1246malewhitectcl nost3diffuse*equivocal10non - crnon - crnon - cr1353femalewhitectcl nost310%no17crcrnon - cr aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reportstable 2patient characteristicscovariatecd30 status p valuecd30 ( n = 54)cd30 + ( n = 14)sex , n ( % ) female26 ( 47.3)7 ( 53.9)0.67 male29 ( 52.7)6 ( 46.2)race , n ( % ) white27 ( 50.9)6 ( 46.2)0.76 african - american26 ( 49.1)7 ( 53.9)age , mean52.456.2time ( months ) from diagnosis to rtsei median16.835.40.30 mean27.235.8pre - rtsei tumor ( t ) stage , n ( % ) t218 ( 32.8)0 ( 0.0 ) 0.01 t329 ( 52.7)8 ( 61.5 ) t48 ( 14.6)5 ( 38.5)pre - rtsei ctcl treatmentprevious use of topical agents , n ( % ) no25 ( 45.5)5 ( 38.5)0.65 yes30 ( 54.6)8 ( 61.5)previous use of systemic antineoplastic agents , n ( % ) no27 ( 49.1)6 ( 46.2)0.85 yes28 ( 50.9)7 ( 53.9)previous use of systemic dermatologic agents , n ( % ) no43 ( 78.2)12 ( 92.3)0.44 yes12 ( 21.8)1 ( 7.7)previous use of phototherapy , n ( % ) no29 ( 52.7)6 ( 46.2)0.67 yes26 ( 47.3)7 ( 53.8)maintenance therapy post - rtsei , n ( % ) no35 ( 63.6)7 ( 53.9)0.54 yes20 ( 36.4)6 ( 57.1)recurrence following rtsei , n ( % ) no18 ( 32.7)6 ( 46.2)0.42 yes37 ( 67.3)7 ( 53.9 ) ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiationbold p values are statistically significant ( p < 0.05 ) summary of cd30 + cases aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reports patient characteristics ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiation bold p values are statistically significant ( p < 0.05 ) pathology confirmed 13 ( 19% ) ctcl patients as cd30 + . cd30% expression ranged from 5% to 30% in tumor cells . an example photomicrograph of a cd30 + transformed ctcl case is shown in fig . 1 . for the cd30 + patients , 7 ( 54% ) were female and 6 were male ( 46% ) , and 7 were aa ( 54% ) and 6 were white ( 46% ) . cd30 + patients had a significantly more advanced t stage disease prior to rtsei ( 62% t3 , 39% t4 ) compared to cd30 patients ( 33% t2 , 53% t3 , 15% t4 ; table 2 ) . there were no significant differences between cd30 subgroups in regard to sex , race , age at diagnosis , time from diagnosis to rtsei , treatments prior to rtsei , maintenance therapy , follow - up time , or proportion that recurred after rtsei.fig . 1transformed cutaneous t cell lymphoma ( case # 9 from table 1 ) . a large transformed lymphoma cells with focal epidermotropism ( h&e stain , original magnification 200 ) . b approximately 30% of lymphoma cells are positive for cd30 ( original magnification 200 ) transformed cutaneous t cell lymphoma ( case # 9 from table 1 ) . a large transformed lymphoma cells with focal epidermotropism ( h&e stain , original magnification 200 ) . b approximately 30% of lymphoma cells are positive for cd30 ( original magnification 200 ) table 3 summarizes ccr rates by cd30 status . cd30 + patients had overall ccr rates of 85% at end of rtsei , 85% at 6 weeks cd30 patients had overall ccr rates of 95% at the end of rtsei , 79% at 6 weeks chi - square and fisher s exact tests revealed no significant differences in ccr rate between cd30 status subgroups following end of rtsei ( p = 0.24 ) or six weeks ( p = 0.44 ) , but did trend toward significance at 6 months post - tsei ( p = 0.083 ) . this trend was driven by a significantly poorer response at 6 months for cd30 + t3 disease compared to cd30 t3 patients ( p = 0.04 ) . further comparisons between cd30 status groups across respective t stages at all other follow - up time points showed no significance difference in ccr rates.table 3complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseifollow - up periodend of rtseisix weeks post - rtseisix months post - rtseicd30 statuscd30 statuscd30 statuscd30 + n ( % ) cd30 n ( % ) p valuecd30 + n ( % ) cd30 n ( % ) p valueall t stages11 ( 85)52 ( 95)0.2411 ( 85)42 ( 79)13 ( 23)24 ( 50)0.083t218 ( 100)15 ( 88)5 ( 42)t37 ( 88)27 ( 93)0.537 ( 88)22 ( 79)11 ( 13)16 ( 57 ) 0.044 t44 ( 80)7 ( 88)14 ( 80)5 ( 62.5)12 ( 40)3 ( 38)1bold p values are statistically significant ( p < 0.05 ) complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 status bold p values are statistically significant ( p < 0.05 ) as seen in table 4 , cd30 + patients had a median rfs of 12.1 months ( 8.3 and 13.2 for t3 and t4 disease , respectively , not significantly different ) . cd30 patients had a median rfs of 9.9 months ( 14.3 , 11.3 , and 6.0 months for t2 , t3 , and t4 disease , respectively , with no significant difference between stages t3 and t4 ) . meier rfs curves comparing cd30 groups and cd30 + patients by t stage are shown in fig . 2.table 4rfs and os , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseimedian rfs ( months)median os ( months)cd30cd30 + p valuecd30cd30 + p valueall t stages9.912.10.7574.5103.30.57t214.3t311.38.30.64103.390.70.95t46.013.20.5130.50.10 os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiationfig . 2survival outcomes . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation rfs and os , stratified by t stage pre - rtsei and cd30 status os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation survival outcomes . a rfs comparing cd30 + vs. cd30 patients . b os comparing cd30 + vs. cd30 patients . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation on multivariate analysis , controlling for t stage , cd30 status was not associated with rfs ( p = 0.75 ) . maintenance therapy following rtsei was associated with significantly improved rfs in cd30 group [ hazard ratio ( hr ) = 2.79 ; 95% confidence interval ( ci ) 1.176.66 ; p = 0.021 ] . this association was not observed in the cd30 + group ( p = 0.383 ) . all other covariates such as race , sex , t stage , histology , age at diagnosis , time from diagnosis to start of tsei , and pre - rstei treatment did not have a clinically significant association with rfs on univariate or multivariate analyses in either cd30 + or cd30 groups . as seen in table 4 , cd30 + patients had a median os of 103.3 months overall with no significant difference in os between stages t3 and t4 ( p = 0.65 ) . cd30 patients had a median os of 74.5 months with a trend towards poorer survival for t4 compared to t3 patients ( 103.3 months for t3 , 30.5 months for t4 , p = 0.09 ) . there was no significant difference in os across cd30 status ( p = 0.57 ) , t3 ( p = 0.95 ) , or t4 ( p = 0.10 ) subgroups . meier os curves for comparing cd30 groups and cd30 + patients by t stage are shown in fig . 2 . on univariate analysis , cd30 status was not associated with os ( p = 0.57 ) . in the cd30 + group , non - mf histology ( compared to mf histology ) was associated with poorer os in univariate analysis ( hr = 10.65 ; p = 0.025 ) . however , after controlling for t stage pre - rtsei , this observed effect only trended towards significance ( p = 0.080 ) . in the cd30 group , lower t stage ( hr = 0.26 ; p = 0.049 ) in univariate analysis was associated with improved os . in multivariate analysis , increased time from diagnosis to rtsei ( hr = 0.98 , p = 0.017 ) and younger age at rtsei ( hr = 1.06 ; p = 0.003 ) were associated with a significantly improved os . maintenance therapy failed to show any significant improvement in os in both cd30 + ( p = 0.998 ) and cd30 ( p = 0.242 ) in multivariate analysis controlling for t stage pre - rtsei . medical records of 110 patients treated with rtsei identified from billing records were retrospectively reviewed . after excluding 42 patients who either had a primary diagnosis of leukemia cutis or did not complete conventional dose rtsei , 68 eligible patients remained . patient characteristics of the study cohort , stratified by cd30 status , are summarized in table 2 . sixty - eight patients were treated with conventional dose rtsei ( range 3036 gy ) . median follow - up time for these patients was 80.2 months.table 1summary of cd30 + casescaseage at rtsei ( years)sexracectcl histologyt stage pre - rtseicd30 + lymphocytetransformation statustime from dx to rtsei ( months)response at end of rtseiresponse at 6 weeksresponse at 6 months153femaleaamft410%equivocal35crcrcr241femaleaamft35%no40crcrnon - cr354femalewhitemft3<5%no30crcrnon - cr461femalewhitemft310%no27crcrnon - cr567malewhitelypt3diffuse*unknown14crcrnon - cr671maleaamft4<5%no18crcrcr786maleaamft4cd30+*unknown18crcrnon - cr849femaleaamft45%no3crcrnon - cr954maleaamft430%yes2non - crnon - crnon - cr1051femaleaamft35%no4crcrcr1172malewhitepcptcl nost35%no93crcrnon - cr1246malewhitectcl nost3diffuse*equivocal10non - crnon - crnon - cr1353femalewhitectcl nost310%no17crcrnon - cr aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reportstable 2patient characteristicscovariatecd30 status p valuecd30 ( n = 54)cd30 + ( n = 14)sex , n ( % ) female26 ( 47.3)7 ( 53.9)0.67 male29 ( 52.7)6 ( 46.2)race , n ( % ) white27 ( 50.9)6 ( 46.2)0.76 african - american26 ( 49.1)7 ( 53.9)age , mean52.456.2time ( months ) from diagnosis to rtsei median16.835.40.30 mean27.235.8pre - rtsei tumor ( t ) stage , n ( % ) t218 ( 32.8)0 ( 0.0 ) 0.01 t329 ( 52.7)8 ( 61.5 ) t48 ( 14.6)5 ( 38.5)pre - rtsei ctcl treatmentprevious use of topical agents , n ( % ) no25 ( 45.5)5 ( 38.5)0.65 yes30 ( 54.6)8 ( 61.5)previous use of systemic antineoplastic agents , n ( % ) no27 ( 49.1)6 ( 46.2)0.85 yes28 ( 50.9)7 ( 53.9)previous use of systemic dermatologic agents , n ( % ) no43 ( 78.2)12 ( 92.3)0.44 yes12 ( 21.8)1 ( 7.7)previous use of phototherapy , n ( % ) no29 ( 52.7)6 ( 46.2)0.67 yes26 ( 47.3)7 ( 53.8)maintenance therapy post - rtsei , n ( % ) no35 ( 63.6)7 ( 53.9)0.54 yes20 ( 36.4)6 ( 57.1)recurrence following rtsei , n ( % ) no18 ( 32.7)6 ( 46.2)0.42 yes37 ( 67.3)7 ( 53.9 ) ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiationbold p values are statistically significant ( p < 0.05 ) summary of cd30 + cases aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reports patient characteristics ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiation bold p values are statistically significant ( p < 0.05 ) pathology confirmed 13 ( 19% ) ctcl patients as cd30 + . cd30% expression ranged from 5% to 30% in tumor cells . an example photomicrograph of a cd30 + transformed ctcl case is shown in fig . 1 . for the cd30 + patients , 7 ( 54% ) were female and 6 were male ( 46% ) , and 7 were aa ( 54% ) and 6 were white ( 46% ) . cd30 + patients had a significantly more advanced t stage disease prior to rtsei ( 62% t3 , 39% t4 ) compared to cd30 patients ( 33% t2 , 53% t3 , 15% t4 ; table 2 ) . there were no significant differences between cd30 subgroups in regard to sex , race , age at diagnosis , time from diagnosis to rtsei , treatments prior to rtsei , maintenance therapy , follow - up time , or proportion that recurred after rtsei.fig . 1transformed cutaneous t cell lymphoma ( case # 9 from table 1 ) . a large transformed lymphoma cells with focal epidermotropism ( h&e stain , original magnification 200 ) . b approximately 30% of lymphoma cells are positive for cd30 ( original magnification 200 ) transformed cutaneous t cell lymphoma ( case # 9 from table 1 ) . a large transformed lymphoma cells with focal epidermotropism ( h&e stain , original magnification 200 ) . cd30 + patients had overall ccr rates of 85% at end of rtsei , 85% at 6 weeks cd30 patients had overall ccr rates of 95% at the end of rtsei , 79% at 6 weeks chi - square and fisher s exact tests revealed no significant differences in ccr rate between cd30 status subgroups following end of rtsei ( p = 0.24 ) or six weeks ( p = 0.44 ) , but did trend toward significance at 6 months post - tsei ( p = 0.083 ) . this trend was driven by a significantly poorer response at 6 months for cd30 + t3 disease compared to cd30 t3 patients ( p = 0.04 ) . further comparisons between cd30 status groups across respective t stages at all other follow - up time points showed no significance difference in ccr rates.table 3complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseifollow - up periodend of rtseisix weeks post - rtseisix months post - rtseicd30 statuscd30 statuscd30 statuscd30 + n ( % ) cd30 n ( % ) p valueall t stages11 ( 85)52 ( 95)0.2411 ( 85)42 ( 79)13 ( 23)24 ( 50)0.083t218 ( 100)15 ( 88)5 ( 42)t37 ( 88)27 ( 93)0.537 ( 88)22 ( 79)11 ( 13)16 ( 57 ) 0.044 t44 ( 80)7 ( 88)14 ( 80)5 ( 62.5)12 ( 40)3 ( 38)1bold p values are statistically significant ( p < 0.05 ) complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 status bold p values are statistically significant ( p < 0.05 ) as seen in table 4 , cd30 + patients had a median rfs of 12.1 months ( 8.3 and 13.2 for t3 and t4 disease , respectively , not significantly different ) . cd30 patients had a median rfs of 9.9 months ( 14.3 , 11.3 , and 6.0 months for t2 , t3 , and t4 disease , respectively , with no significant difference between stages t3 and t4 ) . meier rfs curves comparing cd30 groups and cd30 + patients by t stage are shown in fig . 2.table 4rfs and os , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseimedian rfs ( months)median os ( months)cd30cd30 + p valuecd30cd30 + p valueall t stages9.912.10.7574.5103.30.57t214.3t311.38.30.64103.390.70.95t46.013.20.5130.50.10 os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiationfig . 2survival outcomes . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation rfs and os , stratified by t stage pre - rtsei and cd30 status os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation survival outcomes . a rfs comparing cd30 + vs. cd30 patients . b os comparing cd30 + vs. cd30 patients . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation on multivariate analysis , controlling for t stage , cd30 status was not associated with rfs ( p = 0.75 ) . maintenance therapy following rtsei was associated with significantly improved rfs in cd30 group [ hazard ratio ( hr ) = 2.79 ; 95% confidence interval ( ci ) 1.176.66 ; p = 0.021 ] . this association was not observed in the cd30 + group ( p = 0.383 ) . all other covariates such as race , sex , t stage , histology , age at diagnosis , time from diagnosis to start of tsei , and pre - rstei treatment did not have a clinically significant association with rfs on univariate or multivariate analyses in either cd30 + or cd30 groups . as seen in table 4 , cd30 + patients had a median os of 103.3 months overall with no significant difference in os between stages t3 and t4 ( p = 0.65 ) . cd30 patients had a median os of 74.5 months with a trend towards poorer survival for t4 compared to t3 patients ( 103.3 months for t3 , 30.5 months for t4 , p = 0.09 ) . there was no significant difference in os across cd30 status ( p = 0.57 ) , t3 ( p = 0.95 ) , or t4 ( p = 0.10 ) subgroups . meier os curves for comparing cd30 groups and cd30 + patients by t stage are shown in fig . 2 . on univariate analysis , cd30 status was not associated with os ( p = 0.57 ) . in the cd30 + group , non - mf histology ( compared to mf histology ) was associated with poorer os in univariate analysis ( hr = 10.65 ; p = 0.025 ) . however , after controlling for t stage pre - rtsei , this observed effect only trended towards significance ( p = 0.080 ) . in the cd30 group , lower t stage ( hr = 0.26 ; p = 0.049 ) in univariate analysis was associated with improved os . in multivariate analysis , increased time from diagnosis to rtsei ( hr = 0.98 , p = 0.017 ) and younger age at rtsei ( hr = 1.06 ; p = 0.003 ) were associated with a significantly improved os . maintenance therapy failed to show any significant improvement in os in both cd30 + ( p = 0.998 ) and cd30 ( p = 0.242 ) in multivariate analysis controlling for t stage pre - rtsei . management is difficult as there are no standardized treatment regimens , there is prognostic heterogeneity even within group stages , and high - quality trials are lacking due to disease rarity . there is an urgent need to identify patients at high or low risk for disease progression or mortality to aid in clinical decisions about treatment including escalation / de - escalation of radiation dose or initiation / holding of systemic agents . since treatment for ctcl , especially advanced - stage disease , is palliative , improved prognostication would help avoid treatment toxicity in cases at low risk for progression . recently , the prognostic cutaneous lymphoma international prognostic index ( clipi ) was developed for early- and late - stage mf using a cohort of 1502 uk patients and validated in a set of 1221 patients treated at md anderson cancer center ( mdacc ; houston , tx , usa ) ; however , this has yet to be validated in a prospective manner [ 1517 ] . the cutaneous lymphoma international consortium ( clic ) developed a prognostic score for advanced mf stage iib to iv and sezary syndrome ( ss ) with 1394 patients from 29 centers worldwide including the following factors : stage iv disease , age greater than 60 years , large cell transformation , and elevated serum lactate dehydrogenase . cd30 is a member of the tumor necrosis receptor family , and is found on activated t and b cells . a recent study of 47 non - transformed mf cases from the university of pittsburgh ( pittsburgh , pa , usa ) reported that patients were found to have worse survival , higher stage at diagnosis , and higher maximum stage if dermal cells are cd30 + ( > 4.7% ) . a study of 51 patients with large cell transformation of mf at memorial sloan kettering cancer center ( new york , ny , usa ) reported that predominance of cd30 in the epidermis rather than dermis was associated with poorer survival . however , a retrospective analysis of 100 patients with transformed mf from the netherlands showed that cd30 negativity was associated with multivariate analysis with reduced disease - specific survival ( dss ) and os . in a long - term outcomes study , cd30 was found to have no significant effect on os , dss , and progression - free survival ( pfs ) in 1263 mf ss patients at mdacc . the clic reported that when cd30 is scored positive above 10% of tumoral cells , it is not statistically significant for os but is associated with worse os in the subset of patients with t3 disease . finally , cd30 is also targetable with brentuximab vedotin , a monoclonal chimeric antibody conjugated to antitubulin agent monomethyl auristatin e. a recent phase 2 study found that brentuximab vedotin has significant objective global response 70% ( 21/30 ) in treatment patients with refractory or advanced mf / ss who have a range of cd30 expression levels . similarly , another phase 2 trial found overall response of 73% ( 35/48 ) in patients with cd30 + lymphoproliferative disorders treated with brentuximab vedotin . based on the varying reports of the prognostic significance of cd30 , we sought to assess the response and survival of our cohort of patients treated with rtsei . we found 13 of 68 patients were cd30 + ( range 5% to 30% ) ; they presented with significantly higher t stage at time of rtsei and trended towards decreased ccr at 6 months post - rtsei compared with the cd30 group . one patient in our cohort had 1% cd30 + lymphocytes on pathology and was classified as negative for cd30 ; however , when this patient was added to the cd30 + cohort ( data not reported in results ) the ccr was significantly reduced at 6 months ( p = 0.05 ) . regardless , the difference in 6 month ccr was primarily driven by those with t3 stage disease . overall , cd30 + patients had excellent ccr rates of 85% at end of rtsei but most had progressive disease at 6 months . maintenance therapy including topical , systemic antineoplastic , systemic dermatologic , and phototherapies failed to show any significant improvement in rfs in the cd30 + group . additionally , maintenance therapy failed to show any significant improvement in os in both cd30 + and cd30 groups . although direct comparison is not possible without further study , our rates of clinical response are comparable to those recently reported for brentuximab vedotin . additionally , cd30 + patients may benefit from the addition of novel targeted agents such as brentuximab vedotin to radiation , perhaps concurrently or sequentially . recently , the concept of combined radiation and immunotherapy to enhance clinical outcomes has advanced . we feel prospective trial development of combined brentuximab vedotin and radiation for ctcl should proceed based on the results of recent phase 2 trials . our analysis is limited by small sample size ; however , this is the largest report of cd30 + patients , and their response to radiotherapy . more specifically , the small sample size precluded us from examining in more detail the role of maintenance systemic and dermatologic agents . in addition , if a patient did not follow up or did not have a recorded clinical response at the specified time points without record of recurrence by other providers , they were excluded from ccr analysis which further decreased patient numbers in the present analysis . additionally , as this was a retrospective review , many patients had incomplete clinicopathological information , information from outside hospitals , or were lost to follow - up . incomplete data regarding cd30 status increases confounding as patients deemed by clinicians to have aggressive disease or transformation often have more thorough workup . strengths of our study included documentation of clinical response across multiple post - treatment time points , single institution experience with standardized rtsei delivery and dosing , and all cd30 + cases were reviewed by a hematopathologist to confirm cd30 status . in this retrospective study , rtsei for ctcl resulted in excellent ccr at 6 weeks , with a median rfs of 11 months . cd30 + patients receiving rtsei were found to have higher t stage and trended towards decreased ccr at 6 months post - rtsei compared with the cd30 patients . maintenance therapy including topical , systemic antineoplastic , systemic dermatologic , and phototherapies failed to show any significant improvement in rfs in the cd30 + group . future work should examine quality of life metrics , the role of maintenance systemic or targeted treatments , and combination rtsei and brentuximab vedotin in cd30 + patients with ctcl . hasan h. danish , thatcher r. heumann , kyle t. bradley , jeffrey switchenko , natia esiashvili , mary jo lechowicz , christopher r. flowers , and mohammad k. khan have nothing to disclose . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

introductionrotational total skin electron irradiation ( rtsei ) is an effective therapy for cutaneous t cell lymphoma ( ctcl ) . cd30 expression has been identified as a prognostic factor in ctcl . therefore , we investigated cd30 status , treatment response , and survival in our cohort of patients with ctcl treated with rtsei.methodspatients with ctcl treated with rtsei ( 30 gy ) between 2000 and 2013 at our institution were identified , and clinical and pathologic data were retrospectively reviewed . primary outcomes were complete clinical response ( ccr ; > 90% reduction of skin disease burden ) , relapse - free survival ( rfs ) , and overall survival ( os).resultssixty - eight patients with ctcl treated with rtsei were identified . median age at diagnosis was 51 years with median follow - up of 61 months . median os was 76 months and median rfs was 11 months . thirteen patients ( 19% ) had cd30 + lymphocytes on initial pathology . in the cd30 + cohort , there were no t2 , eight t3 , and five t4 cases . in comparison , in the cd30 cohort , there were 18 t2 , 29 t3 , and 8 t4 cases ( p = 0.01 ) . six weeks post - rtsei , ccr was 85% in cd30 + and 81% in cd30 cases ( p = 1 ) . six months post - rtsei , ccr was 23% in cd30 + and 50% in cd30 cases ( p = 0.083).conclusionrtsei resulted in excellent ccr at 6 weeks in our cohort of patients with ctcl , with a median rfs of 11 months . we found cd30 + patients presented with significantly higher t stage at time of rtsei and trended towards decreased ccr at 6 months post - rtsei compared with the cd30 group .

Introduction Methods Patient Population Treatment Technique Covariates Outcome Measures Results Patient Characteristics Clinical Response Rates Relapse-Free Survival Overall Survival Discussion Conclusions Disclosures Compliance with Ethics Guidelines Open Access

given that there have been no previous studies that have examined the response of cd30 + ctcl to rtsei , we investigated the effect of cd30 status on treatment response and survival in our cohort of patients with ctcl treated with conventional dose rtsei . the overall study cohort was compared across the following covariates : sex , age at diagnosis , age at rtsei , histology , t stage at start of rtsei , pre - rtsei ctcl treatments ( therapies received prior to primary rtsei course including topical , systemic antineoplastic , systemic dermatologic , and phototherapies ) , maintenance therapy ( systemic or topical therapy started within 36 months after rtsei ) , time from diagnosis to rtsei , and recurrence . primary outcome measures were complete clinical response ( ccr ) rate , relapse - free survival ( rfs ) , and os . patients considered non - complete responders ( non - cr ) included those with partial response ( > 50% reduction but less than 90% ) , stable disease , recurrence , progression , or death at each of three time points : ( 1 ) end of rtsei treatment , ( 2 ) 6 weeks post - rtsei , and ( 3 ) 6 months post - rtsei . the overall study cohort was compared across the following covariates : sex , age at diagnosis , age at rtsei , histology , t stage at start of rtsei , pre - rtsei ctcl treatments ( therapies received prior to primary rtsei course including topical , systemic antineoplastic , systemic dermatologic , and phototherapies ) , maintenance therapy ( systemic or topical therapy started within 36 months after rtsei ) , time from diagnosis to rtsei , and recurrence . primary outcome measures were complete clinical response ( ccr ) rate , relapse - free survival ( rfs ) , and os . patients considered non - complete responders ( non - cr ) included those with partial response ( > 50% reduction but less than 90% ) , stable disease , recurrence , progression , or death at each of three time points : ( 1 ) end of rtsei treatment , ( 2 ) 6 weeks post - rtsei , and ( 3 ) 6 months post - rtsei . median follow - up time for these patients was 80.2 months.table 1summary of cd30 + casescaseage at rtsei ( years)sexracectcl histologyt stage pre - rtseicd30 + lymphocytetransformation statustime from dx to rtsei ( months)response at end of rtseiresponse at 6 weeksresponse at 6 months153femaleaamft410%equivocal35crcrcr241femaleaamft35%no40crcrnon - cr354femalewhitemft3<5%no30crcrnon - cr461femalewhitemft310%no27crcrnon - cr567malewhitelypt3diffuse*unknown14crcrnon - cr671maleaamft4<5%no18crcrcr786maleaamft4cd30+*unknown18crcrnon - cr849femaleaamft45%no3crcrnon - cr954maleaamft430%yes2non - crnon - crnon - cr1051femaleaamft35%no4crcrcr1172malewhitepcptcl nost35%no93crcrnon - cr1246malewhitectcl nost3diffuse*equivocal10non - crnon - crnon - cr1353femalewhitectcl nost310%no17crcrnon - cr aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reportstable 2patient characteristicscovariatecd30 status p valuecd30 ( n = 54)cd30 + ( n = 14)sex , n ( % ) female26 ( 47.3)7 ( 53.9)0.67 male29 ( 52.7)6 ( 46.2)race , n ( % ) white27 ( 50.9)6 ( 46.2)0.76 african - american26 ( 49.1)7 ( 53.9)age , mean52.456.2time ( months ) from diagnosis to rtsei median16.835.40.30 mean27.235.8pre - rtsei tumor ( t ) stage , n ( % ) t218 ( 32.8)0 ( 0.0 ) 0.01 t329 ( 52.7)8 ( 61.5 ) t48 ( 14.6)5 ( 38.5)pre - rtsei ctcl treatmentprevious use of topical agents , n ( % ) no25 ( 45.5)5 ( 38.5)0.65 yes30 ( 54.6)8 ( 61.5)previous use of systemic antineoplastic agents , n ( % ) no27 ( 49.1)6 ( 46.2)0.85 yes28 ( 50.9)7 ( 53.9)previous use of systemic dermatologic agents , n ( % ) no43 ( 78.2)12 ( 92.3)0.44 yes12 ( 21.8)1 ( 7.7)previous use of phototherapy , n ( % ) no29 ( 52.7)6 ( 46.2)0.67 yes26 ( 47.3)7 ( 53.8)maintenance therapy post - rtsei , n ( % ) no35 ( 63.6)7 ( 53.9)0.54 yes20 ( 36.4)6 ( 57.1)recurrence following rtsei , n ( % ) no18 ( 32.7)6 ( 46.2)0.42 yes37 ( 67.3)7 ( 53.9 ) ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiationbold p values are statistically significant ( p < 0.05 ) summary of cd30 + cases aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reports patient characteristics ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiation bold p values are statistically significant ( p < 0.05 ) pathology confirmed 13 ( 19% ) ctcl patients as cd30 + . cd30 + patients had overall ccr rates of 85% at end of rtsei , 85% at 6 weeks cd30 patients had overall ccr rates of 95% at the end of rtsei , 79% at 6 weeks chi - square and fisher s exact tests revealed no significant differences in ccr rate between cd30 status subgroups following end of rtsei ( p = 0.24 ) or six weeks ( p = 0.44 ) , but did trend toward significance at 6 months post - tsei ( p = 0.083 ) . further comparisons between cd30 status groups across respective t stages at all other follow - up time points showed no significance difference in ccr rates.table 3complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseifollow - up periodend of rtseisix weeks post - rtseisix months post - rtseicd30 statuscd30 statuscd30 statuscd30 + n ( % ) cd30 n ( % ) p valuecd30 + n ( % ) cd30 n ( % ) p valueall t stages11 ( 85)52 ( 95)0.2411 ( 85)42 ( 79)13 ( 23)24 ( 50)0.083t218 ( 100)15 ( 88)5 ( 42)t37 ( 88)27 ( 93)0.537 ( 88)22 ( 79)11 ( 13)16 ( 57 ) 0.044 t44 ( 80)7 ( 88)14 ( 80)5 ( 62.5)12 ( 40)3 ( 38)1bold p values are statistically significant ( p < 0.05 ) complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 status bold p values are statistically significant ( p < 0.05 ) as seen in table 4 , cd30 + patients had a median rfs of 12.1 months ( 8.3 and 13.2 for t3 and t4 disease , respectively , not significantly different ) . 2.table 4rfs and os , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseimedian rfs ( months)median os ( months)cd30cd30 + p valuecd30cd30 + p valueall t stages9.912.10.7574.5103.30.57t214.3t311.38.30.64103.390.70.95t46.013.20.5130.50.10 os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiationfig . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation rfs and os , stratified by t stage pre - rtsei and cd30 status os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation survival outcomes . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation on multivariate analysis , controlling for t stage , cd30 status was not associated with rfs ( p = 0.75 ) . median follow - up time for these patients was 80.2 months.table 1summary of cd30 + casescaseage at rtsei ( years)sexracectcl histologyt stage pre - rtseicd30 + lymphocytetransformation statustime from dx to rtsei ( months)response at end of rtseiresponse at 6 weeksresponse at 6 months153femaleaamft410%equivocal35crcrcr241femaleaamft35%no40crcrnon - cr354femalewhitemft3<5%no30crcrnon - cr461femalewhitemft310%no27crcrnon - cr567malewhitelypt3diffuse*unknown14crcrnon - cr671maleaamft4<5%no18crcrcr786maleaamft4cd30+*unknown18crcrnon - cr849femaleaamft45%no3crcrnon - cr954maleaamft430%yes2non - crnon - crnon - cr1051femaleaamft35%no4crcrcr1172malewhitepcptcl nost35%no93crcrnon - cr1246malewhitectcl nost3diffuse*equivocal10non - crnon - crnon - cr1353femalewhitectcl nost310%no17crcrnon - cr aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reportstable 2patient characteristicscovariatecd30 status p valuecd30 ( n = 54)cd30 + ( n = 14)sex , n ( % ) female26 ( 47.3)7 ( 53.9)0.67 male29 ( 52.7)6 ( 46.2)race , n ( % ) white27 ( 50.9)6 ( 46.2)0.76 african - american26 ( 49.1)7 ( 53.9)age , mean52.456.2time ( months ) from diagnosis to rtsei median16.835.40.30 mean27.235.8pre - rtsei tumor ( t ) stage , n ( % ) t218 ( 32.8)0 ( 0.0 ) 0.01 t329 ( 52.7)8 ( 61.5 ) t48 ( 14.6)5 ( 38.5)pre - rtsei ctcl treatmentprevious use of topical agents , n ( % ) no25 ( 45.5)5 ( 38.5)0.65 yes30 ( 54.6)8 ( 61.5)previous use of systemic antineoplastic agents , n ( % ) no27 ( 49.1)6 ( 46.2)0.85 yes28 ( 50.9)7 ( 53.9)previous use of systemic dermatologic agents , n ( % ) no43 ( 78.2)12 ( 92.3)0.44 yes12 ( 21.8)1 ( 7.7)previous use of phototherapy , n ( % ) no29 ( 52.7)6 ( 46.2)0.67 yes26 ( 47.3)7 ( 53.8)maintenance therapy post - rtsei , n ( % ) no35 ( 63.6)7 ( 53.9)0.54 yes20 ( 36.4)6 ( 57.1)recurrence following rtsei , n ( % ) no18 ( 32.7)6 ( 46.2)0.42 yes37 ( 67.3)7 ( 53.9 ) ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiationbold p values are statistically significant ( p < 0.05 ) summary of cd30 + cases aa african - american , cr complete response ( < 90% reduction in skin disease ) , ctcl cutaneous t cell lymphoma , lyp lymphomatoid papulosis , non - cr non - complete response , nos not otherwise specified , pcptcl primary cutaneous peripheral t cell lymphoma * slides not available for review , cd30 + description based off previous pathology reports patient characteristics ctcl cutaneous t cell lymphoma , rtsei rotational total skin electron irradiation bold p values are statistically significant ( p < 0.05 ) pathology confirmed 13 ( 19% ) ctcl patients as cd30 + . cd30 + patients had overall ccr rates of 85% at end of rtsei , 85% at 6 weeks cd30 patients had overall ccr rates of 95% at the end of rtsei , 79% at 6 weeks chi - square and fisher s exact tests revealed no significant differences in ccr rate between cd30 status subgroups following end of rtsei ( p = 0.24 ) or six weeks ( p = 0.44 ) , but did trend toward significance at 6 months post - tsei ( p = 0.083 ) . further comparisons between cd30 status groups across respective t stages at all other follow - up time points showed no significance difference in ccr rates.table 3complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 statust stage pre - rtseifollow - up periodend of rtseisix weeks post - rtseisix months post - rtseicd30 statuscd30 statuscd30 statuscd30 + n ( % ) cd30 n ( % ) p valueall t stages11 ( 85)52 ( 95)0.2411 ( 85)42 ( 79)13 ( 23)24 ( 50)0.083t218 ( 100)15 ( 88)5 ( 42)t37 ( 88)27 ( 93)0.537 ( 88)22 ( 79)11 ( 13)16 ( 57 ) 0.044 t44 ( 80)7 ( 88)14 ( 80)5 ( 62.5)12 ( 40)3 ( 38)1bold p values are statistically significant ( p < 0.05 ) complete clinical response rates ( > 90% reduction in tumor burden ) , stratified by t stage pre - rtsei and cd30 status bold p values are statistically significant ( p < 0.05 ) as seen in table 4 , cd30 + patients had a median rfs of 12.1 months ( 8.3 and 13.2 for t3 and t4 disease , respectively , not significantly different ) . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation rfs and os , stratified by t stage pre - rtsei and cd30 status os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation survival outcomes . os overall survival , rfs relapse - free survival , rtsei rotational total skin electron irradiation on multivariate analysis , controlling for t stage , cd30 status was not associated with rfs ( p = 0.75 ) . we found 13 of 68 patients were cd30 + ( range 5% to 30% ) ; they presented with significantly higher t stage at time of rtsei and trended towards decreased ccr at 6 months post - rtsei compared with the cd30 group . one patient in our cohort had 1% cd30 + lymphocytes on pathology and was classified as negative for cd30 ; however , when this patient was added to the cd30 + cohort ( data not reported in results ) the ccr was significantly reduced at 6 months ( p = 0.05 ) . in this retrospective study , rtsei for ctcl resulted in excellent ccr at 6 weeks , with a median rfs of 11 months . cd30 + patients receiving rtsei were found to have higher t stage and trended towards decreased ccr at 6 months post - rtsei compared with the cd30 patients .

the hypothesis is that primates have evolved a sophisticated set of facial muscles that now allows them to convey emotions to observers . to date , most research has focused on the study of six emotions typically seen in most cultures : happiness , surprise , sadness , anger , fear , and disgust . previous research has focused on how the facial expressions of these six emotions are produced . the consistent and differential facial muscle articulations ( typically referred to as action units or aus ) associated with each emotion category have been identified . an au is a set of facial muscle articulations that results in a unique visible image feature . when the frontalis ( pars medialis ) muscle contracts , there is a visible rising of the inner section of the eyebrows , and this is denoted au 1 . the six emotion categories listed in the preceding paragraph are sometimes called basic . by basic it is generally meant that emotions are discrete and have evolved to help us adapt to environmental challenges , ie , the response to a set of events is unique and common to everyone because it is an evolved behavior . with regard to facial expressions of emotion , this means that an automatic response to events of the same emotion category should result in a unique and consistent production ( ie , the aus used to produce that expression are the same across populations but differential between categories ) . in particular , we wish to address the following question : which , and how many , facial expressions of emotion are consistently and differentially produced by humans ? to date , most studies have focused on facial expressions defined by a single component emotion , eg , happy . first , emotion categories need not be associated with a single feeling . while the feeling of joy can be defined as a component emotion ( ie , happiness ) , the feeling of being joyfully surprised can not . experiencing a happily surprising event results in very different behaviors than those experienced when we are happy but not surprised . similarly , happily surprised is different from happily disgusted , even though joy is a common denominator in these emotions . to properly address the question posed in the preceding paragraph ( ie , which and how many facial expressions ) , we need to identify facial constructs that yield a consistent au response pattern across populations and a differential response between categories , regardless of whether these are defined by single or multiple feelings . what is an emotion then ? in our examples above , happiness clearly defines a joyful state and , if of short duration , is considered an emotion . but what is happily disgusted ? at first , this might seem contradictory , yet it is perfectly normal . think about the behavior of someone after hearing a really funny but very disgusting joke ( eg , some readers may be familiar with the 2005 film the aristocrats ) . thus , compound emotions are also emotion categories . both component and compound emotions are discrete entities that identify a unique cognitive state and associated behavior . key to understanding compound emotions is to note that these categories are as important as any other , ie , happily disgusted is a discrete category with a unique facial expression , as is the component emotion fear . herein , we review and expand on our previous studies of compound emotions . in particular , we introduce two new , previously undefined facial expressions of compound emotions , show results on spontaneous expressions , and illustrate the importance of intensity of au activation to define and differentiate these emotion categories . our results support the view that at least 17 compound emotions are associated with unique and differential facial expressions . we then provide a discussion of these results in the study , diagnosis , and treatment of psychopathologies . we called them compound because these emotions are composed of two or more component categories . these 15 compound emotions are : happily surprised , happily disgusted , sadly fearful , sadly angry , sadly surprised , sadly disgusted , angrily fearful , fearfully surprised , fearfully disgusted , angrily surprised , angrily disgusted , disgustedly surprised , appalled , hatred , and awed . figure 1 two additional compound emotions that were not studied in our previous work , but that we will include in our analysis below are : happily fearful and happily sad figure 2 . these discrete categories are a response to a set of common events , situations , or actions . let us define each of them in more detail : happily surprised : this is an emotion we feel when we receive good or joyful unexpected news / outcomes : eg , when you applied for college , your favorite school accepted you even though you were convinced they would deny your application , or when a group of friends organize a surprise birthday party . happily disgusted : you feel joyful about an event , but repulsion regarding what it entails or what you see , hear , smell , or experience ; eg , when someone tells you a very funny yet disgusting joke , or when you see someone making a funny but repulsive gesture . happily sad : you feel happy about an event that is tainted with sadness ( ie , bittersweet ) ; eg , when your children leave for college you feel happy and proud , but sad because they are leaving the nest . sadly fearful : a fearful event or action makes you sad ; eg , you are threatened by someone emotionally close to you , or you are really disappointed when something nonthreatening is making you fearful . sadly angry : an event or action that makes you angry and leaves you disappointed or sad ; eg , despite all your efforts to help , your son fails an important test . sadly surprised : a surprising event makes you sad ; eg , your favorite soccer team is about to win a championship but loses to a last - minute goal . sadly disgusted : something morally or sensorially distasteful that makes you sad ; eg , when society or someone you care about disapproves of your lifestyle using vulgar language . fearfully angry : a fearful event or action makes you angry ; eg , you are driving and you make a mistake that almost causes a serious accident , or you are told you might be fired because of something that you did not do . fearfully surprised : a surprising event makes you fearful ; eg , while you are driving , a deer you had not seen crosses the road and almost causes a collision , or while hiking in the rocky mountains you suddenly see a mountain lion 15 metres ahead . fearfully disgusted : a fearful event or action that is also morally or sensorially unpleasant ; eg , an accident results in a life - threatening wound that is visually revolting . angrily surprised : a surprising event or action makes you angry ; eg , unexpectedly and without provocation , a friend insults you . angrily disgusted : an event or action that makes you angry and is morally or sensorially unpleasant ; eg , someone unjustly accuses you of doing something you did not do and that violates your moral code , or an elite athlete loses to a competitor using performance - enhancing drugs . disgustedly surprised : a surprising event or action that is morally or sensorially disgusting ; eg , you are about to eat a piece of fruit that looks great but are surprised when a revolting odor reaches your nose . appalled : horrified or angry and morally disgusted ; eg , you are appalled at the incompetence of an expert or colleague . hatred : extreme dislike or ill will ; eg , racial or sexual hatred . awed : a feeling of reverential respect including the simultaneous feeling of fear and wonder ; eg , you look in awe at the vastness and the unknown in the jungle at night . furthermore , the facial expressions of these emotion categories might have evolved to help us better solve specific behavioral or social problems . for example similarly , compound emotions such as awe might serve as a social glue in large groups of people through communal events , such as those involving the arts , natural phenomena , and religion . first , as can be seen from the list of emotions given above , some compound emotions are identified by a single word in english . however , these categories are still defined as compounds because they include two emotional feelings . for example , hateful and appalled are defined as the compound feeling of anger and disgust , with the emphasis on anger when hateful and on disgust when appalled . awe is the compound feeling of fear and wonder ( surprise ) , with the emphasis on the latter ( eg , horripilation in awe is believed to be a residual behavior of fear ) . the fact that these compound emotions are defined as single words does not mean they are more or less relevant than the others . this is a characteristic of the english language and may not be the same in other languages . for example , in albanian , awe is described as an abrupt or sudden fear and , hence , requires multiple words to define it . the same holds true for compound words : not all languages use two or more words to define them . for example , in mandarin chinese , happily surprised is written as a single word . finally , emotions associated with a single feeling may be named using one or more words . for instance , in igbo ( a language spoken in the southeastern region of nigeria ) , emotions such as happy are generally described using multiple words . it is important to note that cognitive categories need not match language ; that is , a compound emotion may be given by a single word in one 's language , while a component emotion may be described with multiple words . the important difference is in the simultaneous feelings one experiences , not how the emotion category is written . when we experience a single feeling ( eg , joy ) , we will talk about component emotions ( eg , happy ) . when we experience multiple feelings ( eg , joy and surprise ) , we will talk about compound emotions ( eg , happily surprised ) . as mentioned in the introduction of this paper , an emotion that is externalized should have a facial expression that is produced with the same au combination by people of different cultures but differential from those au patterns employed to display other emotion categories . we thus wish to know whether compound emotions are displayed with unique and differential facial expressions . in other words , when , in a situation that yields a compound emotion , is human behavior consistent and differential ? we denote the six emotion categories studied in the past ( ie , happiness , surprise , anger , sadness , disgust , and fear ) component emotions . the other 17 emotion categories introduced in the preceding section are denoted as compound emotions . the very first thing we wish to know is if these 17 compound emotions yield facial expressions that are consistent across people but differential between categories . this had already been shown to be the case for component emotions , but not for compounds . to study this , we analyzed the facial expressions of compound emotions made by people of different cultures . specifically , we identified the aus that are consistently used by a large majority of people ( > 70% ) . as can be seen in this table , all categories yield a unique facial expression . this is to be expected , because these three emotion categories include the same component categories the simultaneous feeling of anger and disgust . angrily disgusted , we feel equally angry and disgusted , whereas in hatred the emphasis is on anger and in appalled the emphasis is on disgust . as can be seen in table i , this results in slightly different expressions . in angrily disgusted there is the consistent use of three aus ( 4 , 10 , and 17 ) with aus 7 and 9 playing a secondary role . in contrast , hatred mainly uses aus 4 and 10 , with 7 and 17 playing the secondary role . and , finally , in appalled , people primarily use aus 4 and 10 , plus the likely addition of 9 and/or 17 . another way that compound categories of closely related emotional states are differentiated is in the intensity of the activation of their aus . as mentioned earlier in this paper , aus correspond to visual image changes caused by the activation of a specific set of facial muscles . this image change can , however , be more or less visible . when the contraction of the associated muscles is stronger , we generally observe a large image change . typically , we use five values to denote the intensity of activation of an au . they are denoted , from smallest to largest intensity , a through e. figure 3 shows the percentage of times people use a given intensity ( or larger ) in each au for each of the 23 emotion categories . as is made clear by this figure , the au patterns of activation used by people to express each emotion are not only different ; the intensities of au activation are also distinct this is important because it further illustrates the many ways in which the expressions of different discrete emotion categories are consistently produced by people of different cultures . in figure 3 , we also see that the aus used to express a compound emotion are consistent with the aus used to express its component ( subordinate ) categories . that is , the resulting au pattern is unique to that compound emotion ( a fact that allows us to differentiate this facial expression from the others ) , but the resulting expression is also consistent with the au patterns used to display its component categories . note how some aus are emphasized more in the compound than in the subordinate categories ( ie , intensity of production is increased ) , whereas others are deemphasized . this difference in production further facilitates the visual distinction of each facial expression while leaving the active set of aus consistent across compound and subordinate categories . behavioral experiments by our group show that people are quite good at visually discriminating these facial expressions of emotion . our studies have tested people 's ability to name and visually discriminate these expressions of emotion . for instance , in a cognitively taxing task , participants were shown an image of one of the component or compound facial expressions and given a list of 21 possible labels ( ie , happy , happily surprised , sad , sadly angry , etc ) . westerners and east asian participants were able to correctly name 19 of the emotion categories introduced in the present paper . most importantly , the results in these two groups ( westerners and east asians ) were statistically identical . and , notably , a set of eight emotion categories was recognized more readily than the rest by both groups . in another experiment , participants were instructed to indicate by key press whether the two images shown on the screen belonged to the same emotion category or not . all emotion categories were visually discriminated , but eight of them were not only discriminated more accurately , but more rapidly too . one group is hypothesized to play a major role in cognitive and/or social tasks , and are thus more readily recognized . the other expressions are also universally recognized by people of different cultures , but seem to play a less central role in our daily cognitive and social tasks . an alternative hypothesis is that evolution has selected these few emotions because they provided some survival advantage . our group is currently working on trying to better understand these differences and its implications in cognition , behavior , and psychopathologies . thus far , we have presented studies of facial expressions of emotion collected in the laboratory . next , we wish to know if spontaneous expressions are produced with the same aus identified in the laboratory . this is important because the neural systems involved in the production of posed and spontaneous expressions are believed to differ . to answer this question our search was based on keywords ( eg , happy , happily disgusted , happy and disgust , etc ) . the guardian ) and television channel sites ( eg , cnn , cbs news ) and also used google images to identify these expressions . our search started with a qualitative analysis . when an image was visually identified as appearing to express one of the 17 compound emotions listed above by one of the two authors we found that the au pattern of activation of spontaneous expressions is identical to those seen in the laboratory . we are so accustomed to interacting with others nonverbally that replicating this in the laboratory does not pose a major problem . ( surprisingly , happiness might actually be the one major exception to this rule . ) second , when collecting images in the laboratory , we were very careful to instruct the subjects on how to provide as naturalistic an expression as possible . then , participants had the opportunity to practice in front of a mirror to see if their expression matched what they intended before pictures were taken . this procedure yielded the same results we have now observed in spontaneous expressions , further validating the results summarized in table i and figure 3 . most people have felt upbeat or beaten up emotionally at some point in their lives . when these occurrences are transitory ( hours or a few days ) , they change our mood . involuntary long - lasting effects may develop in psychopathologies , for example , clinical depression or post - traumatic stress disorder ( ptsd ) , with genetics believed to be a contributing factor in this transition . other psychopathologies are believed to be developmental , as is the case in autism spectrum disorder ( asd ) . understanding how many facial expressions of emotion neurotypicals produce and recognize is essential to further our study of these disorders , as well as for the development of coping mechanisms and treatment options . the very first hurdle that needs to be cleared in these cases is to provide a correct and accurate diagnosis . for instance , ptsd is generally diagnosed after lengthy interviews with a knowledgeable psychiatrist who usually administers the clinician - administered ptsd scale ( caps ) . caps diagnosis is based on the presence of several symptoms described in the diagnostic and statistical manual of mental disorders ( dsm ) rather than pathophysiologically . to be able to resolve this issue , we need to better understand the differences in production and visual recognition of facial expressions of emotion . our general hypothesis is that pathophysiological distinctive disorders result in a different production and/ or visual interpretation of facial expressions of emotion . before we can fully address this hypothesis though , we need to identify all component and compound facial expressions of emotion . to date , we have identified 23 facial expressions of emotion , but there could be more . some researchers have suggested that contempt is a component emotion . if this were confirmed , it might be possible to compound contempt with other component categories such as happiness or disgust , to yield categories such as derision . for the time being , we believe physicians and medical personnel should learn to quickly identify the known 23 facial expressions of emotion in patients . as we have seen above , we are all quite good at visually identifying these facial constructs , but we believe medical professionals should be proficient at this task . to learn to identify these expressions , one can pay close attention to the results given in the table and figures above . our research laboratory at the ohio state university is also developing an interactive ( web - based ) application to train medical personnel to become better at visually recognizing these expressions . identifying these in patients could prove as valuable as listening closely to what they have to say . our research group is also developing computer algorithms that can recognize these facial expressions automatically . this technology would free the medical professional from having to analyze the face and would eliminate or , at least facilitate , training . all that will be needed is a camera and computer , and the system will provide real - time information to the physician . our current system recognizes expressions of emotion with > 75% accuracy , which is better than untrained individuals can do . improvements on this technology are sure to revolutionize clinical practice in the next few years . advances in face detection , for instance , are fueling improvements in the recognition of facial expressions . another likely difference between the production of facial expressions of emotion in neurotypicals and individuals with psychopathologies is variations in the intensity of activation of aus . for example , are aus in sadness ( including its compounds , eg , sadly angry , sadly surprised , etc ) displayed with larger or smaller au intensity in clinical depression ? characterizing these differences will allow us to develop protocols for evaluating the automatic annotations given by the computer software outlined in the preceding paragraph . past research has shown differences in the visual recognition of facial expressions of component emotions , but there is not yet any research that has studied the perception of compound emotions in the clinical population . this is likely to be a productive area of research in clinical and translational medicine . the set of six emotions used in the research studies listed above is likely to be insufficient to describe all psychopathologies defined in the dsm . there is limited variability that can be readily and consistently observed in the facial expressions of joy , surprise , sadness , anger , disgust , and fear across psychopathologies . by studying a much larger number of emotions ( ie , variables ) , for example , clinical depression might result in an increased production of compound expressions with a sad component ( eg , sadly angry , sadly fearful , sadly disgusted ) , even in the absence of additional facial expressions of sadness . basic research is needed to study this , but psychiatrists and other medical professionals should also report what is observed in their practice . these observations can prove invaluable to researchers , and can serve as hypotheses for future studies . this would require that medical professionals become proficient in the visual interpretation of all facial expressions of emotion . this may seem contradictory at first because happiness and fear seem polar opposites , yet these are common compound emotions people experience . our research shows that the facial expressions that accompany these internally felt emotions are consistent across people and differential between emotion categories . furthermore , the associated facial constructs seem to be universally visually recognized by observers , even in challenging conditions . in our previous work we defined 21 compound facial expressions of emotion . herein , we have further extended this to 23 ( table i ; figures 1 , 2 ) . much still needs to be done to fully understand compound emotions and their facial expressions , however . for instance , we do not yet know how many emotion categories there are . specifically , if contempt ( or others ) were found to also be consistently produced by people of distinct cultures , then many more compound categories would be possible . for example , it is likely that in some circumstances one could feel fearful , surprised and happy , eg , while riding in one of the attractions in an amusement park , it is common to experience surprises that make us fearful and happy . but it is unknown whether some of these compounds result in a consistent and differential facial expression . also , our results show that , while almost all the facial expressions of emotion discussed above are visually recognized by people , there is a small set of expressions we seem to be better at recognizing . current results suggest this distinction is biological , since people of different cultural upbringings show the same recognition ability . but we do not yet know why this difference exists , or what its implications are . the open questions discussed above are of high importance in understanding human cognition and behavior and are essential to further our understanding of psychopathologies . for example , as already mentioned , we believe that medical professionals should familiarize themselves with these expressions and their meanings . ideally , physicians working with populations at risk ( eg , psychiatrists , medical doctors working in the emergency room ) would become proficient in recognizing these emotions . this would provide much - needed information , not only to diagnose , but also to detect potential risks ( eg , risk of suicide , depression , ptsd ) . we are currently working on the design of a web - based training system for professionals that will help fill in this gap . a related priority of our research group is to develop computer vision algorithms that can recognize these facial expressions of emotion automatically , so even untrained professionals can detect potential problems . it will also be necessary for medical doctors and researchers to develop protocols on how to interpret and respond to these observed behaviors .

emotions are sometimes revealed through facial expressions . when these natural facial articulations involve the contraction of the same muscle groups in people of distinct cultural upbringings , this is taken as evidence of a biological origin of these emotions . while past research had identified facial expressions associated with a single internally felt category ( eg , the facial expression of happiness when we feel joyful ) , we have recently studied facial expressions observed when people experience compound emotions ( eg , the facial expression of happy surprise when we feel joyful in a surprised way , as , for example , at a surprise birthday party ) . our research has identified 17 compound expressions consistently produced across cultures , suggesting that the number of facial expressions of emotion of biological origin is much larger than previously believed . the present paper provides an overview of these findings and shows evidence supporting the view that spontaneous expressions are produced using the same facial articulations previously identified in laboratory experiments . we also discuss the implications of our results in the study of psychopathologies , and consider several open research questions .

Introduction Compound facial expressions of emotion Consistent and differential action units Spontaneous facial expressions Clinical applications Discussion

the hypothesis is that primates have evolved a sophisticated set of facial muscles that now allows them to convey emotions to observers . to date , most research has focused on the study of six emotions typically seen in most cultures : happiness , surprise , sadness , anger , fear , and disgust . previous research has focused on how the facial expressions of these six emotions are produced . the consistent and differential facial muscle articulations ( typically referred to as action units or aus ) associated with each emotion category have been identified . an au is a set of facial muscle articulations that results in a unique visible image feature . when the frontalis ( pars medialis ) muscle contracts , there is a visible rising of the inner section of the eyebrows , and this is denoted au 1 . the six emotion categories listed in the preceding paragraph are sometimes called basic . by basic it is generally meant that emotions are discrete and have evolved to help us adapt to environmental challenges , ie , the response to a set of events is unique and common to everyone because it is an evolved behavior . with regard to facial expressions of emotion , this means that an automatic response to events of the same emotion category should result in a unique and consistent production ( ie , the aus used to produce that expression are the same across populations but differential between categories ) . in particular , we wish to address the following question : which , and how many , facial expressions of emotion are consistently and differentially produced by humans ? to date , most studies have focused on facial expressions defined by a single component emotion , eg , happy . first , emotion categories need not be associated with a single feeling . while the feeling of joy can be defined as a component emotion ( ie , happiness ) , the feeling of being joyfully surprised can not . experiencing a happily surprising event results in very different behaviors than those experienced when we are happy but not surprised . similarly , happily surprised is different from happily disgusted , even though joy is a common denominator in these emotions . to properly address the question posed in the preceding paragraph ( ie , which and how many facial expressions ) , we need to identify facial constructs that yield a consistent au response pattern across populations and a differential response between categories , regardless of whether these are defined by single or multiple feelings . think about the behavior of someone after hearing a really funny but very disgusting joke ( eg , some readers may be familiar with the 2005 film the aristocrats ) . thus , compound emotions are also emotion categories . both component and compound emotions are discrete entities that identify a unique cognitive state and associated behavior . key to understanding compound emotions is to note that these categories are as important as any other , ie , happily disgusted is a discrete category with a unique facial expression , as is the component emotion fear . herein , we review and expand on our previous studies of compound emotions . in particular , we introduce two new , previously undefined facial expressions of compound emotions , show results on spontaneous expressions , and illustrate the importance of intensity of au activation to define and differentiate these emotion categories . our results support the view that at least 17 compound emotions are associated with unique and differential facial expressions . we then provide a discussion of these results in the study , diagnosis , and treatment of psychopathologies . we called them compound because these emotions are composed of two or more component categories . these 15 compound emotions are : happily surprised , happily disgusted , sadly fearful , sadly angry , sadly surprised , sadly disgusted , angrily fearful , fearfully surprised , fearfully disgusted , angrily surprised , angrily disgusted , disgustedly surprised , appalled , hatred , and awed . figure 1 two additional compound emotions that were not studied in our previous work , but that we will include in our analysis below are : happily fearful and happily sad figure 2 . let us define each of them in more detail : happily surprised : this is an emotion we feel when we receive good or joyful unexpected news / outcomes : eg , when you applied for college , your favorite school accepted you even though you were convinced they would deny your application , or when a group of friends organize a surprise birthday party . happily disgusted : you feel joyful about an event , but repulsion regarding what it entails or what you see , hear , smell , or experience ; eg , when someone tells you a very funny yet disgusting joke , or when you see someone making a funny but repulsive gesture . happily sad : you feel happy about an event that is tainted with sadness ( ie , bittersweet ) ; eg , when your children leave for college you feel happy and proud , but sad because they are leaving the nest . sadly fearful : a fearful event or action makes you sad ; eg , you are threatened by someone emotionally close to you , or you are really disappointed when something nonthreatening is making you fearful . sadly angry : an event or action that makes you angry and leaves you disappointed or sad ; eg , despite all your efforts to help , your son fails an important test . sadly surprised : a surprising event makes you sad ; eg , your favorite soccer team is about to win a championship but loses to a last - minute goal . fearfully surprised : a surprising event makes you fearful ; eg , while you are driving , a deer you had not seen crosses the road and almost causes a collision , or while hiking in the rocky mountains you suddenly see a mountain lion 15 metres ahead . fearfully disgusted : a fearful event or action that is also morally or sensorially unpleasant ; eg , an accident results in a life - threatening wound that is visually revolting . angrily surprised : a surprising event or action makes you angry ; eg , unexpectedly and without provocation , a friend insults you . angrily disgusted : an event or action that makes you angry and is morally or sensorially unpleasant ; eg , someone unjustly accuses you of doing something you did not do and that violates your moral code , or an elite athlete loses to a competitor using performance - enhancing drugs . appalled : horrified or angry and morally disgusted ; eg , you are appalled at the incompetence of an expert or colleague . hatred : extreme dislike or ill will ; eg , racial or sexual hatred . awed : a feeling of reverential respect including the simultaneous feeling of fear and wonder ; eg , you look in awe at the vastness and the unknown in the jungle at night . furthermore , the facial expressions of these emotion categories might have evolved to help us better solve specific behavioral or social problems . for example similarly , compound emotions such as awe might serve as a social glue in large groups of people through communal events , such as those involving the arts , natural phenomena , and religion . first , as can be seen from the list of emotions given above , some compound emotions are identified by a single word in english . for example , hateful and appalled are defined as the compound feeling of anger and disgust , with the emphasis on anger when hateful and on disgust when appalled . awe is the compound feeling of fear and wonder ( surprise ) , with the emphasis on the latter ( eg , horripilation in awe is believed to be a residual behavior of fear ) . the fact that these compound emotions are defined as single words does not mean they are more or less relevant than the others . this is a characteristic of the english language and may not be the same in other languages . for example , in albanian , awe is described as an abrupt or sudden fear and , hence , requires multiple words to define it . the same holds true for compound words : not all languages use two or more words to define them . for example , in mandarin chinese , happily surprised is written as a single word . finally , emotions associated with a single feeling may be named using one or more words . for instance , in igbo ( a language spoken in the southeastern region of nigeria ) , emotions such as happy are generally described using multiple words . it is important to note that cognitive categories need not match language ; that is , a compound emotion may be given by a single word in one 's language , while a component emotion may be described with multiple words . the important difference is in the simultaneous feelings one experiences , not how the emotion category is written . when we experience a single feeling ( eg , joy ) , we will talk about component emotions ( eg , happy ) . when we experience multiple feelings ( eg , joy and surprise ) , we will talk about compound emotions ( eg , happily surprised ) . as mentioned in the introduction of this paper , an emotion that is externalized should have a facial expression that is produced with the same au combination by people of different cultures but differential from those au patterns employed to display other emotion categories . we thus wish to know whether compound emotions are displayed with unique and differential facial expressions . in other words , when , in a situation that yields a compound emotion , is human behavior consistent and differential ? we denote the six emotion categories studied in the past ( ie , happiness , surprise , anger , sadness , disgust , and fear ) component emotions . the other 17 emotion categories introduced in the preceding section are denoted as compound emotions . the very first thing we wish to know is if these 17 compound emotions yield facial expressions that are consistent across people but differential between categories . to study this , we analyzed the facial expressions of compound emotions made by people of different cultures . specifically , we identified the aus that are consistently used by a large majority of people ( > 70% ) . as can be seen in this table , all categories yield a unique facial expression . this is to be expected , because these three emotion categories include the same component categories the simultaneous feeling of anger and disgust . angrily disgusted , we feel equally angry and disgusted , whereas in hatred the emphasis is on anger and in appalled the emphasis is on disgust . as can be seen in table i , this results in slightly different expressions . in angrily disgusted there is the consistent use of three aus ( 4 , 10 , and 17 ) with aus 7 and 9 playing a secondary role . another way that compound categories of closely related emotional states are differentiated is in the intensity of the activation of their aus . as mentioned earlier in this paper , aus correspond to visual image changes caused by the activation of a specific set of facial muscles . when the contraction of the associated muscles is stronger , we generally observe a large image change . typically , we use five values to denote the intensity of activation of an au . they are denoted , from smallest to largest intensity , a through e. figure 3 shows the percentage of times people use a given intensity ( or larger ) in each au for each of the 23 emotion categories . as is made clear by this figure , the au patterns of activation used by people to express each emotion are not only different ; the intensities of au activation are also distinct this is important because it further illustrates the many ways in which the expressions of different discrete emotion categories are consistently produced by people of different cultures . in figure 3 , we also see that the aus used to express a compound emotion are consistent with the aus used to express its component ( subordinate ) categories . that is , the resulting au pattern is unique to that compound emotion ( a fact that allows us to differentiate this facial expression from the others ) , but the resulting expression is also consistent with the au patterns used to display its component categories . note how some aus are emphasized more in the compound than in the subordinate categories ( ie , intensity of production is increased ) , whereas others are deemphasized . this difference in production further facilitates the visual distinction of each facial expression while leaving the active set of aus consistent across compound and subordinate categories . behavioral experiments by our group show that people are quite good at visually discriminating these facial expressions of emotion . our studies have tested people 's ability to name and visually discriminate these expressions of emotion . for instance , in a cognitively taxing task , participants were shown an image of one of the component or compound facial expressions and given a list of 21 possible labels ( ie , happy , happily surprised , sad , sadly angry , etc ) . westerners and east asian participants were able to correctly name 19 of the emotion categories introduced in the present paper . most importantly , the results in these two groups ( westerners and east asians ) were statistically identical . in another experiment , participants were instructed to indicate by key press whether the two images shown on the screen belonged to the same emotion category or not . one group is hypothesized to play a major role in cognitive and/or social tasks , and are thus more readily recognized . the other expressions are also universally recognized by people of different cultures , but seem to play a less central role in our daily cognitive and social tasks . our group is currently working on trying to better understand these differences and its implications in cognition , behavior , and psychopathologies . thus far , we have presented studies of facial expressions of emotion collected in the laboratory . next , we wish to know if spontaneous expressions are produced with the same aus identified in the laboratory . this is important because the neural systems involved in the production of posed and spontaneous expressions are believed to differ . to answer this question our search was based on keywords ( eg , happy , happily disgusted , happy and disgust , etc ) . the guardian ) and television channel sites ( eg , cnn , cbs news ) and also used google images to identify these expressions . our search started with a qualitative analysis . when an image was visually identified as appearing to express one of the 17 compound emotions listed above by one of the two authors we found that the au pattern of activation of spontaneous expressions is identical to those seen in the laboratory . we are so accustomed to interacting with others nonverbally that replicating this in the laboratory does not pose a major problem . second , when collecting images in the laboratory , we were very careful to instruct the subjects on how to provide as naturalistic an expression as possible . then , participants had the opportunity to practice in front of a mirror to see if their expression matched what they intended before pictures were taken . this procedure yielded the same results we have now observed in spontaneous expressions , further validating the results summarized in table i and figure 3 . when these occurrences are transitory ( hours or a few days ) , they change our mood . involuntary long - lasting effects may develop in psychopathologies , for example , clinical depression or post - traumatic stress disorder ( ptsd ) , with genetics believed to be a contributing factor in this transition . other psychopathologies are believed to be developmental , as is the case in autism spectrum disorder ( asd ) . understanding how many facial expressions of emotion neurotypicals produce and recognize is essential to further our study of these disorders , as well as for the development of coping mechanisms and treatment options . for instance , ptsd is generally diagnosed after lengthy interviews with a knowledgeable psychiatrist who usually administers the clinician - administered ptsd scale ( caps ) . caps diagnosis is based on the presence of several symptoms described in the diagnostic and statistical manual of mental disorders ( dsm ) rather than pathophysiologically . to be able to resolve this issue , we need to better understand the differences in production and visual recognition of facial expressions of emotion . our general hypothesis is that pathophysiological distinctive disorders result in a different production and/ or visual interpretation of facial expressions of emotion . before we can fully address this hypothesis though , we need to identify all component and compound facial expressions of emotion . to date , we have identified 23 facial expressions of emotion , but there could be more . for the time being , we believe physicians and medical personnel should learn to quickly identify the known 23 facial expressions of emotion in patients . as we have seen above , we are all quite good at visually identifying these facial constructs , but we believe medical professionals should be proficient at this task . to learn to identify these expressions , one can pay close attention to the results given in the table and figures above . our research group is also developing computer algorithms that can recognize these facial expressions automatically . this technology would free the medical professional from having to analyze the face and would eliminate or , at least facilitate , training . all that will be needed is a camera and computer , and the system will provide real - time information to the physician . our current system recognizes expressions of emotion with > 75% accuracy , which is better than untrained individuals can do . improvements on this technology are sure to revolutionize clinical practice in the next few years . advances in face detection , for instance , are fueling improvements in the recognition of facial expressions . another likely difference between the production of facial expressions of emotion in neurotypicals and individuals with psychopathologies is variations in the intensity of activation of aus . for example , are aus in sadness ( including its compounds , eg , sadly angry , sadly surprised , etc ) displayed with larger or smaller au intensity in clinical depression ? characterizing these differences will allow us to develop protocols for evaluating the automatic annotations given by the computer software outlined in the preceding paragraph . past research has shown differences in the visual recognition of facial expressions of component emotions , but there is not yet any research that has studied the perception of compound emotions in the clinical population . this is likely to be a productive area of research in clinical and translational medicine . the set of six emotions used in the research studies listed above is likely to be insufficient to describe all psychopathologies defined in the dsm . there is limited variability that can be readily and consistently observed in the facial expressions of joy , surprise , sadness , anger , disgust , and fear across psychopathologies . by studying a much larger number of emotions ( ie , variables ) , for example , clinical depression might result in an increased production of compound expressions with a sad component ( eg , sadly angry , sadly fearful , sadly disgusted ) , even in the absence of additional facial expressions of sadness . these observations can prove invaluable to researchers , and can serve as hypotheses for future studies . this would require that medical professionals become proficient in the visual interpretation of all facial expressions of emotion . this may seem contradictory at first because happiness and fear seem polar opposites , yet these are common compound emotions people experience . our research shows that the facial expressions that accompany these internally felt emotions are consistent across people and differential between emotion categories . furthermore , the associated facial constructs seem to be universally visually recognized by observers , even in challenging conditions . in our previous work we defined 21 compound facial expressions of emotion . herein , we have further extended this to 23 ( table i ; figures 1 , 2 ) . much still needs to be done to fully understand compound emotions and their facial expressions , however . for instance , we do not yet know how many emotion categories there are . specifically , if contempt ( or others ) were found to also be consistently produced by people of distinct cultures , then many more compound categories would be possible . for example , it is likely that in some circumstances one could feel fearful , surprised and happy , eg , while riding in one of the attractions in an amusement park , it is common to experience surprises that make us fearful and happy . but it is unknown whether some of these compounds result in a consistent and differential facial expression . also , our results show that , while almost all the facial expressions of emotion discussed above are visually recognized by people , there is a small set of expressions we seem to be better at recognizing . current results suggest this distinction is biological , since people of different cultural upbringings show the same recognition ability . the open questions discussed above are of high importance in understanding human cognition and behavior and are essential to further our understanding of psychopathologies . for example , as already mentioned , we believe that medical professionals should familiarize themselves with these expressions and their meanings . ideally , physicians working with populations at risk ( eg , psychiatrists , medical doctors working in the emergency room ) would become proficient in recognizing these emotions . this would provide much - needed information , not only to diagnose , but also to detect potential risks ( eg , risk of suicide , depression , ptsd ) . we are currently working on the design of a web - based training system for professionals that will help fill in this gap . a related priority of our research group is to develop computer vision algorithms that can recognize these facial expressions of emotion automatically , so even untrained professionals can detect potential problems .

pseudouridine ( ) is the most abundant post - transcriptional rna base modification and the enzymes responsible for this isomerization of uridine and the positions modified in the rnas , are conserved ( 14 ) . this modification is important in the structure and function of small rnas ( 511 ) , including small nuclear rnas ( snrnas ) , cofactors in pre - mrna splicing ( 1216 ) . one pathway is involved in the formation of in rrna and snrna and requires dyskerin or its homologs ( cbf5p in yeast for example ) and rnp cofactors [ most often h / aca small nucleolar ribonucleoprotein particles ( snornps ) ] that enable one enzyme to recognize many different sites for modification on different substrates ( 1725 ) . the other pathway for formation employs site - specific synthases that require no cofactors to recognize and modify the rna substrate . a number of enzymes have been identified in this pathway and are grouped in six families that all share a common basic structure ( 4 ) . guided pathway has received a great deal of attention because of its similarity to aspects of rna editing , but the site - specific pseudouridine synthases accomplish the same task , on more substrates , with fewer tools , arguably making them more interesting from an enzymatic perspective . one of the most studied site - specific enzymes is pseudouridine synthase 1 ( pus1p ) ( 2635 ) . pus1p modifies uridines in several regions of many different trnas ( 27,33,34 ) and one position in yeast u2 snrna ( 36 ) . it is a transcriptional coactivator in nuclear receptor signaling , modifying steroid receptor activator rna ( sra ) ( 37,38 ) , and when mutated or missing , leads to the disorder mitochondrial myopathy and sideroblastic anemia with lactic acidemia ( mlasa ) ( 3942 ) . in vivo and in vitro pus1p modifies positions 1 , 26 , 27 and 28 in many trnas and positions 34 and 36 in the anticodons of certain intron - containing trnas ( 2628,33 ) . in addition , yeast pus1p has been shown to modify positions 65 and 67 in vivo ( 34 ) and mouse ( mpus1p ) and human pus1p ( hpus1p ) modify position 30 in trnas , whereas yeast pus1p does not ( 29,33 ) . the presence of the intron in yeast pre - trna(uau ) is required for the formation of at positions 34 and 36 in the anticodon by pus1p in vitro ( 26,34 ) . the intron allows for the formation of a stem that contains the uridines to be modified ( 26,34,43 ) . residues that are modified in trna by pus1p are part of a base - paired stem , suggesting rna secondary structure is important for recognition by pus1p ( 26,27,3335 ) . even with all of the studies on pus1p , the only sequence consensus ( not requirement ) that has been proposed for pus1p is the presence of a pyrimidine nucleotide 3 of the site of modification ( 44 ) . interestingly , yeast pus1p has been shown to modify position 44 on yeast u2 snrna , a position that is considered to be in a non - base paired region of u2 snrna ( 36 ) . when mouse pus1p is expressed in a pus1 deleted yeast strain , the mouse enzyme can also modify yeast u2 snrna at the same position ( 33 ) . however , the modification of u2 snrna in caenorhabditis elegans does not require pus1p , since the loss of the pus-1 gene product in worms results in the absence of at known pus1p modification sites on trna , but has no effect on the modification of u2 snrna or other snrnas ( 28,45 ) . however , the structure of a member of the same synthase family , bacterial trua , in the presence of trna substrates , has been revealed ( 46,47 ) . trua modifies uridines at positions 3840 in many trnas and the equivalent activity in eukaryotes is associated with pseudouridine synthase 3 , another member of the trua family [ pus3p ; ( 48,49 ) ] . the substrate trna binds across a dimer of trua ( 46,47 ) . the anticodon stem - loop ( asl ) of the trna , as well as the dihydrouridine ( d ) stem and the elbow where the d and tc loops interact , contact the synthase ( 47 ) . trua did not interact with the variable ( v ) stem - loop or the acceptor stem of the trna substrate ( 47 ) . in order to begin to understand how pus1p can modify several positions in trnas and also modify other rnas such as u2 snrna and sra , we undertook an investigation of the sequence and structural requirements for hpus1p activity on human trna . the activity of hpus1p with wild - type and point and deletion mutants of the trna substrate indicates there are structural requirements in the formation of at position 28 in trna . a minimum level of base pairing in the asl ( the location of the modification ) and the presence of an additional 3 stem - loop , constitute the minimal substrate for hpus1p . even when all nucleotides in the asl stem other than u28 were changed in a single minimal substrate , but base pairing was retained , a near wild - type level of modification was observed . site - directed mutagenesis of human trna ( uga ) was performed with a quikchange ii kit ( stratagene ) , utilizing primers containing the mutations and the phts plasmid [ human trna(uga ) ; gift of h. gross ( 50 ) ] to create the mutants . the resulting constructs have a t7 rna polymerase promoter at the 5-end of the trna gene and a bstn1 site at the 3-end of the gene . plasmid dna digested with bstn1 will yield trnas with cca at the 3-terminus after transcription ( 29,50 ) . the primers used for mutagenesis are given below with just the forward primers listed ; the reverse primers are the inverse complement . the forward primer for : 27c was 5cgagtggttaaggcgctggacttgaaatcca,27 g was 5cgagtggttaaggcggtggacttgaaatcca,28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42g,29c was 5gagtggttaaggcgatcgacttgaaatccatt,30c was 5gtggttaaggcgatgcacttgaaatccattg,31c was 5gtggttaaggcgatggccttgaaatccattgg,39 g was 5cgatggacttgaaagccattggggtc,40 g was 5gatggacttgaaatgcattggggtctc,41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template,42c was 5ggacttgaaatcccttggggtctcc,42 g was 5ggacttgaaatccgttggggtctcc,43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template,30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template,31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template, d stem - loop was 5gtagtcgtggcgatggacttgaaatcc, d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . 27c was 5cgagtggttaaggcgctggacttgaaatcca , 27 g was 5cgagtggttaaggcggtggacttgaaatcca , 28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42 g , 29c was 5gagtggttaaggcgatcgacttgaaatccatt , 30c was 5gtggttaaggcgatgcacttgaaatccattg , 31c was 5gtggttaaggcgatggccttgaaatccattgg , 39 g was 5cgatggacttgaaagccattggggtc , 40 g was 5gatggacttgaaatgcattggggtctc , 41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template , 42c was 5ggacttgaaatcccttggggtctcc , 42 g was 5ggacttgaaatccgttggggtctcc , 43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template , 30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template , 31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template , d stem - loop was 5gtagtcgtggcgatggacttgaaatcc , d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . the digested plasmid dnas were transcribed with t7 rna polymerase in the presence of 25 ci [ 5-h]-utp ( 11.1 or 23.9 ci / mmol , moravek ) or 25 ci [ 5,6-h]-utp ( 38 ci / mmol , moravek ) with 100 m cold utp and 1.0 mm , non - labeled , atp , gtp and ctp ( 51 ) . additional rna substrates were synthesized using a sense t7 dna oligonucleotide , an antisense template oligonucleotide and high concentration t7 rna polymerase , as described ( 52,53 ) . the oligodeoxynucleotides used for this type of synthesis are listed below , note that the oligodeoxynucleotides that serve as the template for synthesis are antisense and have antisense t7 primer sequence at the 3-end . the template for the : asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta, tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta, v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta.asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta , v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta . asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a g , allows for efficient synthesis of rnas in vitro . the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . before incubation with hpus1p , the rna substrates were heated to 78c for 3 min and allowed to cool slowly to at least 37c ( 3040 min ) . substrate reactions with the synthases were carried out in 50 mm ammonium chloride , 5 mm dtt , 25 mm tris ( ph 7.5 ) and 1 mm mgcl2 . the amount of synthase used was ~200 ng at 37c for the times indicated . norit a charcoal binds all of the h counts in the sample except those that have been released to water in the process of forming . the released counts are separated from the charcoal - bound counts using a spin - x column ( costar ) , the filtrate is mixed with scintillation fluid , and the released h counts are measured on a scintillation counter . the equilibrium binding assays were carried out as described ( 32 ) using hpus1p concentrations of 0 , 50 , 100 , 200 , 400 and 800 nm in 50 l reactions , with the h - labeled rna substrates at 500 pm . the rnas were synthesized as described above except that the cold utp concentration in the synthesis reaction was 250 m . a slot - blot apparatus ( biorad ) was used for filtration of the samples , the resulting filters were air dried , cut into appropriate pieces and counted in scintillation fluid . site - directed mutagenesis of human trna ( uga ) was performed with a quikchange ii kit ( stratagene ) , utilizing primers containing the mutations and the phts plasmid [ human trna(uga ) ; gift of h. gross ( 50 ) ] to create the mutants . the resulting constructs have a t7 rna polymerase promoter at the 5-end of the trna gene and a bstn1 site at the 3-end of the gene . plasmid dna digested with bstn1 will yield trnas with cca at the 3-terminus after transcription ( 29,50 ) . the primers used for mutagenesis are given below with just the forward primers listed ; the reverse primers are the inverse complement . the forward primer for : 27c was 5cgagtggttaaggcgctggacttgaaatcca,27 g was 5cgagtggttaaggcggtggacttgaaatcca,28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42g,29c was 5gagtggttaaggcgatcgacttgaaatccatt,30c was 5gtggttaaggcgatgcacttgaaatccattg,31c was 5gtggttaaggcgatggccttgaaatccattgg,39 g was 5cgatggacttgaaagccattggggtc,40 g was 5gatggacttgaaatgcattggggtctc,41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template,42c was 5ggacttgaaatcccttggggtctcc,42 g was 5ggacttgaaatccgttggggtctcc,43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template,30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template,31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template, d stem - loop was 5gtagtcgtggcgatggacttgaaatcc, d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . 27c was 5cgagtggttaaggcgctggacttgaaatcca , 27 g was 5cgagtggttaaggcggtggacttgaaatcca , 28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42 g , 29c was 5gagtggttaaggcgatcgacttgaaatccatt , 30c was 5gtggttaaggcgatgcacttgaaatccattg , 31c was 5gtggttaaggcgatggccttgaaatccattgg , 39 g was 5cgatggacttgaaagccattggggtc , 40 g was 5gatggacttgaaatgcattggggtctc , 41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template , 42c was 5ggacttgaaatcccttggggtctcc , 42 g was 5ggacttgaaatccgttggggtctcc , 43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template , 30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template , 31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template , d stem - loop was 5gtagtcgtggcgatggacttgaaatcc , d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . the digested plasmid dnas were transcribed with t7 rna polymerase in the presence of 25 ci [ 5-h]-utp ( 11.1 or 23.9 ci / mmol , moravek ) or 25 ci [ 5,6-h]-utp ( 38 ci / mmol , moravek ) with 100 m cold utp and 1.0 mm , non - labeled , atp , gtp and ctp ( 51 ) . additional rna substrates were synthesized using a sense t7 dna oligonucleotide , an antisense template oligonucleotide and high concentration t7 rna polymerase , as described ( 52,53 ) . the oligodeoxynucleotides used for this type of synthesis are listed below , note that the oligodeoxynucleotides that serve as the template for synthesis are antisense and have antisense t7 primer sequence at the 3-end . the template for the : asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta, tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta, v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta.asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta , v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta . asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a g , allows for efficient synthesis of rnas in vitro . the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . before incubation with hpus1p , the rna substrates were heated to 78c for 3 min and allowed to cool slowly to at least 37c ( 3040 min ) . substrate reactions with the synthases were carried out in 50 mm ammonium chloride , 5 mm dtt , 25 mm tris ( ph 7.5 ) and 1 mm mgcl2 . the amount of synthase used was ~200 ng at 37c for the times indicated . norit a charcoal binds all of the h counts in the sample except those that have been released to water in the process of forming . the released counts are separated from the charcoal - bound counts using a spin - x column ( costar ) , the filtrate is mixed with scintillation fluid , and the released h counts are measured on a scintillation counter . the equilibrium binding assays were carried out as described ( 32 ) using hpus1p concentrations of 0 , 50 , 100 , 200 , 400 and 800 nm in 50 l reactions , with the h - labeled rna substrates at 500 pm . the rnas were synthesized as described above except that the cold utp concentration in the synthesis reaction was 250 m . a slot - blot apparatus ( biorad ) was used for filtration of the samples , the resulting filters were air dried , cut into appropriate pieces and counted in scintillation fluid . as an initial investigation into the substrate requirements for hpus1p , it was important to choose a known pus1p substrate trna that has only one site modified by the enzyme ( 29,35 ) , to simplify the analysis of the activity . a trna that is typical in structure and has a v stem - loop that was > 6 nt , was preferred in order to be able to test the contribution of all structural components to substrate recognition by hpus1p . in addition , a trna with traditional watson - crick base - pairing in the asl stem was desired . the trna sequence database , trnadb ( 56,57 ) , was consulted to compile consensus sequences for the asl stem where a or u is found at position 28 in sequenced trnas from the fungi and metazoan group in trnadb . at least 36 trna sequences were compiled to generate each consensus shown in figure 1b . first , with a at position 28 , g30c40 is the most conserved base pair ( > 90% conserved ) but when a u is found at position 28 , this sequence is not as well conserved . therefore , one might expect that disruption of this base pair or replacement with the inverse ( c30g40 ) will affect modification . second , for sequenced trnas that have a at 28 , the preferred 2743 bp is u27a43 , whereas with those trnas with a u at 28 , the preferred base pair is c27g43 . third , a a31u39 base pair is more common in those asl stems that have a at position 28 than those with a u at position 28 . finally , the base pairs 2941 and 3139 , below the 2842 bp , are watson - crick only but the rest of the base pairs in the stem can have g - u pairing in either consensus . in the selection of a trna , a good match to this consensus was preferred , and human trna(uga ) meets most of the criteria . the only difference is that this trna has an a27u43 base pair instead of a u a base pair at that position ( figure 1a ) . ( a ) the sequence and proposed secondary structure of human trna(uga ) ( 50 ) . the major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the variable stem - loop . the single site of pus1p modification , position 28 , is boxed . ( b ) consensus sequences for sequenced trnas from the fungi and metazoa group in the trnadb ( 57 ) that have a or a u at position 28 . a solid line between nucleotides indicates watson - crick pairs only and a dash line indicates g u pairs ( < 50% ) are also found at this position . ( a ) the sequence and proposed secondary structure of human trna(uga ) ( 50 ) . the major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the variable stem - loop . the single site of pus1p modification , position 28 , is boxed . ( b ) consensus sequences for sequenced trnas from the fungi and metazoa group in the trnadb ( 57 ) that have a or a u at position 28 . the figure is presented in the output style from trnadb . a solid line between nucleotides indicates watson - crick pairs only and a dash line indicates g u pairs ( < 50% ) are also found at this position . the point mutations in the asl of full - length trna are diagrammed in figure 2h and were obtained by site - directed mutagenesis of the plasmid containing wild - type human trna [ phts , gift from h. gross ; ( 50 ) ] followed by in vitro transcription of linear templates ( see materials and methods section ) . time course analyses of the in vitro activity of the substrate rnas and hpus1p are presented in figure 2 ( panels a g ) , with the same wild - type trna curve in all panels ( figure 2a g ) . the formation of in this trna by hpus1p is reduced to background levels ( 0.02 mol /mol rna ) if the u at position 28 is changed to c and a42 is changed to g to preserve base pairing ( table 2 , second experiment ) . this shows that the only u converted to by hpus1p is the one at position 28 . the panels in figure 2 each contain the wild - type trna curve and either two or three curves generated with trna mutant substrates . this style allows for a simplification of the presentation and for the focus in most panels to be on 1 bp at a time . figure 2.time course experiments with single and double mutants in the asl of human trna . ( a g ) , h - labeled rnas were incubated with hpus1p for the times indicated and the amount of formed assayed . each panel has the same time course of wild - type ( wt ) trna values . each mutant is plotted separately and standard deviation ( sd ) bars are displayed for all time points . ( h ) diagram of asl point mutations made in the context of full - length trna that are presented in panels a ( a g ) , h - labeled rnas were incubated with hpus1p for the times indicated and the amount of formed assayed . each panel has the same time course of wild - type ( wt ) trna values . each mutant is plotted separately and standard deviation ( sd ) bars are displayed for all time points . ( h ) diagram of asl point mutations made in the context of full - length trna that are presented in panels a when the a at 27 is changed to c or g ( figure 2a ) , the levels of formed are not significantly different from wild - type trna . the curve that results when the u at 43 is changed to g is distinct from the wild - type trna curve but the difference is not statistically significant because of the relatively large error bars ( sd ) . mutation of the a at the position across from u28 to either c or g has little effect on time course of formed ( figure 2b ) . even though the a at position 42 is preferred ( figure 1b ) , the formation of 28 does not require canonical or even u g base pairing , something unanticipated given the consensus sequence ( figure 1b ) . when g29 is changed to c or c41 is changed to g ( figure 2c ) , there is a significant decrease in formation for both mutations in the formation of at 28 . however , when the base pair is reformed , but is now c - g in the case of the double mutant 29c/41 g , the curve obtained is indistinguishable from that of wild - type trna ( figure 2f ) . this introduction of a base - paired pyrimidine 3 to u28 did not enhance the formation of in the substrate , an expectation if a pyrimidine is preferred at position 29 , as suggested in the introduction . the consensus sequence shown in figure 1b revealed the g30c40 base pair to be highly conserved in the putative pus1p substrates with a at position 28 . it was expected that disruption of this base pair would affect the formation of by hpus1p . in fact , the 30c and 40 g mutants have significantly lower amounts of formed than wild - type trna ( figure 2d ) . if the base pair is reformed but inverted , as with the 30c/40 g double mutant , the time course of formed is the same as that of wild - type trna ( figure 2f ) . this result argues against the requirement for a g at position 30 , even though the 28 consensus suggests it ( figure 1b ) . when the base pair at a31u39 is disrupted by either the 31c or 39 g point mutations , the results are interesting . the curve of formation seen with the 31c substrate is significantly different from wild - type trna ( figure 2e ) , but with the 39 g mutant the curve mimics that seen with wild - type trna ( figure 2e ) . reforming the base pair with the 31c/39 g double mutant results in a curve that matches that of wild - type trna ( figure 2f ) . the only difference between the 28 consensus stem ( figure 1b ) and the stem of the trna ( figure 1a ) is the 2743 bp , which is a u in trna . the formation curves for the 27c , 27 g and 43 g mutants were not significantly different from wild - type trna ( figure 2a ) . this would suggest that the enzyme does not require a base pair at 2743 , and may in fact be indifferent to the presence of a base pair . that statement was supported by the formation curve seen with the 27g/43 g double mutant , which was not significantly different from the curve seen with wild - type trna ( figure 2 g ) , even though there is no canonical base pair formed . the results with the double mutant 27c/43 g , which would form a stronger base pair than the wild - type a u , are also not significantly different from wild - type trna ( figure 2 g ) even though this mutant now mimics the u28 consensus in figure 1b . equilibrium dissociation constants ( kd ) were determined for wild - type trna and the single- and double - mutant substrates found in the panels of figure 2 . the means and standard deviations of two separate kd determinations are presented in table 1 and that of wild - type trna ( 240 nm ) agrees with a previous determination of the kd ( 250 nm ) of hpus1p and yeast pre - trna ( 29 ) . all of the mutants have a higher mean kd than wild - type , with all but 41 g and 43 g > 300 nm . there is not a correlation between the formation levels and the kds for the mutants . it is interesting to note that a trna that is not a substrate for hpus1p , such as the 28c/42 g double mutant , binds to the enzyme with a mean kd of 332 nm . this was also as seen previously with yeast pus1p and a non - substrate trna ( 31 ) . table 1.equilibrium dissociation constants ( kd ) of hpus1p with various rnasrna substratekd mean ( sd)wild - type trna240 ( 14 ) nmsingle point mutants in trna 27c328 ( 46 ) nm 27g370 ( 14 ) nm 29c408 ( 11 ) nm 30c335 ( 120 ) nm 31c358 ( 53 ) nm 39g380 ( 28 ) nm 40g305 ( 49 ) nm 41g282 ( 18 ) nm 42c360 ( 57 ) nm 42g338 ( 46 ) nm 43g248 ( 32 ) nmdouble mutants in trna 27c/43g302 ( 39 ) nm 27g/43g375 ( 7 ) nm 28c/42g332 ( 18 ) nm 29c/41g328 ( 74 ) nm 30g/40c345 ( 21 ) nm 31c/39g315 ( 92 ) nmlisted are the means and standard deviations ( sd ) of two separate binding experiments of hpus1p and the substrates listed ( see materials and methods section for details ) . equilibrium dissociation constants ( kd ) of hpus1p with various rnas listed are the means and standard deviations ( sd ) of two separate binding experiments of hpus1p and the substrates listed ( see materials and methods section for details ) . in order to identify additional recognition elements of the hpus1p substrate , stem or stem - loop deletions of , and mini - substrates derived from trna , were tested in the formation assay . these mutant substrates were generated either by site - directed mutagenesis of phts and subsequent in vitro transcription or by in vitro transcription using oligodeoxynucleotide templates [ see materials and methods section ; ( 53 ) ] . it is understood that , with the deletion of whole stem - loops or the creation of mini - substrates , the tertiary structure of the entire trna is affected , but surprisingly , many of these mutants can function as substrates for hpus1p . the results for 120 min incubations are listed in table 2 relative to the activity seen with wild - type trna . if the d stem - loop is missing in the substrate , there is no decrease in hpus1p activity at position 28 . if the u at position 28 in the d stem - loop mutant is changed to c ( and the a at 42 is changed to g ) so that can not be formed at position 28 , there is essentially no formation ( 2% of wild - type trna formation , see table 2 ) . this result indicates the level of formation seen with the d stem - loop mutant is not due to formation at an additional site created by an altered secondary structure in the deletion mutant . if the tc stem - loop is missing from the trna , there is a significant reduction in the formation of at u28 ( 15% of wild - type trna levels , table 2 ) but the reduction is not as substantial when the v stem - loop is missing ( 67% of wild - type , table 2 ) . table 2.activity of hpus1p on trna(uga ) point and deletion mutants and mini - substratesrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type human trna0.60 ( 0.08)100 ( 13) tc stem - loop ( deleted nucleotides 5973)0.09 ( 0.05)15 ( 8) variable stem - loop ( deleted nucleotides 4556)0.40 ( 0.08)67 ( 13)wild - type human trna0.43 ( 0.07)100 ( 16)28c/42 g ( no formation expected)0.02 ( 0)5 ( 0) d stem - loop ( deleted nucleotides 1124)0.44 ( 0.16)102 ( 37) d stem - loop with 28c/42g0.01 ( 0.01)2 ( 2)wild - type human trna0.68 ( 0.02)100 ( 3)asl stem loop alone ( includes nucleotides 2643)0.02 ( 0.01)3 ( 1)asl & d stem - loops ( includes nucleotides 1043)0.07 ( 0.01)10 ( 1)asl & v stem - loops ( includes nucleotides 2647)0.05 ( < 0.01)7 ( 1)asl , d , & v stem - loops ( includes nucleotides 1047)0.14 ( < 0.01)21 ( 1)asl , v , & tc stem - loops ( includes nucleotides 2665)0.48 ( 0.04)71 ( 6)asl , d , v , & tc stem - loops ( includes nucleotides 765)0.64 ( 0.16)94 ( 24)activities are from 2-h incubations , an average of two assays , with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type trna(uga ) with the standard deviation ( sd ) also reported as percent of wild - type . these results are grouped as separate experiments with a separate wild - type control for each experiment . activity of hpus1p on trna(uga ) point and deletion mutants and mini - substrates activities are from 2-h incubations , an average of two assays , with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type trna(uga ) with the standard deviation ( sd ) also reported as percent of wild - type . these results are grouped as separate experiments with a separate wild - type control for each experiment . when a mini - substrate of just the asl stem - loop was incubated with hpus1p , formation was at background levels for 2 h of incubation ( 3% of wild - type levels , table 2 ) . in addition , if either the d stem - loop or the v stem - loop was combined with the asl stem - loop , there was little increase in the amount of formed ( 10% and 7% , respectively , of the formation seen in 120 min with wild - type trna , table 2 ) . this implies that adding a single stem - loop to the 5 or 3 side of the asl stem - loop does not constitute an efficient substrate for hpus1p . if both the d and v stem - loops are added to the asl stem - loop there is a noticeable increase in formed but it is still low ( 21% ) compared to wild - type trna . when the tc stem - loop was combined with the v and asl stem - loops , there was a substantial level of hpus1p activity ( 71% of wild - type trna levels of ) . with just the acceptor stem missing from trna ( asl , d , v and tc stem - loops present ) , the activity is essentially the same as with wild - type trna ( 94% of wild - type formation at 120 min , table 2 ) . these results suggest that the secondary structure of the trna is very important for the modification at position 28 , and a minimal substrate could be created by simply connecting the asl and tc stem - loop as diagrammed ( figure 3a ) . the numbering on the diagram is consistent with full - length trna to allow comparisons with the results reported in figure 2 and table 2 . the level of formation at position 28 in this minimal substrate ( wild - type asl & tc mini - substrate ) was ~0.60 mol /mol rna after 2 h of incubation , a substantial level of modification ( table 3 ) . a mini - substrate incorporating a 28c/42 g double mutation was used in a separate experiment with little or no formation observed ( table 3 ) , providing evidence that in this mini - substrate , the u at position 28 is the only one being converted to , as expected . ( a ) predicted structure of the asl & tc stem loop mini - substrate , retaining the numbering found on the full - length trna and indicating the position of the formed . the structure for the y for y r for r stem mini - substrate mutant and the c28 mutant are also shown . ( b ) diagram of the possible base pairing in the asl stem mutants in the mini - substrate listed in table 3 . the activities in percent of the level observed with wild - type asl & tc stem - loop mini - substrate without the sd are listed above the diagrams . table 3.activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutantsrna substratemole /mole rna ( sd)mole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.60 ( 0.11)0.54 ( 0.16)100 ( 18 ) ( 30)28c/42 g ( no u at expected site of formation)0.01 ( 0.01)2 ( 2)27c/42c0.48 ( 0.04)80 ( 7)27c/39g0.24 ( 0.05)40 ( 8)27c/29c/39g0.10 ( 0.03)17 ( 5)27c/29c/42c0.04 ( 0.01)7 ( 2)27c/39g/40g<0.01 ( 0.01)2 ( 2)27c/29c/39g/40g0.02 ( 0.03)3 ( 5)27c/29c/39g/40g/42c0.02 ( < 0.01)3 ( 2)55 & 560.37 ( 0.10)62 ( 17)55580.17 ( 0.06)28 ( 10)gcaa inserted between 55 and 560.46 ( 0.04)77 ( 7)wild - type asl & tc stem - loop mini - substrate0.30 ( 0.04)100 ( 13)ggacg starting at 610.22 ( 0.03)73 ( 10)aagcuua in tc loop0.34 ( 0.04)113 ( 13)activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . ( a ) predicted structure of the asl & tc stem loop mini - substrate , retaining the numbering found on the full - length trna and indicating the position of the formed . the structure for the y for y r for r stem mini - substrate mutant and the c28 mutant are also shown . ( b ) diagram of the possible base pairing in the asl stem mutants in the mini - substrate listed in table 3 . the activities in percent of the level observed with wild - type asl & tc stem - loop mini - substrate without the sd are listed above the diagrams . activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutants activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . concentrating on the asl stem , a number of mutant substrates were synthesized that pick up where the previous mutations in the full length trna ( figure 2 and table 2 ) left off . the asl stem of trna has five watson - crick base pairs and the mini - substrate mutants listed in table 3 were designed to eliminate two or more of those base pairs in each mutant . the base pairs that may still form in each mutant are diagrammed in figure 3b and the levels of activity , as a percentage of the activity observed with the wild - type asl & tc mini - substrate , are given in table 3 and figure 3b . when there is no base - pairing in the stem there is little formation of , and the possibility of 1 or even 2 bp in the stem , does not substantially elevate the amount of formed . however , with the 27 c/42c mutant substrate , which has 3 bp in the stem below the site of modification , 80% of the level seen with the wild - type asl & tc mini - substrate was observed . the level of formation by hpus1p on the 27c/39 g mutant substrate was only 40% , even though it too has 3 bp in the stem . with respect to the tc stem - loop , if the nucleotides at 55 and 56 are deleted in this asl & tc mini - substrate , there is a decrease in formation relative to the wild - type asl & tc mini - substrate ( 62% of wild - type trna , table 3 ) . if the size of the tc loop is reduced further ( 5558 ) the resulting substrate is a poor one , just 28% of wild - type levels of were observed ( table 3 ) . if 4 nt ( gcaa ) are inserted into the tc loop to make it larger , the substrate still allows for a significant amount of to be formed ( 77% of wild - type asl & tc mini - substrate levels ) . in a separate experiment , if watson - crick base - pairing is disrupted in the tc stem , with the sequence ggacg starting at nucleotide 61 , the level of hpus1p activity was also substantial ( 73% or wild - type asl & tc mini - substrate levels ) , suggesting a stem loop is not required . if the sequence of the tc loop was replaced with its complement ( aagcuua ) , the amount of formed at position 28 was 113% of wild - type asl & tc mini - substrate levels , suggesting that any loop of at least 5 nt will suffice . to summarize the results from the mutation and deletion studies , the levels of formation with the single and double mutants in the context of the full - length trna suggest that the sequence of the asl stem is not an absolute determinant of hpus1p activity , except of course that there must be a u at position 28 to modify ( tables 2 and 3 ) . the preference for a pyrimidine 3 to the site of modification ( at position 29 ; see introduction section ) was not confirmed since even though the 29 c single mutant showed a decreased amount of formed and the double mutant 29 c/41 g exhibited an almost identical curve as wild - type trna , suggesting that a pyrimidine is not preferred , only the base pair . the point mutations in full - length trna show that the base pairing below the modified position ( u28 ) affects formation , whereas the 27 a the results with the asl & tc stem loop mini - substrate mutants also showed that the base pairs below u28 were the most critical . the sequence of the stem nucleotides is of less importance than the presence of base pairs , which agrees with the fact that known substrates of pus1p exhibit no absolute sequence requirement ( 26,27,3335 ) . in terms of the requirements for a particular secondary structure of trna , the d - stem loop and acceptor stem are dispensable for formation at u28 . in fact , a single stem loop , 3 to the asl , is all that is required for substantial formation at position 28 . the sequence , and possibly the structure of this additional rna , is not constrained . given the above summary of the mutation experiments , a mini - substrate where all of the nucleotides in the asl stem have been changed , except of course for the u at position 28 which is modified , should be a substrate for hpus1p . a substrate fitting this description ( figure 3a ) , along with another where the u at position 28 was changed to c , were incubated with hpus1p and assayed for activity . the y for y r for r stem mini - substrate mutant has a replaced by g , g replaced by a , c replaced by u , and u replaced by c when compared with the wild - type asl & tc mini - substrate , except at position 28 . the 28 c mutant has complete replacement of the stem sequence , and should not be modified by hpus1p . the results with these substrates , as well as with the wild - type asl & tc mini - substrate , are presented in table 4 and show that this mutant is modified by hpus1p ( 0.33 mol /mol rna ) at 80% of wild - type levels for this experiment . in addition , the 28c mutant is not modified ( 0.01 mol /mol rna ; 2% of wild - type levels ) , proving that only the u at position 28 is modified . so , even though all the asl stem nucleotides in the mutant are different from the wild - type trna asl stem , and there is a u28g42 base pair in the mutant , the u at position 28 is converted to at near wild - type levels . table 4.activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutantsrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.41 ( 0.05)100 ( 12)y for y r for r stem mini - substrate mutant 27g/29a/30a/31g/39c/40u/41u/42g/43c0.33 ( 0.02)80 ( 5)y for y r for r stem mini - substrate 28c mutant 27g/28c/29a/30a/31g/39c/40u/41u/42g/43c0.01 ( 0.01)2 ( 2)activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutants activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . a number of studies have determined the sites in trnas that are modified by pus1p ( 26,27,29,33,34 ) . yeast and mammalian pus1p are the most extensively studied and in vivo and in vitro , the enzymes are responsible for the formation of at positions 27 and 28 in many trnas , at 34 and 36 in certain intron containing trnas , and at position 1 in a few substrates ( 29,33,34 ) . the yeast enzyme can also modify uridine at positions 26 , 65 and 67 in vivo but not in vitro ( 34 ) . when the mouse enzyme was expressed in a pus1 yeast strain , position 26 was not modified , and the modification states of positions 65 and 67 were not determined ( 33 ) . sequenced human trnas do not have at positions 26 , 65 , or 67 ( 58 ) , so it is unlikely mpus1p or hpus1p exhibit activity at these sites in vivo . the mammalian enzymes do modify position 30 in a few trnas but the yeast enzyme lacks this activity ( 29,33 ) . for the purposes of the discussion of the current results , the modification at positions 27 and 28 will be considered , since these are formed in vitro in a number of substrates by the mammalian enzymes ( 27,33,35 ) . nearly all ( 17 out of 18 ) of the sequenced yeast and human trnas that have a u at position 27 have converted it to ( 58 ) . however , in the six sequenced yeast trnas that have a u at position 28 , the three that have a 27c43 g base pair lack at position 28 , whereas , the other three have a 2743a base pair and a at position 28 ( 58 ) . this completely agrees with the two consensus sequences shown in figure 1b . in the sequenced human trnas with a u at 28 ( eight total ) three of the trnas have a 2743a base pair , two have a at position 28 , but one does not . a human trna with an 27a43u base pair does have a at position 28 ( 58 ) , suggesting that an u a or a u base pair at 2743 allows for formation at 28 , whereas a 27c43 g base pair is not conducive to formation at position 28 . however , with the four remaining trnas , which have a 27c43 g base pair , two have a at position 28 and two do not ( 58 ) . the current finding that the loss of base pairing at , or the presence of , a c g base pair at 2743 does not significantly affect the formation of at position 28 in trna , correlates with the data from human trnas . human mitochondrial trna is known to be a substrate of hpus1p in vivo ( 41 ) but the sequence of the stem only matches the 28 consensus at the 2743 and 2842 bp . finally , the y for y r for r stem mini - substrate mutant is an efficient substrate for hpus1p in vitro , even though the sequence in the stem does not match the consensus at any position except 28 ( figure 3a and table 4 ) . there must be other determinants allowing for or suppressing formation in these other trnas with a u at 28 . the uridine to be modified does not have to participate in watson - crick base - pairing but nucleotides participating in base - pairing 3 to the modified position appear to be required to constitute an efficient substrate . these results correlate well with the observation that , in vivo , yeast pus1p formed at position 26 in yeast trna(cca ) , where the uridine is in the hinge between the d stem - loop and the asl , and is followed by 5 bp in the asl stem ( 33 ) . in addition , both yeast and mouse pus1p modify the uridine in position 1 on yeast trna(acg ) , with base pairing present in the acceptor stem 3 to the position of modification ( 33 ) . and finally , yeast and mammalian pus1p modifies several uridines in the extensively base - paired asl of yeast pre - trna(uau ) in vitro and in vivo ( 27,29,33 ) . the lack of a strict substrate sequence requirement for hpus1p can be contrasted with the rigid recognition motif of yeast pseudouridine synthase 7 ( pus7p ) , another synthase that can recognize more than one site in trna and other types of rna ( 44 ) . with pus7p there is an absolute requirement for a u at 2 and an a at + 1 relative to the uridine modified ( 44 ) . of the sequenced yeast and human trnas , all that have the proscribed pus7p recognition sequence ( 17 total ) the other site - specific synthases modify only one site in trnas ( pseudouridine synthases 4/10 , 6 , 8/9 ) or one small region in many trnas ( pus3p ) ( 48,49,5962 ) . with these latter synthases the local trna structure appears to be a determining factor for substrate recognition when one considers the results of trua ( pus3p homolog ) and trub ( pseudouridine synthase 4 homolog ) structural studies ( 46,47,63,64 ) . given the requirement that the uridine to be modified be in a base - paired stem , why is the uridine at position 44 in yeast u2 snrna modified by pus1p ( 33 ) when that position is not considered to be in a region that is base paired ? yeast pus1p can modify u2 snrna in vitro and in vivo , but mpus1p only modifies u44 on u2 snrna in vivo , in a pus1 strain ( 33,36 ) . an alternative structure , perhaps in the ribonucleoprotein particle where u44 is base - paired , may form in vivo . it is also possible that another rna , such as a snorna , base pairs in the region of u44 and forms a stem that can be recognized by yeast or mouse pus1p . it is interesting to note that formation in u2 snrna in c. elegans is not dependent on pus1p activity ( 28 ) . the results with mutations in the tc stem - loop suggest that there is a minimum size requirement for this loop but the actual sequence , or base pairing in the stem , are not critical . a minimum size requirement may be a reason that the v stem - loop of trna(uga ) does not substitute for the tc stem - loop when combined with the asl . the v stem - loop in trna(uga ) is predicted to have only 3 nt in the loop . the fact that the tc stem - loop is separated from the asl by 4 nt in the mini - substrate and the v stem - loop has no separation , may also be a factor in why the tc stem - loop can participate in creating a mini - substrate that is recognized by hpus1p . the only synthase in the trua family that has been crystallized is bacterial trua ( see above ) and the structures were obtained in the presence of substrate trnas ( 46,47 ) . trua is a dimer and makes contact with the asl ( positions 3840 are modified if a u is present at those sites ) , the d stem , and the elbow where the d and tc loops interact . the v loop or stem - loop and the acceptor stem did not interact with trua ( 47 ) . in this report the d stem - loop of trna(uga ) was found to be dispensable for hpus1p activity , as was the acceptor stem , but the absence of the v stem - loop from the trna does reduce the amount of formed . in this trna substrate , the tc stem - loop appears to be the more critical component for recognition by hpus1p . therefore , even though there is considerable homology between trua and hpus1p , the substrate contacts with the protein are probably different . yeast pus1p has been shown to be a monomer when bound to trna ( 30 ) and it is reasonable to assume the substrate / enzyme interactions will be fundamentally different between trua and pus1p . crystal structures for any of the pus1p proteins in the presence of trna substrates will go a long way towards reconciling these differences . with what appear to be relatively lax requirements for substrate recognition in trna , what prevents other uridines , such as those at 39 , 43 , 44 , 63 , or 70 in trna ( figure 1a ) from being modified by hpus1p in vitro ? modifications in the asl ( 26 , 27 , 28 , 30 ) of all natural substrates of pus1p are only found on the 5 side of the stem . this specificity is most likely due to the presentation of this side of the asl to the enzyme 's active site cleft , in much the same way the opposite side of the stem is presented to trua for modification of positions 3840 ( 47 ) . this flipping of the orientation of the substrate in the active site cleft could explain why the tc stem - loop is a critical part of the recognition motif for hpus1p rather than the d stem - loop . these other sites that have uridines in a stem are not recognized because they are not presented in a conformation compatible with placing the uridine in the active site . additional natural and mini - substrates will need to be studied to refine the parameters of hpus1p substrate recognition to construct a recognition site that can be used to narrow down the possible modification sites in sra , a relatively large , non - trna substrate of pus1p , where most of the actual residues have not been identified ( 37,38 ) , or to identify additional substrates . the known substrates of pus1p may just be the tip of the iceberg of actual rna substrates for this enigmatic enzyme . national institutes of health ( grant number dk074368 - 01 ) ; south carolina honors college at the university of south carolina ( undergraduate research fellowship and thesis preparation grants to b.s.s . ) .

pseudouridine synthase 1 ( pus1p ) is an unusual site - specific modification enzyme in that it can modify a number of positions in trnas and can recognize several other types of rna . no consensus recognition sequence or structure has been identified for pus1p . human pus1p was used to determine which structural or sequence elements of human trnaser are necessary for pseudouridine ( ) formation at position 28 in the anticodon stem - loop ( asl ) . some point mutations in the asl stem of trnaser had significant effects on the levels of modification and compensatory mutation , to reform the base pair , restored a wild - type level of formation . deletion analysis showed that the trnaser tc stem - loop was a determinant for modification in the asl . a mini - substrate composed of the asl and tc stem - loop exhibited significant formation at position 28 and a number of mutants were tested . substantial base pairing in the asl stem ( 3 out of 5 bp ) is required , but the sequence of the tc loop is not required for modification . when all nucleotides in the asl stem other than u28 were changed in a single mutant , but base pairing was retained , a near wild - type level of modification was observed .

INTRODUCTION MATERIALS AND METHODS Isolation of recombinant hPus1p and site directed mutagenesis of tRNA RNA synthesis, the tritium-release assay and binding assays RESULTS DISCUSSION FUNDING

the anticodon stem - loop ( asl ) of the trna , as well as the dihydrouridine ( d ) stem and the elbow where the d and tc loops interact , contact the synthase ( 47 ) . a minimum level of base pairing in the asl ( the location of the modification ) and the presence of an additional 3 stem - loop , constitute the minimal substrate for hpus1p . even when all nucleotides in the asl stem other than u28 were changed in a single minimal substrate , but base pairing was retained , a near wild - type level of modification was observed . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . the point mutations in the asl of full - length trna are diagrammed in figure 2h and were obtained by site - directed mutagenesis of the plasmid containing wild - type human trna [ phts , gift from h. gross ; ( 50 ) ] followed by in vitro transcription of linear templates ( see materials and methods section ) . if the u at position 28 in the d stem - loop mutant is changed to c ( and the a at 42 is changed to g ) so that can not be formed at position 28 , there is essentially no formation ( 2% of wild - type trna formation , see table 2 ) . if the tc stem - loop is missing from the trna , there is a significant reduction in the formation of at u28 ( 15% of wild - type trna levels , table 2 ) but the reduction is not as substantial when the v stem - loop is missing ( 67% of wild - type , table 2 ) . when a mini - substrate of just the asl stem - loop was incubated with hpus1p , formation was at background levels for 2 h of incubation ( 3% of wild - type levels , table 2 ) . in addition , if either the d stem - loop or the v stem - loop was combined with the asl stem - loop , there was little increase in the amount of formed ( 10% and 7% , respectively , of the formation seen in 120 min with wild - type trna , table 2 ) . when the tc stem - loop was combined with the v and asl stem - loops , there was a substantial level of hpus1p activity ( 71% of wild - type trna levels of ) . these results suggest that the secondary structure of the trna is very important for the modification at position 28 , and a minimal substrate could be created by simply connecting the asl and tc stem - loop as diagrammed ( figure 3a ) . the level of formation at position 28 in this minimal substrate ( wild - type asl & tc mini - substrate ) was ~0.60 mol /mol rna after 2 h of incubation , a substantial level of modification ( table 3 ) . table 3.activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutantsrna substratemole /mole rna ( sd)mole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.60 ( 0.11)0.54 ( 0.16)100 ( 18 ) ( 30)28c/42 g ( no u at expected site of formation)0.01 ( 0.01)2 ( 2)27c/42c0.48 ( 0.04)80 ( 7)27c/39g0.24 ( 0.05)40 ( 8)27c/29c/39g0.10 ( 0.03)17 ( 5)27c/29c/42c0.04 ( 0.01)7 ( 2)27c/39g/40g<0.01 ( 0.01)2 ( 2)27c/29c/39g/40g0.02 ( 0.03)3 ( 5)27c/29c/39g/40g/42c0.02 ( < 0.01)3 ( 2)55 & 560.37 ( 0.10)62 ( 17)55580.17 ( 0.06)28 ( 10)gcaa inserted between 55 and 560.46 ( 0.04)77 ( 7)wild - type asl & tc stem - loop mini - substrate0.30 ( 0.04)100 ( 13)ggacg starting at 610.22 ( 0.03)73 ( 10)aagcuua in tc loop0.34 ( 0.04)113 ( 13)activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutants activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . concentrating on the asl stem , a number of mutant substrates were synthesized that pick up where the previous mutations in the full length trna ( figure 2 and table 2 ) left off . in a separate experiment , if watson - crick base - pairing is disrupted in the tc stem , with the sequence ggacg starting at nucleotide 61 , the level of hpus1p activity was also substantial ( 73% or wild - type asl & tc mini - substrate levels ) , suggesting a stem loop is not required . if the sequence of the tc loop was replaced with its complement ( aagcuua ) , the amount of formed at position 28 was 113% of wild - type asl & tc mini - substrate levels , suggesting that any loop of at least 5 nt will suffice . to summarize the results from the mutation and deletion studies , the levels of formation with the single and double mutants in the context of the full - length trna suggest that the sequence of the asl stem is not an absolute determinant of hpus1p activity , except of course that there must be a u at position 28 to modify ( tables 2 and 3 ) . the point mutations in full - length trna show that the base pairing below the modified position ( u28 ) affects formation , whereas the 27 a the results with the asl & tc stem loop mini - substrate mutants also showed that the base pairs below u28 were the most critical . given the above summary of the mutation experiments , a mini - substrate where all of the nucleotides in the asl stem have been changed , except of course for the u at position 28 which is modified , should be a substrate for hpus1p . so , even though all the asl stem nucleotides in the mutant are different from the wild - type trna asl stem , and there is a u28g42 base pair in the mutant , the u at position 28 is converted to at near wild - type levels . table 4.activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutantsrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.41 ( 0.05)100 ( 12)y for y r for r stem mini - substrate mutant 27g/29a/30a/31g/39c/40u/41u/42g/43c0.33 ( 0.02)80 ( 5)y for y r for r stem mini - substrate 28c mutant 27g/28c/29a/30a/31g/39c/40u/41u/42g/43c0.01 ( 0.01)2 ( 2)activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . the results with mutations in the tc stem - loop suggest that there is a minimum size requirement for this loop but the actual sequence , or base pairing in the stem , are not critical . the fact that the tc stem - loop is separated from the asl by 4 nt in the mini - substrate and the v stem - loop has no separation , may also be a factor in why the tc stem - loop can participate in creating a mini - substrate that is recognized by hpus1p .

cryptococcosis presents in three forms cutaneous , pulmonary , and meningococcal and is a life - threatening fungal disease . although the genus cryptococcus contains more than 50 species of free - living basidiomycete fungi , only two , c. neoformans and c. gattii , are significant pathogens of humans [ 1 , 2 ] . disease is typically caused in the immunocompromised , such as hiv / aids or organ transplant patients , and is usually attributable to c. neoformans . however , incidences of cryptococcosis in otherwise healthy individuals have been rising , and c. gattii is primarily responsible for these cases [ 2 , 3 ] . cryptococcal infection is acquired through inhalation of basidiospores or desiccated yeast cells into the lungs , from where cryptococci can potentially disseminate to all organs but with a predilection to the brain [ 1 , 48 ] . cryptococcal meningitis is estimated to kill 600,000 people annually worldwide , with more than 80% of deaths occurring in sub - saharan africa . in the course of infection through airways to lungs and from lungs to brain , cryptococcus must overcome two major barriers : the innate and adaptive immune mechanisms of the host . the former consists of anatomical or physical barriers such as the mucosal or lung epithelium , the blood - brain barrier of the cns , and phagocytic cells such as neutrophils , monocytes / macrophages , and dendritic cells . successful evasion of the host defences results in cryptococcal colonization of host tissues and hence cryptococcosis . to better understand the pathogenesis of this disease , numerous in vivo and in vitro models have been developed to investigate features of cryptococcosis and address questions such as the course of cryptococcal infection , invasion of cellular barriers , and interactions with and evasion of the immune response . the aim of this review is to present all reported experimental models of cryptococcosis and summarise recent and/or prolific discoveries using these . this will hopefully provide an evaluation of how different models can aid cryptococcal research and give food for thought on how current and new models could be utilised in novel ways . macrophages detect , phagocytose and kill extracellular microorganisms , and present antigen to t cells [ 10 , 11 ] . macrophage phagocytosis of nonopsonised cryptococcal cells is very poor , but dramatically enhanced by complement or immunoglobulin - based opsonins . dysfunctional phagocytic apparatus cripples the immune response ; for example monocytes from hiv / aids patients that were unable to phagocytose cryptococci failed to induce lymphoproliferative response in a macrophage lymphocyte coculture system . once cryptococcus cells have been engulfed , macrophages can present cryptococcal antigen and induce il-1 expression and t - cell proliferation in vitro [ 14 , 15 ] . however , cryptococci show a remarkable ability to survive and proliferate within macrophages , an adaptation that has been explored using both live imaging ( figure 1 ) [ 1619 ] and gene expression [ 20 , 21 ] approaches in macrophage cell lines ( including j774 and raw ) and primary cells ( including bone - marrow - derived murine cells and peripheral blood monocyte - derived human cells ) [ 16 , 17 , 20 , 2224 ] . a noteworthy consideration when using in vitro models is that the cryptococcus - cell interaction can differ depending on the type of macrophage used . for example , cryptococcal expulsion rates were found to be significantly higher in human primary macrophages compared to the j774 cell line . however , despite the fact that different macrophage types are known to vary significantly in their behaviour in vivo , this aspect has yet to be extensively investigated in the context of cryptococcosis . dendritic cells ( dcs ) constitute vital mediators of the initiation of adaptive immune response and are regarded as professional antigen presenters . although less well studied than macrophages , several aspects of dc function have been documented in vitro . phagocytosis of live and heat - killed cryptococci and antigen presentation have been demonstrated in both human peripheral blood mononuclear cells ( pbmcs ) , derived dcs and mouse bone - marrow - derived dendritic cells ( bmdcs ) [ 27 , 28 ] . internalization of live c. neoformans induced minimal tnf - alpha production by human monocyte - derived dcs and none in mouse - derived bmdcs , whereas human dcs incubated with acapsular cryptococci produced significantly higher amounts of tnf - alpha . while it is clear from these studies that dcs can internalize and process both dead and live cryptococci , it is not known whether both dead and live antigen induce the same type and intensity of cytokine response . in addition , it is as yet undetermined whether intracellular cryptococci are eradicated by dcs or whether they survive , proliferate , and escape as seen in macrophages . neutrophils make up the largest population of phagocytes and are the first to be recruited to areas of infection . however their short lifespan means that they are a challenging cell type used in in vitro experimentation . despite this limitation , investigations have shown that neutrophils are able to kill cryptococci in vitro [ 29 , 30 ] . although neutrophil cryptococci killing is typically considered to be mediated by the oxidative burst , neutrophils retain partial anticryptococcal function even when treated with respiratory burst inhibitors . unlike macrophages , neutrophils are also able to control extracellular cryptococci , although cryptococci produce mannitol , superoxide dismutase , and other ros scavengers which help protect against extracellular killing by the respiratory burst [ 32 , 33 ] . to date , however , it is not known whether cryptococci can persist to any degree within neutrophils , or how this interaction is modulated by the presence of macrophages at the infection site . eosonophils , mostly associated with inflammatory response to helminthic parasites , have also been shown to be capable of phagocytosing and killing c. neoformans [ 34 , 35 ] . eosonophil interactions have been studied using primary rat peritoneal eosinophils , in which it has been demonstrated that uptake of cryptococcal cells is strongly enhanced by antibody opsonization and is mediated by fcrii and cd18 [ 34 , 35 ] . garro and colleagues further showed that coincubation of c. neoformans - stimulated eosinophils with cd4 + and cd8 + t cells from infected mice resulted in proliferation of t cells in an mhc 1- and 2-dependent manner , and hence eosinophils can act as antigen presenters and induce adaptive immune response to cryptococcal infection . lymphocytes , particularly cd4 + t cells , are important in the cell - mediated immune response to cryptococcosis . cryptococcus interaction models have used either primary mouse - derived or human pbmc - derived t lymphocytes and , to a lesser extent , immortalized t - cell lines . both t cells and nk cells attach to cryptococcus and directly inhibit its growth in vitro [ 36 , 37 ] . cryptococcus conjugate formation was enhanced when lymphocytes from mice immunized with heat killed c. neoformans were coincubated with cryptococcal yeast cells , suggesting that prior exposure activates this process , presumably through antigen processing and presentation by phagocytes to lymphocytes . intriguingly , both cd4 + and cd8 + t lymphocytes can kill extracellular cryptococci via granulysin , nk cells utilise perforin to achieve the same ends [ 4043 ] . the inhalation route of infection makes the lung the first internal organ to be colonized by cryptococcus . the interaction between cryptococcus and epithelial cells lining the alveolar spaces is thus critical in regulating cryptococcal entry into the circulation system . initial investigation showed that glucuronoxylomannan is an important factor permitting the attachment of yeast to host cell and subsequent infiltration of , and damage to , the host intracellular environment . in vitro experiments have also provided insight into how the capsule virulence factor promotes the ability of cryptococcus to cause infection at the lung barrier . human lung epithelial cells can internalize both encapsulated and acapsular strains of c. neoformans . the lung surfactant protein - d ( sp - d ) was shown to bind to and promote phagocytosis of both encapsulated and acapsular c. neoformans by macrophages in vitro . however , encapsulated cryptococci are not efficiently opsonized by sp - d and those that were phagocytosed resisted intracellular killing by macrophages . in addition , the lung is the site of granuloma formation during pulmonary cryptococcosis ( and potentially during latent infections ) . to date , however , no models have been established to address the role of lung epithelial cells in granuloma formation . modelling the blood - brain barrier ( bbb ) with the appropriate neurovascular properties has been a prominent challenge in cryptococcosis research . isolation of human , rat and mouse brain microvascular endothelial cells ( bmecs ) , which can be propagated in vitro to monolayers with bbb - like properties , has greatly improved the study of cryptococcus bbb interactions ( figure 2 ) . both human and mouse immortalized bmec cell lines are commercially available and have been used in several studies , although freshly isolated bmecs are not so readily accessible . several studies using either primary or immortalized human , rat and mouse bmec as bbb models have shown that bmec can associate with and internalize yeast cells [ 39 , 4850 ] . transwell chamber systems with bmec monolayers have been used in some of the studies to demonstrate the occurrence of transcytosis across this cell layer [ 39 , 50 ] . however in vivo the bbb is a complex tissue in which bmecs are supported by pericytes and astrocytes , a feature which can not at present be recreated in vitro . in this context , the recent application of intravital imaging to examine cryptococcal traversal across the bbb in vivo represents a major advance for the field . in vivo models can complement results of in vitro studies and also stand alone as valuable tools with which to make new discoveries and test out hypotheses . in this context , it is important to make the distinction between models that are used because they are vectors or carriers of human disease , and those that are used purely for their amenability in an experimental context . those that are studied because they naturally harbour cryptococcus , for example the koala , will not be discussed here . instead we concentrate below on the range of model organisms exploited to undertake a whole - organism approach to studying cryptococcus . in addition , the use of invertebrate models is an increasingly popular approach in many areas of biomedical research , including that of human fungal pathogens . we place a relatively large focus on these novel , emerging models over the more established and well - used vertebrate systems in order to highlight potential avenues for exploiting new model systems for cryptococcal research . advantages over their mammalian counterparts include reduced maintenance costs , fewer ethical restrictions , relatively short reproduction times , and large brood sizes . a key argument for the use of invertebrate models is that features of their immune systems allow rigorous investigation into human disease . whereas vertebrates have evolved innate and adaptive immunity , invertebrates possess only the innate system , the most ancient form of pathogen defence . the basic underlying mechanisms of immune response can therefore be studied without potential confusion from adaptive immunity , which can be very species- or even individual - specific . a well - rehearsed argument that doubts the degree to which invertebrate models can be of value in biomedical research centres on the concept that the human immune system is vastly different from the invertebrate system . indeed , adaptive immunity allows hosts to recognise molecular details of different invading pathogens , construct specific proteins to fight them , and maintain these proteins to protect against subsequent infections . however despite its lack of immune memory , the innate system of invertebrates is highly sophisticated and intricate and does not simply involve the same stereotypical response to every pathogen it encounters [ 53 , 54 ] . studies from organisms that use only innate immunity have demonstrated the complexity of this immune response [ 55 , 56 ] , and how it can vary according to a range of biotic and abiotic factors . in addition , many human pathogens probably evolved their virulence mechanisms in more primitive organisms [ 5760 ] . in the case of cryptococcus sp . adaptations that allowed the pathogens to escape natural invertebrate predators could have been translated into virulence factors for infecting mammalian hosts , and therefore studying interactions between the invertebrates and microbes could elucidate valuable information about disease . invertebrates are also simple and efficient models in which to study fungal virulence factors , such as capsule growth and melanin production [ 59 , 61 ] ; morphological dimorphism ; phospholipase production . antifungal activity can also be tested rapidly and economically using invertebrate models , due to their amenability to high - throughput screens of chemical libraries that can measure key factors in drug development including host immune response , efficacy , and toxicity [ 64 , 65 ] . caenorhabditis elegans has been employed for immunological study of many pathogens , and the past decade has seen it established as a model for cryptococcus neoformans infection . in this system a culture of c. neoformans in ypd liquid media is spread onto agar plates , usually with the addition of antibiotics , and incubated overnight . when c. elegans are transferred to these plates they acquire the pathogen orally , leading to fatal disease . critical similarities exist between pathogenicity in c. elegans and mammalian hosts , for example strains of cryptococcus with reduced virulence in mammalian hosts generally show similar attenuation in c. elegans . these parallels support the case for c. elegans constituting a sound model for investigating basic mechanisms underlying cryptococcus infection , disease , and treatment . the molecular basis of infection and disease can be rigorously investigated in c. elegans , due to its amenability as a genetic model , and consequently a wealth of knowledge has been accumulated concerning immunity at the gene level . the potential to screen large numbers of mutant pathogens in c. elegans and identify genes important for virulence demonstrates a particular strength for the nematode model . a recent investigation identified mutations in c. neoformans that caused reduced survival in ex vivo cerebral spinal fluid and commensurate attenuation in c. elegans and the rabbit . in addition , genes important for c. neoformans virulence in mammalian systems have been shown to be instrumental in causing death in c. elegans killing assays , highlighting a parallel between the immunity and pathogenesis of the cryptococcus - human and cryptococcus - worm model systems . for example , c. neoformans genes associated with virulence in both mammalian and nematode models include those associated with signal transduction pathways ( gpa1 , pka1 , pkr1 , ras1 ) , laccase production ( lac1 ) , and the { alpha } mating type . c. elegans can use nonpathogenic forms of cryptococcus , for example c. laurentii and c. kuetzingii , as a food source in the laboratory . this lends support to the theory that the nematode is a natural predator of cryptococcus and could therefore be an ideal model in which to investigate the evolution of virulence factors that have enabled certain strains of cryptococcus to become pathogenic to worms and other hosts . however , a profound limitation of c. elegans as a model for investigating pathogenesis of mammalian disease is that the mode of infection is completely different to that in mammals . the nematode ingests the pathogen , and cryptococcal growth is restricted to the intestine , in marked contrast to the lung inhalation and subsequent dissemination that is characteristic of human infections . along with the limitations associated with this entirely different mode of infection , it is not possible to administer an exact pathogen dose in this model , which could severely constrain the scope of certain experiments . in addition , phagocytes are not present in c. elegans , so in terms of studying phagocytosis in a whole organism model a more favourable invertebrate model may be an insect or amoeba . soil amoebae are environmental reservoirs of human pathogens and therefore show promise as model hosts in the laboratory setting . amoebae feed by phagocytosis of microorganisms , in a process that is essentially similar to the phagocytosis of microbes by human macrophages . amoebae therefore provide a simple model in which to investigate aspects of this fundamental immune response . additionally , there are a number of characteristics that make these organisms valuable experimental models , primarily with respect to genetic tools . acantamoeba castellaniiin terms of using invertebrate hosts to study evolution of human pathogen virulence , the interaction between cryptococcus sp . and a. castellanii provides interesting and unique opportunity for investigation . cryptococcus can cause amoebae to lyse following phagocytosis , and a popular theory postulates that this adaptation to overcome predation could have been a precursor to the escape from mammalian phagocytes that cryptococci demonstrate in vitro . in support of this , characteristics that promoted cryptococcus survival in a. castellanii were also identified as virulence factors that enhanced cryptococcus parasitism in macrophages . although a. castellanii may not be as genetically well characterised as the amoeboid model d. discoideum , the former has the advantage of remaining viable in laboratory conditions above 25c , therefore more accurately simulating conditions of human infection , which occur in the critical 3237c window .an important feature of the a. castellanii model is that this organism is killed by cryptococcus . on a basic level this suggests that cryptococcus readily acts as a pathogen in amoebae , which could confer an advantage over another invertebrate , such as d. melanogaster , which is not killed by injection of cryptococcus and therefore may not be such a valuable a model of pathogenesis . in terms of using invertebrate hosts to study evolution of human pathogen virulence , the interaction between cryptococcus sp . and a. castellanii provides interesting and unique opportunity for investigation . cryptococcus can cause amoebae to lyse following phagocytosis , and a popular theory postulates that this adaptation to overcome predation could have been a precursor to the escape from mammalian phagocytes that cryptococci demonstrate in vitro . in support of this , characteristics that promoted cryptococcus survival in a. castellanii were also identified as virulence factors that enhanced cryptococcus parasitism in macrophages . although a. castellanii may not be as genetically well characterised as the amoeboid model d. discoideum , the former has the advantage of remaining viable in laboratory conditions above 25c , therefore more accurately simulating conditions of human infection , which occur in the critical 3237c window . an important feature of the a. castellanii model is that this organism is killed by cryptococcus . on a basic level this suggests that cryptococcus readily acts as a pathogen in amoebae , which could confer an advantage over another invertebrate , such as d. melanogaster , which is not killed by injection of cryptococcus and therefore may not be such a valuable a model of pathogenesis . dictyostelium discoideum has proved a useful tool for studying intracellular proliferation of human pathogens including cryptococcus . the small haploid genome of d. discoideum has been sequenced , and is particularly amenable to genetic manipulation [ 68 , 74].important parallels in terms of cryptococcal virulence are present between d. discoideum and human hosts . for example the fungal capsule is important for c. neoformans infection of d. discoideum , as acapsular mutants did not replicate in the amoebae . another important finding in d. discoideum showed that c. neoformans caused disease with increased efficiency following growth in the amoeboid model , indicating that adaptations for survival in the host translated into virulence factors . dictyostelium discoideum has proved a useful tool for studying intracellular proliferation of human pathogens including cryptococcus . the small haploid genome of d. discoideum has been sequenced , and is particularly amenable to genetic manipulation [ 68 , 74 ] . important parallels in terms of cryptococcal virulence are present between d. discoideum and human hosts . for example the fungal capsule is important for c. neoformans infection of d. discoideum , as acapsular mutants did not replicate in the amoebae . another important finding in d. discoideum showed that c. neoformans caused disease with increased efficiency following growth in the amoeboid model , indicating that adaptations for survival in the host translated into virulence factors . after pathogen recognition , the insect immune response follows a characteristic sequence of events involving two major classes of effector systems cellular and humoral [ 75 , 76 ] . the humoral immune response concerns the production of antimicrobial peptides and induction of enzyme cascades which function to minimise harm caused by the pathogen . circulating haemocytes within the insect haemocoel produce the cellular response and rapidly fight the pathogen using three key cellular defences : phagocytosis , nodule formation , and encapsulation [ 77 , 78 ] . assays to measure insect immune response can be applied to a variety of species and employed in a high - throughput manner . for example , in vivo phagocytosis can be assessed by nondestructive extraction of haemolymph containing phagocytosed pathogens from insects and then analysed using in vitro assays . given the importance of phagocytosis in cryptococcosis research , insects may therefore prove a valuable experimental tool . despite the many advantages of insect models for human disease research insects lack most organs found in humans , such as lungs , kidneys , and hearts . given that organs such as the lungs are particularly important in cryptococcus infection and disease , this highlights an obvious limitation of insects rather than mammalian models for this fungal pathogen . in a similar vein , the blood - brain barrier is a particularly important area of investigation for cryptococcus research , but the lack of blood capillaries in the insect body means that this aspect of the disease can not be studied in insect hosts . galleria mellonellathe larvae of the greater wax moth , galleria mellonella , have been used as whole - organism virulence models for various species of pathogenic fungi . this organism can live at mammalian body temperature , thereby allowing temperature - sensitive investigation of pathogenesis . the larva also provides a good infection model because it is easy to inoculate with specific doses of fungal pathogen , owing largely to its size ( approximately 1.52.5 cm in length ) , which contrasts with the smaller size of insects such as drosophila , in which controlling dose per insect is challenging . to date , the g. mellonella model has primarily been used to assess virulence and/or antifungal activity . inoculation of microbes into the haemocoel is minimally invasive because piercing of the haemocoel is not required . instead applying gentle pressure can open up the base of the proleg , and a needle is inserted . this nonpiercing method means that immune response directed at wound repair does not affect the results of experiments , which would be the case if insects were injected through the cuticle . g. mellonella has been advocated as a particularly good model for diseases that disseminate through the body via the bloodstream , such as cryptococcus neoformans and candida albicans . the development of g. mellonella as a model for cryptococcus neoformans was first achieved by mylonakis and colleagues in 2005 . all c. neoformans strains tested caused death of the insect host , despite effective phagocytosis by insect haemocytes . this suggests that cryptococcus virulence may rely on the same mechanisms in g. mellonella as it does in in vitro assays , during which the phagocytic immune response is evaded and even exploited by the pathogen.recently the g. mellonella system was employed to investigate whether the antifungal activity of fluconazole could be enhanced with the addition of other drugs . fluconazole proved to have greater beneficial effect for g. mellonella survival when administered in combination with the antihistamine astemizole and a closely related analog ( a2 ) . the results of this in vivo study were supported and enhanced by an in vitro experiment , in which the usually fungistatic fluconazole became fungicidal when combined with astemizole and a2 . this investigation demonstrates how survival data from an insect model that is relatively easy to infect and monitor in a high - throughput manner can support in vitro mechanistic discovery . the larvae of the greater wax moth , galleria mellonella , have been used as whole - organism virulence models for various species of pathogenic fungi . this organism can live at mammalian body temperature , thereby allowing temperature - sensitive investigation of pathogenesis . the larva also provides a good infection model because it is easy to inoculate with specific doses of fungal pathogen , owing largely to its size ( approximately 1.52.5 cm in length ) , which contrasts with the smaller size of insects such as drosophila , in which controlling dose per insect is challenging . to date , the g. mellonella model has primarily been used to assess virulence and/or antifungal activity . inoculation of microbes into the haemocoel is minimally invasive because piercing of the haemocoel is not required . instead applying gentle pressure can open up the base of the proleg , and a needle is inserted . this nonpiercing method means that immune response directed at wound repair does not affect the results of experiments , which would be the case if insects were injected through the cuticle . g. mellonella has been advocated as a particularly good model for diseases that disseminate through the body via the bloodstream , such as cryptococcus neoformans and candida albicans . the development of g. mellonella as a model for cryptococcus neoformans was first achieved by mylonakis and colleagues in 2005 . all c. neoformans strains tested caused death of the insect host , despite effective phagocytosis by insect haemocytes . this suggests that cryptococcus virulence may rely on the same mechanisms in g. mellonella as it does in in vitro assays , during which the phagocytic immune response is evaded and even exploited by the pathogen . recently the g. mellonella system was employed to investigate whether the antifungal activity of fluconazole could be enhanced with the addition of other drugs . fluconazole proved to have greater beneficial effect for g. mellonella survival when administered in combination with the antihistamine astemizole and a closely related analog ( a2 ) . the results of this in vivo study were supported and enhanced by an in vitro experiment , in which the usually fungistatic fluconazole became fungicidal when combined with astemizole and a2 . this investigation demonstrates how survival data from an insect model that is relatively easy to infect and monitor in a high - throughput manner can support in vitro mechanistic discovery . drosophila melanogasterinfection of d. melanogaster with c. neoformans can be achieved by a variety of methods , and both dissemination of disease and local infection can be investigated . injection into the haemocoel is done using a small needle inserted into the thorax or abdomen ( e.g. , ) . a limitation of this method is that it is far removed from the natural pathway of infection , as microorganisms such as cryptococcus would not be able to cross the insect cuticle unless an opening was already present . an alternative method is to administer the pathogen by incorporating it into the fly food.interest in d. melanogaster as a model of human disease has grown in recent years , with the continuing realisation that immune signalling pathways are highly conserved between fly and human [ 82 , 83 ] . of particular relevance to fungal disease is the finding that toll receptor activation downstream of fungal infection leads to the production of antifungal peptides by d. melanogaster .wild - type d. melanogaster shows resistance to fungal pathogens administered by injection into the haemocoel including candida albicans , aspergillus fumigatus , and cryptococcus neoformans . experiments have shown that the toll pathway is crucial in this resistance [ 84 , 85 ] . in contrast , the toll pathway is not involved in pathogen defence when the method of cryptococcus neoformans infection is ingestion . this research using drosophila has suggested that cryptococcus neoformans may elicit different responses in the systemic- and digestive - related immunity , in this host at least . infection of d. melanogaster with c. neoformans can be achieved by a variety of methods , and both dissemination of disease and local infection can be investigated . injection into the haemocoel is done using a small needle inserted into the thorax or abdomen ( e.g. , ) . a limitation of this method is that it is far removed from the natural pathway of infection , as microorganisms such as cryptococcus would not be able to cross the insect cuticle unless an opening was already present . an alternative method is to administer the pathogen by incorporating it into the fly food . interest in d. melanogaster as a model of human disease has grown in recent years , with the continuing realisation that immune signalling pathways are highly conserved between fly and human [ 82 , 83 ] . of particular relevance to fungal disease is the finding that toll receptor activation downstream of fungal infection leads to the production of antifungal peptides by d. melanogaster . wild - type d. melanogaster shows resistance to fungal pathogens administered by injection into the haemocoel including candida albicans , aspergillus fumigatus , and cryptococcus neoformans . experiments have shown that the toll pathway is crucial in this resistance [ 84 , 85 ] . in contrast , the toll pathway is not involved in pathogen defence when the method of cryptococcus neoformans infection is ingestion . this research using drosophila has suggested that cryptococcus neoformans may elicit different responses in the systemic- and digestive - related immunity , in this host at least . for certain elements of fungal pathogen research , the benefit of the adaptive immune system of vertebrates will always be a feature that renders them more valuable as models than invertebrates . vertebrate models have been extensively covered in other reviews ( e.g. , [ 86 , 87 ] ) but are discussed briefly below . the majority of cryptococcus - related work utilising mammalian systems has been carried out using mice . due to the fact that murine models are well established as valuable study systems in many research areas including fungal pathogenesis , an in - depth discussion of mice in cryptococcus research is not within the scope of the present review . briefly , however , mouse models are a popular choice because they are well established and characterised in medical research , and a variety of genetic backgrounds are available . infection with cryptococcus can be achieved by a variety of methods , including intranasally , intraperitoneally , intracerebrally , intravenously , intratracheally , and via inhalation [ 88 , 89 ] , which opens up a range of experimental opportunities . in addition , the vast array of information available on the mouse immune system means that parallels and pitfalls can be readily identified , allowing for the design of highly refined experiments . the slightly larger body size allows a variety of experimental manipulations to be achieved with relative ease , including endotracheal intubation , bronchoalveolar lavage , serial venepuncture , csf collection , radiography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) . the rat has also been reported to develop chronic pulmonary cryptococcosis in the wild , suggesting this organism as a potentially valuable disease model . a model mimicking pulmonary cryptococcosis establishment , induced by c. gattii intratracheal inoculation in immunocompetent rat hosts , this opens up the opportunity to use this model to study more complex elements of cryptococcal etiology , such as long - term latency or the emergence of c. gattii as a primary pathogen . the rat model has been very useful in research into mammalian host responses to cryptococcus . for example expression of acidic mammalian chitinase ( amcase ) in response to infection by cryptococcus , which contains chitin in its cell wall , could be a potential mediator of asthma . the guinea pig model was first established for cryptococcal disease relatively recently by kirkpatrick et al . . the larger size of guinea pigs compared to mice means they are ideal for more sensitive experimental manipulation . another benefit of the guinea pig model for fungal research is that oral doses of antifungals sufficient to control fungal infections are similar to the doses in humans . an example of the utilisation of the guinea pig model is the investigation into the effectiveness of an intravenous delivery of the antifungal itraconazole in fighting disseminated fungal infections including cryptococcosis . the rabbit has been advocated as a model for cryptococcal meningitis mainly due to its size in comparison to other mammalian study systems . for example , steen et al . chose the rabbit model because its relatively large body size enables the study of yeast at the site of infection , which is more difficult in smaller mammals such as mice and guinea pigs . historically the rabbit model was not an appealing choice for cryptococcal disease research because the organism appeared to be naturally resistant to this pathogen . however the potential for this model increased when pretreatment with corticosteroids and subsequent cryptococcus inoculation successfully resulted in the development of chronic cryptococcal meningitis . a recent utilisation of this model involved measuring survival of cryptococcus mutants in rabbits to establish whether mutations causing reduced attenuation in cerebral spinal fluid showed corresponding reduced virulence in vivo . this is an example of the use of a whole - organism model to validate hypotheses of disease attenuation developed from in vitro experiments . the zebrafish is emerging as an attractive model system for a variety of human diseases . mutagenesis and screening can be done on a large scale with the zebrafish , which is a fairly unique feature in vertebrate models . another benefit to this model is that it can be maintained at relatively low cost , with few ethical restraints . thus the zebrafish in many ways has the economical advantages of an invertebrate model whilst also possessing all of the vertebrate immune system features that researchers in fungal pathogenesis require . the zebrafish has not yet been used to investigate infection and disease in relation to cryptococcus this model has been used for other human fungal pathogens , lending hope to the idea that a disease model for cryptococcus could soon be developed . for example , a comprehensive infection model of the zebrafish with candida albicans has been established , in which pathogen morphogenesis and gene expression , and host immune response , were monitored . fish were killed by c. albicans in a dose - dependent manner , and infection was established at a number of different sites , indicating that this organism could be a valuable tool in fungal research . ex vivo organ culture could be considered a way of achieving a balance between the advantages of in vitro and in vivo experimental models . the use of perfused organs provides a relatively authentic physiological environment and permits the study of pathogens crossing membranes , disseminating through host tissue , and migrating to the bloodstream processes not captured by in vitro experimentation . organs from higher animals , which would otherwise carry enormous ethical and economic implications , can be studied when perfused organs are chosen over whole animal models . for example , haemoperfused liver from pigs was recently established as a model for c. albicans investigation . this model could be adapted for the study of other human fungal pathogens including cryptococcus . an ex vivo model has also been established for swine trachea , and since cryptococcus infects the lungs , this model could be of particular interest to adapt for investigation of this pathogen . cryptococcal research utilises a diverse range of experimental model systems , spanning from individual cells to unicellular whole organisms to mammalian models . progress from hereon may benefit from different research disciplines exchanging knowledge and skills so that models already established in one field , such as drug development , can be adapted for another area of research such as pathogen evolution .

cryptococcosis is a life - threatening fungal disease that infects around one million people each year . establishment and progression of disease involves a complex interplay between the fungus and a diverse range of host cell types . over recent years , numerous cellular , tissue , and animal models have been exploited to probe this host - pathogen interaction . here we review the range of experimental models that are available for cryptococcosis research and compare the relative advantages and limitations of the different systems .

1. Introduction 2. 3. 4. 5. 6. Closing Remarks

cryptococcosis presents in three forms cutaneous , pulmonary , and meningococcal and is a life - threatening fungal disease . disease is typically caused in the immunocompromised , such as hiv / aids or organ transplant patients , and is usually attributable to c. neoformans . however , incidences of cryptococcosis in otherwise healthy individuals have been rising , and c. gattii is primarily responsible for these cases [ 2 , 3 ] . in the course of infection through airways to lungs and from lungs to brain , cryptococcus must overcome two major barriers : the innate and adaptive immune mechanisms of the host . the former consists of anatomical or physical barriers such as the mucosal or lung epithelium , the blood - brain barrier of the cns , and phagocytic cells such as neutrophils , monocytes / macrophages , and dendritic cells . successful evasion of the host defences results in cryptococcal colonization of host tissues and hence cryptococcosis . to better understand the pathogenesis of this disease , numerous in vivo and in vitro models have been developed to investigate features of cryptococcosis and address questions such as the course of cryptococcal infection , invasion of cellular barriers , and interactions with and evasion of the immune response . the aim of this review is to present all reported experimental models of cryptococcosis and summarise recent and/or prolific discoveries using these . this will hopefully provide an evaluation of how different models can aid cryptococcal research and give food for thought on how current and new models could be utilised in novel ways . macrophages detect , phagocytose and kill extracellular microorganisms , and present antigen to t cells [ 10 , 11 ] . once cryptococcus cells have been engulfed , macrophages can present cryptococcal antigen and induce il-1 expression and t - cell proliferation in vitro [ 14 , 15 ] . dendritic cells ( dcs ) constitute vital mediators of the initiation of adaptive immune response and are regarded as professional antigen presenters . although less well studied than macrophages , several aspects of dc function have been documented in vitro . phagocytosis of live and heat - killed cryptococci and antigen presentation have been demonstrated in both human peripheral blood mononuclear cells ( pbmcs ) , derived dcs and mouse bone - marrow - derived dendritic cells ( bmdcs ) [ 27 , 28 ] . internalization of live c. neoformans induced minimal tnf - alpha production by human monocyte - derived dcs and none in mouse - derived bmdcs , whereas human dcs incubated with acapsular cryptococci produced significantly higher amounts of tnf - alpha . in addition , it is as yet undetermined whether intracellular cryptococci are eradicated by dcs or whether they survive , proliferate , and escape as seen in macrophages . although neutrophil cryptococci killing is typically considered to be mediated by the oxidative burst , neutrophils retain partial anticryptococcal function even when treated with respiratory burst inhibitors . unlike macrophages , neutrophils are also able to control extracellular cryptococci , although cryptococci produce mannitol , superoxide dismutase , and other ros scavengers which help protect against extracellular killing by the respiratory burst [ 32 , 33 ] . eosonophil interactions have been studied using primary rat peritoneal eosinophils , in which it has been demonstrated that uptake of cryptococcal cells is strongly enhanced by antibody opsonization and is mediated by fcrii and cd18 [ 34 , 35 ] . garro and colleagues further showed that coincubation of c. neoformans - stimulated eosinophils with cd4 + and cd8 + t cells from infected mice resulted in proliferation of t cells in an mhc 1- and 2-dependent manner , and hence eosinophils can act as antigen presenters and induce adaptive immune response to cryptococcal infection . cryptococcus interaction models have used either primary mouse - derived or human pbmc - derived t lymphocytes and , to a lesser extent , immortalized t - cell lines . both t cells and nk cells attach to cryptococcus and directly inhibit its growth in vitro [ 36 , 37 ] . the interaction between cryptococcus and epithelial cells lining the alveolar spaces is thus critical in regulating cryptococcal entry into the circulation system . initial investigation showed that glucuronoxylomannan is an important factor permitting the attachment of yeast to host cell and subsequent infiltration of , and damage to , the host intracellular environment . in addition , the lung is the site of granuloma formation during pulmonary cryptococcosis ( and potentially during latent infections ) . to date , however , no models have been established to address the role of lung epithelial cells in granuloma formation . modelling the blood - brain barrier ( bbb ) with the appropriate neurovascular properties has been a prominent challenge in cryptococcosis research . isolation of human , rat and mouse brain microvascular endothelial cells ( bmecs ) , which can be propagated in vitro to monolayers with bbb - like properties , has greatly improved the study of cryptococcus bbb interactions ( figure 2 ) . both human and mouse immortalized bmec cell lines are commercially available and have been used in several studies , although freshly isolated bmecs are not so readily accessible . several studies using either primary or immortalized human , rat and mouse bmec as bbb models have shown that bmec can associate with and internalize yeast cells [ 39 , 4850 ] . transwell chamber systems with bmec monolayers have been used in some of the studies to demonstrate the occurrence of transcytosis across this cell layer [ 39 , 50 ] . however in vivo the bbb is a complex tissue in which bmecs are supported by pericytes and astrocytes , a feature which can not at present be recreated in vitro . in this context , it is important to make the distinction between models that are used because they are vectors or carriers of human disease , and those that are used purely for their amenability in an experimental context . those that are studied because they naturally harbour cryptococcus , for example the koala , will not be discussed here . instead we concentrate below on the range of model organisms exploited to undertake a whole - organism approach to studying cryptococcus . advantages over their mammalian counterparts include reduced maintenance costs , fewer ethical restrictions , relatively short reproduction times , and large brood sizes . the basic underlying mechanisms of immune response can therefore be studied without potential confusion from adaptive immunity , which can be very species- or even individual - specific . indeed , adaptive immunity allows hosts to recognise molecular details of different invading pathogens , construct specific proteins to fight them , and maintain these proteins to protect against subsequent infections . studies from organisms that use only innate immunity have demonstrated the complexity of this immune response [ 55 , 56 ] , and how it can vary according to a range of biotic and abiotic factors . adaptations that allowed the pathogens to escape natural invertebrate predators could have been translated into virulence factors for infecting mammalian hosts , and therefore studying interactions between the invertebrates and microbes could elucidate valuable information about disease . invertebrates are also simple and efficient models in which to study fungal virulence factors , such as capsule growth and melanin production [ 59 , 61 ] ; morphological dimorphism ; phospholipase production . antifungal activity can also be tested rapidly and economically using invertebrate models , due to their amenability to high - throughput screens of chemical libraries that can measure key factors in drug development including host immune response , efficacy , and toxicity [ 64 , 65 ] . caenorhabditis elegans has been employed for immunological study of many pathogens , and the past decade has seen it established as a model for cryptococcus neoformans infection . in this system a culture of c. neoformans in ypd liquid media is spread onto agar plates , usually with the addition of antibiotics , and incubated overnight . these parallels support the case for c. elegans constituting a sound model for investigating basic mechanisms underlying cryptococcus infection , disease , and treatment . the molecular basis of infection and disease can be rigorously investigated in c. elegans , due to its amenability as a genetic model , and consequently a wealth of knowledge has been accumulated concerning immunity at the gene level . a recent investigation identified mutations in c. neoformans that caused reduced survival in ex vivo cerebral spinal fluid and commensurate attenuation in c. elegans and the rabbit . in addition , genes important for c. neoformans virulence in mammalian systems have been shown to be instrumental in causing death in c. elegans killing assays , highlighting a parallel between the immunity and pathogenesis of the cryptococcus - human and cryptococcus - worm model systems . for example , c. neoformans genes associated with virulence in both mammalian and nematode models include those associated with signal transduction pathways ( gpa1 , pka1 , pkr1 , ras1 ) , laccase production ( lac1 ) , and the { alpha } mating type . this lends support to the theory that the nematode is a natural predator of cryptococcus and could therefore be an ideal model in which to investigate the evolution of virulence factors that have enabled certain strains of cryptococcus to become pathogenic to worms and other hosts . the nematode ingests the pathogen , and cryptococcal growth is restricted to the intestine , in marked contrast to the lung inhalation and subsequent dissemination that is characteristic of human infections . additionally , there are a number of characteristics that make these organisms valuable experimental models , primarily with respect to genetic tools . and a. castellanii provides interesting and unique opportunity for investigation . cryptococcus can cause amoebae to lyse following phagocytosis , and a popular theory postulates that this adaptation to overcome predation could have been a precursor to the escape from mammalian phagocytes that cryptococci demonstrate in vitro . although a. castellanii may not be as genetically well characterised as the amoeboid model d. discoideum , the former has the advantage of remaining viable in laboratory conditions above 25c , therefore more accurately simulating conditions of human infection , which occur in the critical 3237c window .an important feature of the a. castellanii model is that this organism is killed by cryptococcus . and a. castellanii provides interesting and unique opportunity for investigation . cryptococcus can cause amoebae to lyse following phagocytosis , and a popular theory postulates that this adaptation to overcome predation could have been a precursor to the escape from mammalian phagocytes that cryptococci demonstrate in vitro . an important feature of the a. castellanii model is that this organism is killed by cryptococcus . the small haploid genome of d. discoideum has been sequenced , and is particularly amenable to genetic manipulation [ 68 , 74].important parallels in terms of cryptococcal virulence are present between d. discoideum and human hosts . dictyostelium discoideum has proved a useful tool for studying intracellular proliferation of human pathogens including cryptococcus . the small haploid genome of d. discoideum has been sequenced , and is particularly amenable to genetic manipulation [ 68 , 74 ] . circulating haemocytes within the insect haemocoel produce the cellular response and rapidly fight the pathogen using three key cellular defences : phagocytosis , nodule formation , and encapsulation [ 77 , 78 ] . given the importance of phagocytosis in cryptococcosis research , insects may therefore prove a valuable experimental tool . despite the many advantages of insect models for human disease research insects lack most organs found in humans , such as lungs , kidneys , and hearts . in a similar vein , the blood - brain barrier is a particularly important area of investigation for cryptococcus research , but the lack of blood capillaries in the insect body means that this aspect of the disease can not be studied in insect hosts . galleria mellonellathe larvae of the greater wax moth , galleria mellonella , have been used as whole - organism virulence models for various species of pathogenic fungi . inoculation of microbes into the haemocoel is minimally invasive because piercing of the haemocoel is not required . instead applying gentle pressure can open up the base of the proleg , and a needle is inserted . the development of g. mellonella as a model for cryptococcus neoformans was first achieved by mylonakis and colleagues in 2005 . all c. neoformans strains tested caused death of the insect host , despite effective phagocytosis by insect haemocytes . fluconazole proved to have greater beneficial effect for g. mellonella survival when administered in combination with the antihistamine astemizole and a closely related analog ( a2 ) . the larvae of the greater wax moth , galleria mellonella , have been used as whole - organism virulence models for various species of pathogenic fungi . inoculation of microbes into the haemocoel is minimally invasive because piercing of the haemocoel is not required . instead applying gentle pressure can open up the base of the proleg , and a needle is inserted . this nonpiercing method means that immune response directed at wound repair does not affect the results of experiments , which would be the case if insects were injected through the cuticle . all c. neoformans strains tested caused death of the insect host , despite effective phagocytosis by insect haemocytes . fluconazole proved to have greater beneficial effect for g. mellonella survival when administered in combination with the antihistamine astemizole and a closely related analog ( a2 ) . the results of this in vivo study were supported and enhanced by an in vitro experiment , in which the usually fungistatic fluconazole became fungicidal when combined with astemizole and a2 . drosophila melanogasterinfection of d. melanogaster with c. neoformans can be achieved by a variety of methods , and both dissemination of disease and local infection can be investigated . an alternative method is to administer the pathogen by incorporating it into the fly food.interest in d. melanogaster as a model of human disease has grown in recent years , with the continuing realisation that immune signalling pathways are highly conserved between fly and human [ 82 , 83 ] . of particular relevance to fungal disease is the finding that toll receptor activation downstream of fungal infection leads to the production of antifungal peptides by d. melanogaster .wild - type d. melanogaster shows resistance to fungal pathogens administered by injection into the haemocoel including candida albicans , aspergillus fumigatus , and cryptococcus neoformans . in contrast , the toll pathway is not involved in pathogen defence when the method of cryptococcus neoformans infection is ingestion . this research using drosophila has suggested that cryptococcus neoformans may elicit different responses in the systemic- and digestive - related immunity , in this host at least . infection of d. melanogaster with c. neoformans can be achieved by a variety of methods , and both dissemination of disease and local infection can be investigated . interest in d. melanogaster as a model of human disease has grown in recent years , with the continuing realisation that immune signalling pathways are highly conserved between fly and human [ 82 , 83 ] . of particular relevance to fungal disease is the finding that toll receptor activation downstream of fungal infection leads to the production of antifungal peptides by d. melanogaster . wild - type d. melanogaster shows resistance to fungal pathogens administered by injection into the haemocoel including candida albicans , aspergillus fumigatus , and cryptococcus neoformans . this research using drosophila has suggested that cryptococcus neoformans may elicit different responses in the systemic- and digestive - related immunity , in this host at least . for certain elements of fungal pathogen research , the benefit of the adaptive immune system of vertebrates will always be a feature that renders them more valuable as models than invertebrates . vertebrate models have been extensively covered in other reviews ( e.g. due to the fact that murine models are well established as valuable study systems in many research areas including fungal pathogenesis , an in - depth discussion of mice in cryptococcus research is not within the scope of the present review . briefly , however , mouse models are a popular choice because they are well established and characterised in medical research , and a variety of genetic backgrounds are available . infection with cryptococcus can be achieved by a variety of methods , including intranasally , intraperitoneally , intracerebrally , intravenously , intratracheally , and via inhalation [ 88 , 89 ] , which opens up a range of experimental opportunities . the slightly larger body size allows a variety of experimental manipulations to be achieved with relative ease , including endotracheal intubation , bronchoalveolar lavage , serial venepuncture , csf collection , radiography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) . another benefit of the guinea pig model for fungal research is that oral doses of antifungals sufficient to control fungal infections are similar to the doses in humans . an example of the utilisation of the guinea pig model is the investigation into the effectiveness of an intravenous delivery of the antifungal itraconazole in fighting disseminated fungal infections including cryptococcosis . this is an example of the use of a whole - organism model to validate hypotheses of disease attenuation developed from in vitro experiments . mutagenesis and screening can be done on a large scale with the zebrafish , which is a fairly unique feature in vertebrate models . thus the zebrafish in many ways has the economical advantages of an invertebrate model whilst also possessing all of the vertebrate immune system features that researchers in fungal pathogenesis require . for example , a comprehensive infection model of the zebrafish with candida albicans has been established , in which pathogen morphogenesis and gene expression , and host immune response , were monitored . fish were killed by c. albicans in a dose - dependent manner , and infection was established at a number of different sites , indicating that this organism could be a valuable tool in fungal research . ex vivo organ culture could be considered a way of achieving a balance between the advantages of in vitro and in vivo experimental models . the use of perfused organs provides a relatively authentic physiological environment and permits the study of pathogens crossing membranes , disseminating through host tissue , and migrating to the bloodstream processes not captured by in vitro experimentation . organs from higher animals , which would otherwise carry enormous ethical and economic implications , can be studied when perfused organs are chosen over whole animal models . for example , haemoperfused liver from pigs was recently established as a model for c. albicans investigation . an ex vivo model has also been established for swine trachea , and since cryptococcus infects the lungs , this model could be of particular interest to adapt for investigation of this pathogen . cryptococcal research utilises a diverse range of experimental model systems , spanning from individual cells to unicellular whole organisms to mammalian models .

curcumin ( 17-bis ( 4-hydroxy-3-methoxyphenyl)-1,6-hepta - diene-3,5-dione ) extracted from curcuma longa l. is generally used as a spice and as a coloring agent in food [ 1 , 2 ] . curcumin has anti - inflammatory and antioxidant effects and downregulates chemokine expression in inflammatory cells [ 1 , 4 , 5 ] . curcumin affects kinase reactions , such as those of map kinase , and pkc , c - jun / ap-1 [ 6 , 7 ] . furthermore , several researchers , including our group , have reported that curcumin blocks nf - kb activity during transcription [ 8 , 9 ] . the molecular mechanism of nf - kb inhibition involves the blocking of ikb phosphorylation in a human nontransforming epithelial cell line [ 9 , 10 ] . although the inhibitory effect of curcumin on inflammatory responses is well known [ 11 , 12 ] , the contribution of curcumin to the signal transduction pathway in the proinflammatory cytokine response remains unclear . il-8 affects the functions of human neutrophils , including enhanced chemotaxis , enzyme release , and expression of surface adhesion molecules . these receptors are seven - transmembrane guanine nucleotide - binding protein - coupled receptors ( gpcrs ) . il-8 receptors are membrane - bound proteins expressed in large amount in primary cultured neutrophils . humans have two high - affinity receptors for il-8 , namely , cxcr1 and cxcr2 . the sequences of these receptors show 77% homology [ 13 , 14 ] . receptor - mediated signal transduction is initiated by coupling with heterotrimeric g proteins , and this results in an increase in the cytosolic free ca concentration due to activation by il-8 [ 14 , 15 ] . cxcr1 has limited activators , such as il-8 ; however , cxcr2 has numerous activator cytokines , that is , cxc chemokines such as nap-2 and gro [ 16 , 17 ] . recent evidence has suggested that il-8 receptors internalize via clathrin - coated vesicle upon agonist binding [ 18 , 19 ] . after the release of the agonist , the il-8 receptors are transported from the cytoplasm to the nucleus ; some of the receptors are transported back to the cell surface via the recycling endosomes . cxcr1 is recycled to the cell surface more rapidly than cxcr2 [ 17 , 1922 ] . it was reported that cxcr1 is the dominant mediator of the neutrophil chemotactic response to il-8 . thus , cxcr1 is involved in the crucial process of neutrophil - induced inflammation . we previously reported that il-8 and its receptors were constitutively produced by pancreatic cancer cells . in addition , the autocrine growth effect of il-8 is blocked by curcumin . in this process , curcumin contributes not only to the inhibition of il-8 production but also to the inhibition of signal transduction via il-8 receptors . these data suggested that curcumin is a potent agent that blocks the il-8 receptor - mediated neutrophil responses . in this study , we investigated the effect of curcumin on neutrophil chemotaxis mediated by il-8 and determined whether curcumin treatment inhibits signal transduction from the il-8 receptor in human primary neutrophils . we designed the study in order to test the hypothesis that curcumin treatment has an effect on the endosomal trafficking pathway of il-8 receptors . therefore , the endosomal vesicles that participate in the trafficking of il-8 receptors may play an important role in the curcumin - mediated regulation . the rab proteins ras - related small guanosine-5-triphosphatases ( gtpases ) might be essential since they are involved in the mechanisms of trafficking intracellular vesicles to the target organs [ 2426 ] . more than 60 types of rab proteins were found , and they play a role in regulating some of the steps involved in the intracellular trafficking of target molecules . considerable evidence showed that cxcr2 and rab11 coexist in the recycling step of cxcr2-transfected cells [ 27 , 28 ] . these data suggested that rab11 might be a key mediator in the distribution and trafficking pathway of il-8 receptors upon curcumin treatment . in this study , our data showed that curcumin treatment regulated the recycling of il-8 receptors and the amount of cell surface il-8 receptors . this change may be closely associated with the anti - inflammatory response caused by the interference of the chemotactic activity of il-8 . intraprep permeabilization reagent was obtained from immunotech ( marseille , france ) , ril-8 from r&d systems inc . ( minneapolis , minn , usa ) , diff - quick from midori juji ( osaka , japan ) , fura red - am from molecular probes ( eugene , ore , usa ) . anti - cxcr1 mouse monoclonal igg1 was purchased from santa cruz biotechnologies ( santa cruz , calif , usa ) . anti - cxcr2 mouse monoclonal igg1 , mouse igg1 , allophycocyanin ( apc)-conjugated rat antimouse igg1 monoclonal antibody , and purified mouse igg1 monoclonal igg standard were purchased from bd pharmingen ( san diego , calif , usa ) . heparinized whole blood was layered onto polymorphoprep ( axis - shield poc as ) and centrifuged at 500 xg for 35 minutes . the supernatant and the peripheral blood mononuclear cell ( pbmc ) layers were discarded . the polymorphonuclear neutrophil ( pmn ) layer was removed , and hyposmotic hemolysis was performed for the remaining erythrocytes . since nh4cl , a lysosomotrophic agent , may inhibit receptor trafficking in human neutrophils , the neutrophils used in these experiments were prepared without nh4cl . the pmns were resuspended in rpmi 1640 medium containing 10% fetal calf serum ( fcs ) . the purity of the neutrophil preparations was routinely found to be greater than 95% with viability greater than 98% , as observed by trypan blue exclusion test . curcumin was dissolved in dimethyl sulfoxide ( dmso ) on the day of use , and was added to the cells at a final concentration of 0.5100 m . trypan blue solution ( 0.4% ) was added to the cell suspension ( final concentration , 0.2% ) . after treatment with curcumin ( 10100 m ) for 2 hours , the number of positive cells was counted in triplicate . chemotaxis was assessed by a modification of a previously described method ; 24-well microchemotaxis chambers ( becton dickinson , cowley , uk ) were used , and these were covered with polyethylene terephthalate membranes with pore size of 3 m [ 29 , 30 ] . the lower wells were filled with 700 l of medium with various concentrations of recombinant human il-8 ( hril-8 ) ( 10 10000 ng / ml ) or medium without hril-8 . subsequently , 700 l of a suspension containing 1 10 pmns was added to each well of the upper chamber . after incubation under conditions of 90% humidity and 5% co2 for 1 hour at 37c , the cells that passed through the filter to the lower wells were stained with diff - quick and counted in 5 high - power fields ( hpf , 40 10 ) using a calibrated grating . neutrophil chemotactic activity was determined in triplicates and expressed as the average number of migrating neutrophils/5 hpf . next , to determine the effect of curcumin on neutrophil chemotaxis , the neutrophils were preincubated with various concentrations of curcumin ( 10 100 m ) at 37c for 2 hours . the preincubated neutrophils were added to each well of the upper chamber and incubated for 1 hour , as described above . the cells that passed through the filter to the lower wells were stained and counted . the obtained neutrophils were colored yellow by curcumin that has a bimodal peak of absorption at 210 nm and 425 nm . the color causes fluorescence noise when using optical instruments . to avoid interference due to the fluorescence noise , we used fura red because it shows decreased red fluorescence that is not interfered by curcumin . neutrophils ( 1 10 cells / ml ) were loaded with 13 m fura red - am in hepes - buffered saline ( 135 mm nacl , 4.6 mm kcl , 1.2 mm mgcl2 , 11 mm hepes , 11 mm glucose , 1.5 mm cacl2 , at ph 7.4 ) for 90 minutes at 37c in a co2 incubator . the loaded neutrophils were then analyzed using a facs calibur system ( beckton dickinson , usa ) for analyzing red fluorescence ( 640 nm ) following excitation at 488 nm using an argon laser . the mean channel of fluorescence was detected by cellquest software ( beckton dickinson ) [ 31 , 32 ] . the measurements of the mean channel of fluorescence can be used for statistical comparison of the cells before and after hril-8 treatment . the data obtained showed differences between the mean channel value before and after il-8 stimulation . internalization analysis was performed as previously described [ 8 , 15 ] . in brief , neutrophils were preincubated with rpmi 1640 medium containing 10% fcs supplemented with 50 m curcumin or without curcumin at 37c for 2 hours . the cells were washed twice in pbs and once in bovine serum albumin medium ( bsa medium = rpmi 1640 medium containing 1% bovine serum albumin and 25 mm hepes ) . aliquots of 5 10 cells were then removed and supplemented with 50 ng / ml hril-8 diluted in bsa medium , while no hril-8 was added to the control tubes . the cells were incubated at 37c for 2 hours and then incubated for an additional 10 minutes on ice . the cells were washed using the cell sorter buffer ( csb : pbs containing 1% fcs , 0.02% nan3 , and 25 mm hepes ) and incubated at 4c for 15 minutes with human igg to block the fc receptors . they were washed twice with csb and incubated with either monoclonal mouse anti - cxcr1 or anti - cxcr2 antibody and mouse - igg1a as the control . following the 15-minute incubation , the cells were washed twice in csb , and apc - conjugated rat antimouse igg antibody was added and incubated at 4c for 15 minutes . since apc emission is collected in a detector for fluorescence wavelengths between 640 nm and 680 nm , in these experiments , interference by curcumin fluorescence should be avoided . the cells were then washed once in csb and resuspended in pbs containing 0.02% nan3 . the neutrophils were permeabilized using the intraprep permeabilization reagent , including reagents 1 and 2 , according to the manufacturer 's instructions . in brief , to stain cxcr present on the surface of the cell membrane , the neutrophils were incubated with a primary antibody at 4c for 15 minutes , washed twice with csb , and stained with the secondary antibody for 15 minutes . after vigorous vortexing , the cells were incubated for 15 minutes at room temperature . after one wash with pbs , 100 l of intraprep reagent 2 was added , and then the mixture was incubated for 5 minutes at room temperature for permeabilization . next , the neutrophils were incubated with the primary antibody at 4c for 15 minutes , and the intracellular il-8 receptors were incubated with the secondary antibody for 15 minutes . following these incubations , the cells were washed once with csb and resuspended in pbs containing 1% formaldehyde . the pretreated neutrophils were lysed in a lysis buffer comprising 50 mm hepes ( ph 7.4 ) , 150 mm nacl , 1 mm edta , 1% triton x-100 , 10% glycerol , 1 mm naf , 2 mm na orthovandate , 0.1 nm phenylmethylsulfonyl fluoride , 5 g / ml leupeptin , 4.6 g / ml aprotinin , and 3.5 ng / ml pepstatin a. proteins in the cell lysates were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) under reducing conditions and then blotted onto nitrocellulose membranes ( hybond c , amersham pharmacia biotech , uk ) . the membranes were blocked in net buffer ( 50 mm tris - hcl , ph 7.5 , 150 mm nacl , 5 mm edta , 0.05% triton x-100 ) containing 1% gelatin and then incubated sequentially with the respective antibodies and rabbit or mouse igg trueblot ( ebioscience , san diego , calif , usa ) . the protein bands were visualized with an enhanced chemiluminescence ( ecl ) detection kit ( amersham pharmacia biotech ) . the lysates were precleared by incubation for 1 hour with trueblot antimouse ig ip beads ( ebioscience ) . the primary antibody and trueblot antimouse ig ip beads were added to the precleared lysates . the beads were washed in a washing buffer [ 50 mm hepes ( ph 7.4 ) , 150 mm nacl , 0.1% triton x-100 , 10% glycerol ] and resuspended in the sds sample buffer . the procedure used to determine receptor recycling was similar to that used to determine receptor internalization . the samples were allowed to undergo a receptor recovery process that was performed as previously described . in brief , internalization was induced by exposure to 1000 ng / ml hril-8 for 30 minutes at 37c . in order to allow receptor recovery , the cells were washed once and resuspended in bsa medium at 37c for the specified time periods . the neutrophils were incubated with the specified concentrations of curcumin during the il-8 receptor recovery process . to reduce the contribution of de novo protein synthesis to receptor reappearance on the cell membrane , cell samples undergoing receptor recovery following incubation , the cells were washed and labeled at 4c with anti - cxcr1 or anti - cxcr2 antibody and mouse igg , as described above . we observed that neutrophils rapidly passed through the membranes toward the wells of the lower chamber containing hril-8 . after 1 hour of incubation , the neutrophils that passed through the filter to the lower wells were counted . in figures 1(a ) and 1(b ) triplicate results of the average number of migrating neutrophils/5 hpf are shown . these data revealed that the chemotactic activity of neutrophils was stimulated by hril-8 in a dose - dependent manner . to determine the effect of curcumin on neutrophil chemotaxis , the neutrophils were incubated with various concentrations of curcumin added to the wells of the upper chamber . as shown in figures 1(c ) and 1(d ) , curcumin significantly inhibited cell migration induced by hril-8 ( 10000 pg / ml ) at concentrations of 10100 m in a dose - dependent manner . the obtained neutrophils were cultured in the presence of various doses of curcumin for 2 hours . curcumin did not affect neutrophil viability at a concentration up to 100 m when compared with the control treatment ( data not shown ) . in these experiments , agonist stimulation at 37c did not cause a significant decrease in the cell number or reduction in cell viability ( > 99% by trypan blue exclusion ) . to determine the effect of curcumin on intracellular signaling due to il-8-mediated il-8 receptor stimulation , we investigated the effects of changes in the intracellular calcium concentration . approximately 1 10 neutrophils treated with various concentrations of curcumin for 2 hours were loaded in a volume of 100 l in culture tubes containing 13 m fura red - am in hepes - buffered saline ( 135 mm nacl , 4.6 mm kcl , 1.2 mm mgcl2 , 11 mm hepes , 11 mm glucose , 1.5 mm cacl2 at ph 7.4 ) for 90 minutes at 37c in a co2 incubator . as shown in figure 2 , hril-8 ( 50 ng / ml ) increased the intracellular calcium concentration , and this phenomenon was demonstrated as a decrease in the fura red fluorescence . curcumin significantly inhibited the increase in the intracellular calcium concentrations in a dose - dependent manner . these data suggested that curcumin reduced neutrophil chemotaxis by affecting signal transduction mediated by il-8 via the il-8 receptors . to investigate the status of il-8 receptors after curcumin treatment , we examined the il-8 receptor on the cell surface by facscan using the cxcr1 and cxcr2 monoclonal antibodies . as shown in figure 3 , both cxcr1 and cxcr2 present on the cell surface were downregulated by hril-8 ( 50 ng / ml ) treatment due to receptor internalization . in addition , both il-8 receptors present on the surface of neutrophils treated with curcumin were downregulated by ril-8 ( 50 ng / ml ) to a greater extent . these data revealed that the amounts of intracellular cxcr1 and cxcr2 are relatively increased by curcumin treatment . these findings indicated that the regulation of neutrophil chemotaxis may depend on the decreased amounts of cxcr1 and cxcr2 present on the cell surface . to determine whether the production of il-8 receptors in neutrophils has influenced the curcumin treatment , we also evaluated the change of il-8 receptors without de novo synthesis which are transported to the cell surface . neutrophils were incubated with cycloheximide , a strong protein synthesis inhibitor , at a concentration of 10 ng / ml . facscan analysis showed the same decrease pattern of the amount of il-8 receptor present on the cell surface . cycloheximide treatment did not alter the reduction of il-8 receptors after hril-8 treatment in curcumin - treated neutrophils ( see figure 4 ) . these findings suggested that de novo synthesis of il-8 receptors may not be associated with the changes due to curcumin treatment of il-8 receptors present on the cell surface . to investigate the influence of curcumin on the degradation of il-8 receptors the expression of total cellular amount of il-8 receptors revealed no significant difference between the curcumin - treated and the control neutrophils ( see figure 5 ) . however , in the curcumin - treated neutrophils , the il-8 receptors on the cell surface showed obviously decrease than the control ( see figures 3(e ) and 3(f ) ) . taken together , these data suggested that curcumin changes the cellular distribution of il-8 receptors . the increment of the intercellular il-8 receptors may account for the decrement of il-8 receptors on the cell surface after curcumin treatment . in order to investigate the impact on the recycling pathway , the obtained neutrophils were pretreated with hril-8 ( 1000 ng / ml ) at 37c for 30 minutes . they were resuspended for the specified time periods in hril-8-free medium containing various concentrations of curcumin . representative data of cxcr1 recycling for 30 minutes and cxcr2 recycling for 120 minutes are shown in figure 6 . the recycling of cxcr1 and cxcr2 to the cell membrane was blocked by curcumin in a dose - dependent manner ( see figure 6 ) . in order to assess the interaction of the small gtpase rab11 that functionally regulates cytosolic trafficking of il-8 receptors in human primary neutrophils , we analyzed anti - cxcr1 and anti - cxcr2 immunoprecipitates for the presence of rab11 . western blotting showed that the total amount of the rab11 protein was not altered by curcumin treatment ( see figure 7(a ) ) . immunoprecipitation studies were performed to determine the association of cxcr1 and cxcr2 with rab11 after curcumin treatment . both receptors showed interaction with rab11 ( see figures 7(b ) , 7(c ) , and 7(d ) ) . various inflammations cause biological responses such as neutrophil activation through proinflammatory cytokine receptors ; however , the mechanisms that control these cytokine receptors remain unclear . in this study , we reported that the chemotaxis of primary cultured human neutrophils was significantly inhibited in a dose - dependent manner by curcumin treatment . this inhibition did not result from curcumin - induced cell cytotoxicity , since the total number of cells and the cell viability at the end of the culture period did not differ with the culture conditions . antichemotaxis provided important evidence for the anti - inflammatory responses due to curcumin treatment because neutrophil accumulation is a primary response in local inflammation , and this is followed by activation and infiltration of neutrophils . in particular , il-8 is a strong neutrophil chemoattractant in the local inflammatory site . other researchers , including our group , have already reported that curcumin inhibits the production of il-8 by monocytes , macrophages , and other lymphatic cells . the blockage of il-8 production should be one of the key factors in the regulation of inflammation caused by neutrophils . another way in which the inflammation elicited by neutrophils can be inhibited is by the interception of il-8 promoted signal transduction in neutrophils . in this study , experimental studies have demonstrated that human anti - cxcr monoclonal antibodies blocked il-8-dependent cellular functions including neutrophil chemotaxis directly . our previous study reported that exogenous il-8 could not recover response to il-8 receptor by curcumin treatment . therefore , we examined about il-8 signal transduction through il-8 receptors after curcumin treatment . in our study , a ca mobilization assay confirmed that il-8 signal transduction through il-8 receptors was blocked by curcumin treatment . regarding the mechanisms of il-8 receptor signaling , it is widely accepted that ligand - promoted internalization is one of the most important initiating pathways [ 19 , 21 , 22 , 35 ] . in neutrophils , il-8 stimulated the -arrestin - dependent internalization of the cxcr receptor [ 19 , 36 ] . upon agonist binding , cxcr1 and cxcr2 receptor activation is followed by receptor phosphorylation at multiple serine residues and subsequent desensitization of the receptor to further stimulation [ 17 , 21 , 37 ] . these events are usually accompanied by receptor endocytosis and/or recycling of the receptor . in our study , facscan analysis showed a decrease in the amount of both cxcr1 and cxcr2 present on the cell surface after curcumin treatment . in particular , after il-8 promoted receptor internalization , the amount of il-8 receptors on the neutrophil cell surface is significantly decreased . curcumin has been shown to inhibit nf - kb activity and to block a signal upstream of the nf - kb - inducing kinase and ikk and curcumin is suggested to regulate some transcriptional factors . after blocking new protein synthesis by cycloheximide , our experiments showed the same decrease pattern in the il-8 receptors present on the cell surface . this result suggests that the decrease in the amount of il-8 receptors present on the cell membrane was not only due to degradation . the possibility of the receptor shedding should be considered , but there is no convincible report concerning the shedding of il-8r . moreover , figure 5 demonstrated that the total amount of cxcr is not different after curcumin treatment , we consider that the possibility of the receptor shedding is deniable . this finding was consistent with those of our previous studies , suggesting that curcumin may affect the ligand - promoted trafficking pathway of the il-8 receptor . the trafficking pathway of il-8 receptors is known to comprise two different transport directions including internalization and externalization . either promotion of internalization or inhibition of externalization should induce a decrease in the amount of receptors present on the cell surface . our experiments based on facscan confirmed that curcumin delayed the trafficking pathway in the cytosol , and this resulted in the blockage of the recycling pathway to the cell surface . the rab gtpase family is known to play an essential role in the cellular trafficking pathway . in particular , rab11 is associated with the recycling compartment and trans - golgi network ( tgn ) membranes , and it controls the slow endosomal recycling pathway as well as the traffic from the tgn [ 24 , 26 , 38 ] . the small gtpase rab11 has been reported to regulate the trafficking of cxcr2 through the recycling endosome [ 27 , 28 ] . our immunoprecipitation study also showed the association of cxcr2 and rab11 in primary neutrophils . further analyses are required to determine the mechanism of blockage of il-8 receptor recycling ; however , this is the first report on the association of the il-8 receptor with rab11 in primary cultured neutrophils . following curcumin treatment , the binding of rab11 and cxcr1 and cxcr2 appeared to be more prominent . the recycling process from the endosome to the cell surface remains unclear ; however , stacking of the receptor proteins with the transporter proteins may be responsible for the deposition of il-8 receptors in the endosomes . therefore , it can be considered that these findings are consistent with those of previous studies that showed a reduction in the recycling of il-8 receptors . although our study had several limitations , these data suggest that curcumin might induce the stacking of the rab11 vesicle complex with cxcr1 and cxcr2 in the endocytic pathway . moreover , curcumin is suggested to affect signal transduction from the other transmembrane receptors , such as transferrin receptor and the m4 muscarinic acetylcholine receptor , whose recycling is regulated by rab11 through cytosolic trafficking pathways . since curcumin is also known to be an inhibitory factor for several serine / threonine kinases , such as pkc and ikk , gtpases activity might be inhibited by kinase dephosphorylation , and this could result in an increased stability of the transporter protein association . although phosphorylation is a crucial determinant of il-8 receptor internalization , our western blotting data showed no significant difference in the mobility of the il-8 receptor due to phosphorylation ( data not shown ) . previous studies were unable to completely explain the delays in cxcr1 and cxcr2 recycling to the cell surface by curcumin ; however , it is possible that curcumin has an effect on the transport system due to recycling of the endosomes because the association of the il-8 receptor with rab11 after curcumin treatment was increased . these findings have identified curcumin as a regulator of kinase activities , a regulator of transcriptions , and a modulator of oxidative agent . although there is no evidence about direct interact with membranous receptor , some investigators suggested that curcumin have a unique role in a modification of membranous or cytoskeleton system . previous study can not fully explain about mechanisms of regulation of il-8 receptor by curcumin , but our result is partially consistent with those of prior studies showing modification of cytosol components by curcumin . as for our studies ' limitation , we would have included an inactive analog in our study , if one had been available . there are several analogs and metabolites which have been reported , but almost analogs have a similar and modified effect of curcumin , which are not useful as an inactive control in this study . in conclusion , our data provide some noteworthy evidence that curcumin inhibits neutrophil chemotactic activity caused by il-8 chemokine . curcumin is suggested to inhibit cxcr1 and cxcr2 recycling ; this results in a decrease in neutrophil chemotaxis . in addition , we demonstrated that curcumin treatment changed the intercellular trafficking of the il-8 receptor in neutrophils . these mechanisms may involve inhibition of the endosomal transport pathway by members of the rab11 family . our study suggests that curcumin affects numerous bioactivities that involve signal transduction through the il-8 receptor . therefore , curcumin is able to function as a potent anti - inflammatory agent that regulates the receptor trafficking pathway in cytosol . in vivo use of curcumin might be beneficial for patients with inflammation due to the enhanced production of various proinflammatory cytokines , such as ards and pancreatitis as well as patients with the paraneoplastic syndrome . recently a great deal of evidence has revealed that cytokine overreaction contributes to the severity of the systemic inflammation response syndrome ( sirs ) . therefore , the control of cytokine signal transduction may play an important role in the inflammation therapy . the unique mechanisms by which curcumin regulates the receptor transport system should be further elucidated .

we investigated the impact of curcumin on neutrophils . chemotactic activity via human recombinant il-8 ( hril-8 ) was significantly inhibited by curcumin . curcumin reduced calcium ion flow induced by internalization of the il-8 receptor . we analyzed flow cytometry to evaluate the status of the il-8 receptor after curcumin treatment . the change in the distribution of receptors intracellularly and on the cell surface suggested that curcumin may affect the receptor trafficking pathway intracellulary . rab11 is a low molecular weight g protein associated with the cxcr recycling pathway . following curcumin treatment , immunoprecipitation studies showed that the il-8 receptor was associated with larger amounts of active rab11 than that in control cells . these data suggest that curcumin induces the stacking of the rab11 vesicle complex with cxcr1 and cxcr2 in the endocytic pathway . the mechanism for antiinflammatory response by curcumin may involve unique regulation of the rab11 trafficking molecule in recycling of il-8 receptors .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION

furthermore , several researchers , including our group , have reported that curcumin blocks nf - kb activity during transcription [ 8 , 9 ] . although the inhibitory effect of curcumin on inflammatory responses is well known [ 11 , 12 ] , the contribution of curcumin to the signal transduction pathway in the proinflammatory cytokine response remains unclear . il-8 receptors are membrane - bound proteins expressed in large amount in primary cultured neutrophils . humans have two high - affinity receptors for il-8 , namely , cxcr1 and cxcr2 . recent evidence has suggested that il-8 receptors internalize via clathrin - coated vesicle upon agonist binding [ 18 , 19 ] . after the release of the agonist , the il-8 receptors are transported from the cytoplasm to the nucleus ; some of the receptors are transported back to the cell surface via the recycling endosomes . cxcr1 is recycled to the cell surface more rapidly than cxcr2 [ 17 , 1922 ] . in addition , the autocrine growth effect of il-8 is blocked by curcumin . in this process , curcumin contributes not only to the inhibition of il-8 production but also to the inhibition of signal transduction via il-8 receptors . these data suggested that curcumin is a potent agent that blocks the il-8 receptor - mediated neutrophil responses . in this study , we investigated the effect of curcumin on neutrophil chemotaxis mediated by il-8 and determined whether curcumin treatment inhibits signal transduction from the il-8 receptor in human primary neutrophils . we designed the study in order to test the hypothesis that curcumin treatment has an effect on the endosomal trafficking pathway of il-8 receptors . therefore , the endosomal vesicles that participate in the trafficking of il-8 receptors may play an important role in the curcumin - mediated regulation . the rab proteins ras - related small guanosine-5-triphosphatases ( gtpases ) might be essential since they are involved in the mechanisms of trafficking intracellular vesicles to the target organs [ 2426 ] . more than 60 types of rab proteins were found , and they play a role in regulating some of the steps involved in the intracellular trafficking of target molecules . considerable evidence showed that cxcr2 and rab11 coexist in the recycling step of cxcr2-transfected cells [ 27 , 28 ] . these data suggested that rab11 might be a key mediator in the distribution and trafficking pathway of il-8 receptors upon curcumin treatment . in this study , our data showed that curcumin treatment regulated the recycling of il-8 receptors and the amount of cell surface il-8 receptors . this change may be closely associated with the anti - inflammatory response caused by the interference of the chemotactic activity of il-8 . trypan blue solution ( 0.4% ) was added to the cell suspension ( final concentration , 0.2% ) . the lower wells were filled with 700 l of medium with various concentrations of recombinant human il-8 ( hril-8 ) ( 10 10000 ng / ml ) or medium without hril-8 . next , to determine the effect of curcumin on neutrophil chemotaxis , the neutrophils were preincubated with various concentrations of curcumin ( 10 100 m ) at 37c for 2 hours . the preincubated neutrophils were added to each well of the upper chamber and incubated for 1 hour , as described above . in brief , to stain cxcr present on the surface of the cell membrane , the neutrophils were incubated with a primary antibody at 4c for 15 minutes , washed twice with csb , and stained with the secondary antibody for 15 minutes . next , the neutrophils were incubated with the primary antibody at 4c for 15 minutes , and the intracellular il-8 receptors were incubated with the secondary antibody for 15 minutes . the pretreated neutrophils were lysed in a lysis buffer comprising 50 mm hepes ( ph 7.4 ) , 150 mm nacl , 1 mm edta , 1% triton x-100 , 10% glycerol , 1 mm naf , 2 mm na orthovandate , 0.1 nm phenylmethylsulfonyl fluoride , 5 g / ml leupeptin , 4.6 g / ml aprotinin , and 3.5 ng / ml pepstatin a. proteins in the cell lysates were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) under reducing conditions and then blotted onto nitrocellulose membranes ( hybond c , amersham pharmacia biotech , uk ) . the membranes were blocked in net buffer ( 50 mm tris - hcl , ph 7.5 , 150 mm nacl , 5 mm edta , 0.05% triton x-100 ) containing 1% gelatin and then incubated sequentially with the respective antibodies and rabbit or mouse igg trueblot ( ebioscience , san diego , calif , usa ) . the beads were washed in a washing buffer [ 50 mm hepes ( ph 7.4 ) , 150 mm nacl , 0.1% triton x-100 , 10% glycerol ] and resuspended in the sds sample buffer . the neutrophils were incubated with the specified concentrations of curcumin during the il-8 receptor recovery process . to reduce the contribution of de novo protein synthesis to receptor reappearance on the cell membrane , cell samples undergoing receptor recovery following incubation , the cells were washed and labeled at 4c with anti - cxcr1 or anti - cxcr2 antibody and mouse igg , as described above . these data revealed that the chemotactic activity of neutrophils was stimulated by hril-8 in a dose - dependent manner . to determine the effect of curcumin on neutrophil chemotaxis , the neutrophils were incubated with various concentrations of curcumin added to the wells of the upper chamber . as shown in figures 1(c ) and 1(d ) , curcumin significantly inhibited cell migration induced by hril-8 ( 10000 pg / ml ) at concentrations of 10100 m in a dose - dependent manner . the obtained neutrophils were cultured in the presence of various doses of curcumin for 2 hours . curcumin did not affect neutrophil viability at a concentration up to 100 m when compared with the control treatment ( data not shown ) . in these experiments , agonist stimulation at 37c did not cause a significant decrease in the cell number or reduction in cell viability ( > 99% by trypan blue exclusion ) . to determine the effect of curcumin on intracellular signaling due to il-8-mediated il-8 receptor stimulation , we investigated the effects of changes in the intracellular calcium concentration . as shown in figure 2 , hril-8 ( 50 ng / ml ) increased the intracellular calcium concentration , and this phenomenon was demonstrated as a decrease in the fura red fluorescence . curcumin significantly inhibited the increase in the intracellular calcium concentrations in a dose - dependent manner . these data suggested that curcumin reduced neutrophil chemotaxis by affecting signal transduction mediated by il-8 via the il-8 receptors . to investigate the status of il-8 receptors after curcumin treatment , we examined the il-8 receptor on the cell surface by facscan using the cxcr1 and cxcr2 monoclonal antibodies . as shown in figure 3 , both cxcr1 and cxcr2 present on the cell surface were downregulated by hril-8 ( 50 ng / ml ) treatment due to receptor internalization . in addition , both il-8 receptors present on the surface of neutrophils treated with curcumin were downregulated by ril-8 ( 50 ng / ml ) to a greater extent . these data revealed that the amounts of intracellular cxcr1 and cxcr2 are relatively increased by curcumin treatment . these findings indicated that the regulation of neutrophil chemotaxis may depend on the decreased amounts of cxcr1 and cxcr2 present on the cell surface . to determine whether the production of il-8 receptors in neutrophils has influenced the curcumin treatment , we also evaluated the change of il-8 receptors without de novo synthesis which are transported to the cell surface . facscan analysis showed the same decrease pattern of the amount of il-8 receptor present on the cell surface . cycloheximide treatment did not alter the reduction of il-8 receptors after hril-8 treatment in curcumin - treated neutrophils ( see figure 4 ) . these findings suggested that de novo synthesis of il-8 receptors may not be associated with the changes due to curcumin treatment of il-8 receptors present on the cell surface . to investigate the influence of curcumin on the degradation of il-8 receptors the expression of total cellular amount of il-8 receptors revealed no significant difference between the curcumin - treated and the control neutrophils ( see figure 5 ) . however , in the curcumin - treated neutrophils , the il-8 receptors on the cell surface showed obviously decrease than the control ( see figures 3(e ) and 3(f ) ) . taken together , these data suggested that curcumin changes the cellular distribution of il-8 receptors . the increment of the intercellular il-8 receptors may account for the decrement of il-8 receptors on the cell surface after curcumin treatment . in order to investigate the impact on the recycling pathway , the obtained neutrophils were pretreated with hril-8 ( 1000 ng / ml ) at 37c for 30 minutes . they were resuspended for the specified time periods in hril-8-free medium containing various concentrations of curcumin . the recycling of cxcr1 and cxcr2 to the cell membrane was blocked by curcumin in a dose - dependent manner ( see figure 6 ) . in order to assess the interaction of the small gtpase rab11 that functionally regulates cytosolic trafficking of il-8 receptors in human primary neutrophils , we analyzed anti - cxcr1 and anti - cxcr2 immunoprecipitates for the presence of rab11 . western blotting showed that the total amount of the rab11 protein was not altered by curcumin treatment ( see figure 7(a ) ) . immunoprecipitation studies were performed to determine the association of cxcr1 and cxcr2 with rab11 after curcumin treatment . in this study , we reported that the chemotaxis of primary cultured human neutrophils was significantly inhibited in a dose - dependent manner by curcumin treatment . this inhibition did not result from curcumin - induced cell cytotoxicity , since the total number of cells and the cell viability at the end of the culture period did not differ with the culture conditions . antichemotaxis provided important evidence for the anti - inflammatory responses due to curcumin treatment because neutrophil accumulation is a primary response in local inflammation , and this is followed by activation and infiltration of neutrophils . in particular , il-8 is a strong neutrophil chemoattractant in the local inflammatory site . other researchers , including our group , have already reported that curcumin inhibits the production of il-8 by monocytes , macrophages , and other lymphatic cells . the blockage of il-8 production should be one of the key factors in the regulation of inflammation caused by neutrophils . our previous study reported that exogenous il-8 could not recover response to il-8 receptor by curcumin treatment . therefore , we examined about il-8 signal transduction through il-8 receptors after curcumin treatment . in our study , a ca mobilization assay confirmed that il-8 signal transduction through il-8 receptors was blocked by curcumin treatment . regarding the mechanisms of il-8 receptor signaling , it is widely accepted that ligand - promoted internalization is one of the most important initiating pathways [ 19 , 21 , 22 , 35 ] . in neutrophils , il-8 stimulated the -arrestin - dependent internalization of the cxcr receptor [ 19 , 36 ] . upon agonist binding , cxcr1 and cxcr2 receptor activation is followed by receptor phosphorylation at multiple serine residues and subsequent desensitization of the receptor to further stimulation [ 17 , 21 , 37 ] . these events are usually accompanied by receptor endocytosis and/or recycling of the receptor . in our study , facscan analysis showed a decrease in the amount of both cxcr1 and cxcr2 present on the cell surface after curcumin treatment . in particular , after il-8 promoted receptor internalization , the amount of il-8 receptors on the neutrophil cell surface is significantly decreased . curcumin has been shown to inhibit nf - kb activity and to block a signal upstream of the nf - kb - inducing kinase and ikk and curcumin is suggested to regulate some transcriptional factors . after blocking new protein synthesis by cycloheximide , our experiments showed the same decrease pattern in the il-8 receptors present on the cell surface . this result suggests that the decrease in the amount of il-8 receptors present on the cell membrane was not only due to degradation . the possibility of the receptor shedding should be considered , but there is no convincible report concerning the shedding of il-8r . moreover , figure 5 demonstrated that the total amount of cxcr is not different after curcumin treatment , we consider that the possibility of the receptor shedding is deniable . this finding was consistent with those of our previous studies , suggesting that curcumin may affect the ligand - promoted trafficking pathway of the il-8 receptor . the trafficking pathway of il-8 receptors is known to comprise two different transport directions including internalization and externalization . either promotion of internalization or inhibition of externalization should induce a decrease in the amount of receptors present on the cell surface . our experiments based on facscan confirmed that curcumin delayed the trafficking pathway in the cytosol , and this resulted in the blockage of the recycling pathway to the cell surface . the rab gtpase family is known to play an essential role in the cellular trafficking pathway . in particular , rab11 is associated with the recycling compartment and trans - golgi network ( tgn ) membranes , and it controls the slow endosomal recycling pathway as well as the traffic from the tgn [ 24 , 26 , 38 ] . further analyses are required to determine the mechanism of blockage of il-8 receptor recycling ; however , this is the first report on the association of the il-8 receptor with rab11 in primary cultured neutrophils . following curcumin treatment , the binding of rab11 and cxcr1 and cxcr2 appeared to be more prominent . the recycling process from the endosome to the cell surface remains unclear ; however , stacking of the receptor proteins with the transporter proteins may be responsible for the deposition of il-8 receptors in the endosomes . therefore , it can be considered that these findings are consistent with those of previous studies that showed a reduction in the recycling of il-8 receptors . although our study had several limitations , these data suggest that curcumin might induce the stacking of the rab11 vesicle complex with cxcr1 and cxcr2 in the endocytic pathway . since curcumin is also known to be an inhibitory factor for several serine / threonine kinases , such as pkc and ikk , gtpases activity might be inhibited by kinase dephosphorylation , and this could result in an increased stability of the transporter protein association . although phosphorylation is a crucial determinant of il-8 receptor internalization , our western blotting data showed no significant difference in the mobility of the il-8 receptor due to phosphorylation ( data not shown ) . previous studies were unable to completely explain the delays in cxcr1 and cxcr2 recycling to the cell surface by curcumin ; however , it is possible that curcumin has an effect on the transport system due to recycling of the endosomes because the association of the il-8 receptor with rab11 after curcumin treatment was increased . although there is no evidence about direct interact with membranous receptor , some investigators suggested that curcumin have a unique role in a modification of membranous or cytoskeleton system . previous study can not fully explain about mechanisms of regulation of il-8 receptor by curcumin , but our result is partially consistent with those of prior studies showing modification of cytosol components by curcumin . there are several analogs and metabolites which have been reported , but almost analogs have a similar and modified effect of curcumin , which are not useful as an inactive control in this study . in conclusion , our data provide some noteworthy evidence that curcumin inhibits neutrophil chemotactic activity caused by il-8 chemokine . curcumin is suggested to inhibit cxcr1 and cxcr2 recycling ; this results in a decrease in neutrophil chemotaxis . in addition , we demonstrated that curcumin treatment changed the intercellular trafficking of the il-8 receptor in neutrophils . these mechanisms may involve inhibition of the endosomal transport pathway by members of the rab11 family . our study suggests that curcumin affects numerous bioactivities that involve signal transduction through the il-8 receptor . therefore , curcumin is able to function as a potent anti - inflammatory agent that regulates the receptor trafficking pathway in cytosol . in vivo use of curcumin might be beneficial for patients with inflammation due to the enhanced production of various proinflammatory cytokines , such as ards and pancreatitis as well as patients with the paraneoplastic syndrome .

the statistical model proposed in for one probe set in multiple oligonucleotide arrays has the form it states that the perfect match ( pm)/mismatch ( mm ) difference in array i , probe j of this probe set is the product of model - based expression index ( mbei ) in array i ( i ) and probe - sensitivity index of probe j ( j ) plus random error . fitting the model , we can identify cross - hybridizing probes ( j with large standard error ( se ) , which are excluded during iterative fitting ) and arrays with image contamination at this probe set ( i with large se ) , as well as single outliers ( image spikes ) which are replaced by the fitted values . in effect the estimated expression index i is a weighted average of pm / mm differences : with larger weights given to probes with larger . the image of outliers ( array and single outliers ) identified through model - fitting can be used to assess the quality of an experiment and to identify unexpected problems such as a misaligned corner of a dat file . we have investigated several important properties of the model , including the reliability and stability of the fitted parameters mbei ( ) and probe sensitivity indexes ( ) , the performance of mbei compared to the commonly used average difference ( ad ) , and how the availability of se facilitates downstream comparative and clustering analysis . in practice , in an array experiment , a researcher hybridizes tissue or cell line samples , corresponding to different treatments or conditions , to a batch of arrays . ideally , the probe - sensitivity index ( ) should be independent of the tissue type . this condition , however , may not hold for those probes that have cross - hybridization affinity to non - target genes . nevertheless , assuming that a non - target gene cross - hybridizes only to a few probes of a probe set , and its expression levels across arrays do not correlate with the target gene , the iterative probe - excluding procedure in may be able to exclude cross - hybridizing probes , regardless of the tissue type hybridized . in addition , the relative probe - sensitivity indexes of the good probes called by the model are likely to be similar across sets of arrays hybridizing to different tissue samples . we apply the model ( equation 1 ) independently to six sets of hu6800 arrays ( 21 leukemia , lymphoma and mantle cell samples , 20 prostate cancer cell lines , 17 brain tumor samples , 55 cancer cell lines , 58 brain samples , and 55 lung tumor samples ) . figure 1a shows the values fitted for probe set 6457 ( used in figure 1 and 2 of ) in the six array sets . the patterns resemble each other greatly , showing that the probe - sensitivity index is an inherent property of these non - cross - hybridizing probes and can be consistently identified from different sets of arrays . it is noteworthy that the probe 11 in array set 5 is likely to be cross - hybridizing , making its relative strength ( here mm is consistently larger than pm and this leads to a negative ) dissimilar to the probe 11 in other array sets . the model identifies this probe as a ' probe - outlier ' only for array set 5 and excludes it when calculating mbei ( 0 ) for array set 5 . values estimated for probe sets ( a ) 6457 , ( b ) 1248 , and ( c ) 6571 in six array sets ( shown in panels 16 from left to right for each probe set ) . values ( constrained to have sum square equal to number of probes used in each array set ) are on the y - axis , and probe pairs are labeled 1 to 20 on the x - axis . the title of each panel ( for example , p = 0 ) indicates the proportion of arrays ' present ' for the target gene in the array set . they are stratified by average lower presence proportion in two array sets ( the presence proportion of a probe set is the proportion of arrays in an array set where the target gene is called ' present ' by genechip 's algorithm ) . the average is taken over c(6 , 2 ) = 15 pairwise comparison of two array sets for each probe set , and the correlation is calculated using probes that are not identified as an outlier in both array sets . the range of the average lower presence proportion for the six boxplots are : ( 0 , 0.17 ) , ( 0.17 , 0.34 ) , ( 0.34 , 0.51 ) , ( 0.51 , 0.68 ) , ( 0.68 , 0.85 ) , ( 0.85 , 1 ) . the title of each boxplot is the number of probe sets classified into this boxplot . eleven probe sets with too few non - outlier probes to calculate correlations for all 15 comparisons are not included in the boxplots . the average lower presence proportion and average pairwise correlation for probe sets in figure 1 are ( a ) 1 , 0.95 ; ( b ) , 0.93 , 0.94 ; and ( c ) 0 , 0.86 . in figure 1a , b the target gene is present in most samples of all array sets . for a probe set whose target gene is mostly absent throughout samples ( figure 1c ) , many probes are identified as probe - outliers because of their negative indexes . here , we can not obtain correct probe - sensitivity indexes because of the absence of the target gene . nevertheless , the pm - mm values for these probes are random fluctuations around zero , leading to a correct expression index close to zero . if the target gene becomes available for a future array set , the correct probe - sensitivity indexes will be recovered and these probes will be used for expression calculation . occasionally , a responsive probe set may give rise to very different estimates in two array sets . in figure 1b , probes 8 and 13 have different relative responses in array set 1 and 4 , leading to different probe - response patterns . this might be due to the possibility that the probes in this probe set are differentially cross - hybridized in different array sets , or that the same probe in different batches of arrays may systematically behave differently . identification and flagging such probe sets is desirable and essential if we want to compare arrays hybridized to different tissue samples . figure 2 shows the boxplots of average pairwise correlations of values between two array sets , stratified by average lower presence proportion in the two sets . in general , when a gene is present in many samples of two array sets , the patterns estimated from the two sets are very similar . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . from figure 1 of , one can see that some mm probes may respond poorly to the changes in the expression level of the target gene . this phenomenon raised questions on the efficiency of using mm probes , and led some investigators to design custom arrays that use pm probes exclusively ( r. abagyan and yingyao zhou , personal communication ; b.r . conklin , personal communication ) , and others to calculate fold changes using only pm probes ( f. naef , personal communication ) . this design greatly increases the number of genes that can be studied on one array . to investigate the relative performance of pm / mm versus pm - only designs , we exploited the model to estimate gene expression levels using only pm probes , and compared it to the mbei using both pm and mm probes . the full intensity model ( equation 1 of ) specifies the relationship of pm probe responses and expression level : where vj is the baseline response of probe pair j due to nonspecific hybridization , and ' j is the sensitivity of pm probe of the probe pair j. the parameter estimates can be obtained by iteratively fitting i and vj,'j , regarding the other set as known . the mm probe responses have a similar form as equation 2 except for different probe - sensitivity indexes . we fit a pm - only and an mm - only model to obtain expression values of all 20-probe probe sets using array set 1 . for comparison , we also used half of the probe pairs ( by alternatively picking one out of every two probes ) in a 20-probe probe set to fit to the difference model ( equation 1 ) . for each probe set , these three sets of expression values were compared with the expression values of the original difference model using 20 probes , in terms of correlation of s obtained by two methods across the 21 arrays . we assumed the 20-probe difference model provides the most accurate expression estimates . if , for a probe set , a simplified model ( pm - only , mm - only or 10-probe difference model ) performs reasonably well , we expect its estimates to correlate with that from the 20-probe difference model . figure 3 shows the histogram and figure 4 the boxplot of correlations of s estimated from the 20-probe difference model and s estimated from the 10-probe difference model ( a ) , the 20-probe pm - only model ( b ) and the 20-probe mm - only model ( c ) . for probe sets with high presence proportion , both the 10-probe difference model and the pm - only model correlate well with the 20-probe difference model . we note that this comparison is intrinsically biased in favor of the 10-probe difference model because the ' truth ' is constructed from pm - mm differences . histogram of correlations between model - based expression values estimated using the 20-probe difference model and those estimated using different models . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; ( c ) 20-probe mm - only model . boxplot of correlations between values estimated using the 20-probe difference model and s estimated using different models , stratified by presence proportion . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; and ( c ) 20-probe mm - only model . the number of presence calls for a probe set in the 21 arrays and the subpopulation size for the six boxplots are : 03 , 4,385 ; 47 , 693 ; 811 , 413 ; 1215 , 488 ; 1619 , 497 ; and 2021 , 323 . this comparison corroborates the basic notion of the technology : the pm probes hybridize more strongly to the target signals than mm probes and contain most of the information . we stress that , whereas the above analysis illustrates the applicability of model - based analysis to pm - only arrays , the assessment presented here is only tentative because of the limited information provided by the hu6800 arrays on the comparisons . definitive comparisons of the efficiency of the designs must await the availability of data from pm - only arrays . each pair consists of two arrays hybridizing to samples replicated at total mrna level ( the total mrna sample is split and then amplified and labeled separately , and hybridized to two different arrays ) . the differences between the expression values of the two replicate arrays in a pair are due to the variation introduced in experimental steps after the split , the array manufacturing difference and analytical methods such as normalization and expression calculation . this difference provides a lower bound of biological variation that can be detected between two independently amplified samples , and serves as a good statistic for comparing different analytical methods . for comparison , we also used the method in to calculate ads for all probe sets and plot them in figure 5b ( ad is based on normalized probe values , see methods and materials section for the normalization method . also note that genechip software excludes probes whose pm / mm difference is outside three standard deviations ( sds ) of all probe differences in either of the two arrays in the comparison ; here , as we are comparing multiple arrays at the same time , when calculating ads a probe is excluded if its difference is an outlier in the above sense in any of the arrays , until a minimum of five probes is reached , where all five probes will be used ) . both the mbei and the ad method yielded some expression values differing by more than a factor of two , especially for genes at low expression level . this might be explained by the relatively larger amplification variation for weakly expressed genes , given a constant success rate of amplifying a sequence by a certain fold . log ( base 10 ) expression indexes of a pair of replicate arrays ( array 1 and 2 of array set 5 ) for different statistical methods . only 6,695 ( a ) and 4,696 ( b ) probe sets with positive values in both arrays are used . the center line is y = x , and the flanking lines indicate the difference of a factor of two . researchers often use ' log ratio ' between expression values of a gene in two arrays as the criterion for identifying differentially expressed genes . between duplicate arrays , we expect these log ratios of expression values based on a good expression index ( ad or mbei ) to be close to zero . thus for every probe set we calculated its average absolute log ( base 10 ) ratio of 29 pairs of duplicates as a statistic to compare the variation in expression levels between duplicates using the ad or the mbei method . the average absolute log ratio distribution of the mbei method is significantly lower than that of the ad method when expression level is low ( and thus probe sets have a low proportion of detections of the target gene across arrays ) . as expression level becomes higher ( when the target gene of a probe set is detected in more arrays ) , the ad method shows a rapid improvement in performance , approaching the level of the mbei method . the same boxplots ( figure 7 ) for another set of 60 human u95a arrays consisting of 30 replicate pairs conveys similar information . these results suggest that the mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . boxplots of average absolute log ( base 10 ) ratios between replicate arrays stratified by presence proportion for different statistical methods . ( a ) mbei method ; ( b ) ad method . the number of presence calls for a probe set in the 58 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5058 . the title of each boxplot is the number of probe sets used for the boxplot . log ratios are not calculated for negative expression values or expression values identified as ' array - outliers ' by the mbei method in either array of a replicate pair , and are not used to calculate the average . 744 probe sets are not included as their average absolute log ratios can not be calculated for all the 29 pairs using either method . similar plots as in figure 6 for another set of 30 pairs of duplicated human u95a arrays . ( a ) mbei method ; ( b ) ad method.the number of presence calls for a probe set in the 60 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5060 . the title of each boxplot is the number of probe sets used for the boxplot . after obtaining expression indexes using ad or mbei , fold changes can be calculated between two arrays for every gene and used to identify differentially expressed genes . usually , low or negative expressions are truncated to a small number before calculating fold changes , and genechip also cautions against using fold changes when the baseline expression is absent . the availability of ses for the model - based expression indexes allows us to obtain confidence intervals for fold changes . suppose where 1 and 2 are the real expression levels in the sample , and 1 and 2 are the model - based estimates of expression levels . we substitute the model - based ses for 1 and 2 letting r = 1/2 be the real fold change , then inference on r can be based on the quantity it can be shown that q has a distribution with 1 degree of freedom irrespective of the values of 1 and 2 . thus q is a pivotal quantity involving r. we can use q to construct fixed - level tests and to invert them to obtain confidence intervals ( ci ) for fold changes . table 1 presents the estimated expression indexes ( with ses ) in two arrays and the 90% confidence intervals of the fold changes for 14 genes . although all genes have similar estimated fold changes , the confidence intervals are very different . for example , gene 1 has fold change 2.47 and a tight confidence interval ( 2.06 , 3.02 ) . in contrast , gene 11 has a similar fold change of 2.48 but a much wider confidence interval ( 0.96 , 18.18 ) . thus the fold change around 2.5 for gene 11 is not as trustworthy as that for gene 1 . further examination reveals that this is due to the large ses relative to the expression indexes for gene 11 . this agrees with the intuition that when one or both expression levels are close to zero for one gene , the fold change can not be estimated with much accuracy . in addition , when image contamination results in unreliable expression values with large ses , the fold changes calculated using these expression value are attached with wide cis . in this manner , the measurement accuracy of expression values propagates to the estimation of fold changes . using expression levels and associated ses to determine confidence intervals of fold changes in practice , we find it useful to sort genes by the lower confidence bound ( ' lower cb ' in table 1 ) , which is a conservative estimate of the fold change . when an expression index is negative ( as a result of taking pm / mm differences ) , we do not calculate the confidence intervals . in such a case , it is more helpful to filter genes by presence calls . cluster analysis is a popular method for analyzing the data of a series of microarrays [ 6 , 7 ] . if two genes are co - regulated at the transcription level , their expression values across samples are likely to be correlated . clustering algorithms use these correlations ( or monotone transformation of correlations ) to cluster co - regulated genes together the correlation based on the estimated expression levels may , however , be different from that based on the real but unobserved expression levels . also , the commonly used hierarchical clustering algorithm is an irreversible process : once two genes or nodes are merged , they will stay together , even if later on there is good reason to adjust previous clustering . a global way of using se in hierarchical clustering is to resample or bootstrap the whole ' gene by sample ' data matrix and redo the clustering , then investigate the overall properties emerging from this repertoire of clustering trees . in bittner et al . , the data matrix coming from cdna microarray experiments is resampled using the estimated variation derived from the median sd of log ratios for a gene across samples . as we now have ses for all data points , we can resample each expression value from a normal distribution with mean equal to the estimated expression value and sd equal to the attached se . figure 8a shows a hierarchical clustering tree of 225 selected genes with presence proportion > 0.5 and coefficient of variation ( sd / mean ) > 0.7 across the 20 samples in array set 2 . in trying to interpret this tree , we may be interested in the gene cluster colored in blue and the reliability of the gene members belonging to this cluster . the whole data matrix is resampled , and the clustering is performed again ( figure 8b ) . we notice that some blue genes ( genes in the original cluster are colored blue ) are clustered with other non - blue genes , and some non - blue genes are mixed into the main body of the blue genes . after each resampling , we identify a cluster that contains more than 80% of all the blue genes , but as few non - blue genes as possible ( measured as a percentage of all genes in this cluster ) . this cluster is considered to be the cluster that corresponds to the original one in figure 8a . in figure 8b the root node of the ' corresponding cluster ' is marked with small horizontal line intersecting the vertical line ( representing the range of the cluster ) on the right of the clustering picture . then , for each of all the 225 genes , if it belongs to this ' corresponding cluster ' , we increase its ' in - cluster ' count by 1 . after resampling 30 times , the in - cluster counts are indicated in gray - scale on the left side of the original clustering ( figure 8c ) , with black representing 30 and white representing zero . a high ' in - cluster ' count indicates a gene ' remains ' in the original cluster in most of the resampled clustering trees . gene clustering ( a ) 225 filtered genes are clustered based on their expression profiles across 20 samples . each gene 's expression values are standardized to have mean 0 and sd 1 across 20 samples . although the original ' blue ' genes are scattered to various places , we can still determine where the original cluster is , using the criteria described in the text . ( c ) after resampling 30 times , the reliability of the genes belonging to the original cluster is indicated by the vertical gray - scale bar on the left of the blue - red picture . we can see from figure 8c that most genes in the original cluster are reliable members , whereas a few genes at the bottom of the cluster are not ( in fact they are merged into the original cluster last ) . interestingly , some genes originally not in the original cluster group with the ' corresponding clusters ' during resampling many times and have gray ' in - cluster ' marks . these genes may be related to the original cluster in some way . in summary , this method can help us to distinguish reliable and unreliable gene members of a cluster , as well as draw our attention to related genes originally clustered somewhere else because of the accidental nature of hierarchical clustering . in practice , in an array experiment , a researcher hybridizes tissue or cell line samples , corresponding to different treatments or conditions , to a batch of arrays . ideally , the probe - sensitivity index ( ) should be independent of the tissue type . this condition , however , may not hold for those probes that have cross - hybridization affinity to non - target genes . nevertheless , assuming that a non - target gene cross - hybridizes only to a few probes of a probe set , and its expression levels across arrays do not correlate with the target gene , the iterative probe - excluding procedure in may be able to exclude cross - hybridizing probes , regardless of the tissue type hybridized . in addition , the relative probe - sensitivity indexes of the good probes called by the model are likely to be similar across sets of arrays hybridizing to different tissue samples . we apply the model ( equation 1 ) independently to six sets of hu6800 arrays ( 21 leukemia , lymphoma and mantle cell samples , 20 prostate cancer cell lines , 17 brain tumor samples , 55 cancer cell lines , 58 brain samples , and 55 lung tumor samples ) . figure 1a shows the values fitted for probe set 6457 ( used in figure 1 and 2 of ) in the six array sets . the patterns resemble each other greatly , showing that the probe - sensitivity index is an inherent property of these non - cross - hybridizing probes and can be consistently identified from different sets of arrays . it is noteworthy that the probe 11 in array set 5 is likely to be cross - hybridizing , making its relative strength ( here mm is consistently larger than pm and this leads to a negative ) dissimilar to the probe 11 in other array sets . the model identifies this probe as a ' probe - outlier ' only for array set 5 and excludes it when calculating mbei ( 0 ) for array set 5 . values estimated for probe sets ( a ) 6457 , ( b ) 1248 , and ( c ) 6571 in six array sets ( shown in panels 16 from left to right for each probe set ) . values ( constrained to have sum square equal to number of probes used in each array set ) are on the y - axis , and probe pairs are labeled 1 to 20 on the x - axis . the title of each panel ( for example , p = 0 ) indicates the proportion of arrays ' present ' for the target gene in the array set . they are stratified by average lower presence proportion in two array sets ( the presence proportion of a probe set is the proportion of arrays in an array set where the target gene is called ' present ' by genechip 's algorithm ) . the average is taken over c(6 , 2 ) = 15 pairwise comparison of two array sets for each probe set , and the correlation is calculated using probes that are not identified as an outlier in both array sets . the range of the average lower presence proportion for the six boxplots are : ( 0 , 0.17 ) , ( 0.17 , 0.34 ) , ( 0.34 , 0.51 ) , ( 0.51 , 0.68 ) , ( 0.68 , 0.85 ) , ( 0.85 , 1 ) . the title of each boxplot is the number of probe sets classified into this boxplot . eleven probe sets with too few non - outlier probes to calculate correlations for all 15 comparisons are not included in the boxplots . the average lower presence proportion and average pairwise correlation for probe sets in figure 1 are ( a ) 1 , 0.95 ; ( b ) , 0.93 , 0.94 ; and ( c ) 0 , 0.86 . in figure 1a , b the target gene is present in most samples of all array sets . for a probe set whose target gene is mostly absent throughout samples ( figure 1c ) , many probes are identified as probe - outliers because of their negative indexes . here , we can not obtain correct probe - sensitivity indexes because of the absence of the target gene . nevertheless , the pm - mm values for these probes are random fluctuations around zero , leading to a correct expression index close to zero . if the target gene becomes available for a future array set , the correct probe - sensitivity indexes will be recovered and these probes will be used for expression calculation . occasionally , a responsive probe set may give rise to very different estimates in two array sets . in figure 1b , probes 8 and 13 have different relative responses in array set 1 and 4 , leading to different probe - response patterns . this might be due to the possibility that the probes in this probe set are differentially cross - hybridized in different array sets , or that the same probe in different batches of arrays may systematically behave differently . identification and flagging such probe sets is desirable and essential if we want to compare arrays hybridized to different tissue samples . figure 2 shows the boxplots of average pairwise correlations of values between two array sets , stratified by average lower presence proportion in the two sets . in general , when a gene is present in many samples of two array sets , the patterns estimated from the two sets are very similar . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . from figure 1 of , one can see that some mm probes may respond poorly to the changes in the expression level of the target gene . this phenomenon raised questions on the efficiency of using mm probes , and led some investigators to design custom arrays that use pm probes exclusively ( r. abagyan and yingyao zhou , personal communication ; b.r . conklin , personal communication ) , and others to calculate fold changes using only pm probes ( f. naef , personal communication ) . this design greatly increases the number of genes that can be studied on one array . to investigate the relative performance of pm / mm versus pm - only designs , we exploited the model to estimate gene expression levels using only pm probes , and compared it to the mbei using both pm and mm probes . the full intensity model ( equation 1 of ) specifies the relationship of pm probe responses and expression level : where vj is the baseline response of probe pair j due to nonspecific hybridization , and ' j is the sensitivity of pm probe of the probe pair j. the parameter estimates can be obtained by iteratively fitting i and vj,'j , regarding the other set as known . the same outlier exclusion procedure in is applied . the mm probe responses have a similar form as equation 2 except for different probe - sensitivity indexes . we fit a pm - only and an mm - only model to obtain expression values of all 20-probe probe sets using array set 1 . for comparison , we also used half of the probe pairs ( by alternatively picking one out of every two probes ) in a 20-probe probe set to fit to the difference model ( equation 1 ) . for each probe set , these three sets of expression values were compared with the expression values of the original difference model using 20 probes , in terms of correlation of s obtained by two methods across the 21 arrays . if , for a probe set , a simplified model ( pm - only , mm - only or 10-probe difference model ) performs reasonably well , we expect its estimates to correlate with that from the 20-probe difference model . figure 3 shows the histogram and figure 4 the boxplot of correlations of s estimated from the 20-probe difference model and s estimated from the 10-probe difference model ( a ) , the 20-probe pm - only model ( b ) and the 20-probe mm - only model ( c ) . for probe sets with high presence proportion , both the 10-probe difference model and the pm - only model correlate well with the 20-probe difference model . the mm - only model yields noticeably lower correlations , however . we note that this comparison is intrinsically biased in favor of the 10-probe difference model because the ' truth ' is constructed from pm - mm differences . histogram of correlations between model - based expression values estimated using the 20-probe difference model and those estimated using different models . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; ( c ) 20-probe mm - only model . boxplot of correlations between values estimated using the 20-probe difference model and s estimated using different models , stratified by presence proportion . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; and ( c ) 20-probe mm - only model . the number of presence calls for a probe set in the 21 arrays and the subpopulation size for the six boxplots are : 03 , 4,385 ; 47 , 693 ; 811 , 413 ; 1215 , 488 ; 1619 , 497 ; and 2021 , 323 . this comparison corroborates the basic notion of the technology : the pm probes hybridize more strongly to the target signals than mm probes and contain most of the information . we stress that , whereas the above analysis illustrates the applicability of model - based analysis to pm - only arrays , the assessment presented here is only tentative because of the limited information provided by the hu6800 arrays on the comparisons . definitive comparisons of the efficiency of the designs must await the availability of data from pm - only arrays . each pair consists of two arrays hybridizing to samples replicated at total mrna level ( the total mrna sample is split and then amplified and labeled separately , and hybridized to two different arrays ) . the differences between the expression values of the two replicate arrays in a pair are due to the variation introduced in experimental steps after the split , the array manufacturing difference and analytical methods such as normalization and expression calculation . this difference provides a lower bound of biological variation that can be detected between two independently amplified samples , and serves as a good statistic for comparing different analytical methods . for comparison , we also used the method in to calculate ads for all probe sets and plot them in figure 5b ( ad is based on normalized probe values , see methods and materials section for the normalization method . also note that genechip software excludes probes whose pm / mm difference is outside three standard deviations ( sds ) of all probe differences in either of the two arrays in the comparison ; here , as we are comparing multiple arrays at the same time , when calculating ads a probe is excluded if its difference is an outlier in the above sense in any of the arrays , until a minimum of five probes is reached , where all five probes will be used ) . both the mbei and the ad method yielded some expression values differing by more than a factor of two , especially for genes at low expression level . this might be explained by the relatively larger amplification variation for weakly expressed genes , given a constant success rate of amplifying a sequence by a certain fold . log ( base 10 ) expression indexes of a pair of replicate arrays ( array 1 and 2 of array set 5 ) for different statistical methods . ( a ) mbei method ; ( b ) ad method . only 6,695 ( a ) and 4,696 ( b ) probe sets with positive values in both arrays are used . the center line is y = x , and the flanking lines indicate the difference of a factor of two . researchers often use ' log ratio ' between expression values of a gene in two arrays as the criterion for identifying differentially expressed genes . between duplicate arrays , we expect these log ratios of expression values based on a good expression index ( ad or mbei ) to be close to zero . thus for every probe set we calculated its average absolute log ( base 10 ) ratio of 29 pairs of duplicates as a statistic to compare the variation in expression levels between duplicates using the ad or the mbei method . the average absolute log ratio distribution of the mbei method is significantly lower than that of the ad method when expression level is low ( and thus probe sets have a low proportion of detections of the target gene across arrays ) . as expression level becomes higher ( when the target gene of a probe set is detected in more arrays ) , the ad method shows a rapid improvement in performance , approaching the level of the mbei method . the same boxplots ( figure 7 ) for another set of 60 human u95a arrays consisting of 30 replicate pairs conveys similar information . these results suggest that the mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . boxplots of average absolute log ( base 10 ) ratios between replicate arrays stratified by presence proportion for different statistical methods . ( a ) mbei method ; ( b ) ad method . the number of presence calls for a probe set in the 58 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5058 . the title of each boxplot is the number of probe sets used for the boxplot . log ratios are not calculated for negative expression values or expression values identified as ' array - outliers ' by the mbei method in either array of a replicate pair , and are not used to calculate the average . 744 probe sets are not included as their average absolute log ratios can not be calculated for all the 29 pairs using either method . similar plots as in figure 6 for another set of 30 pairs of duplicated human u95a arrays . ( a ) mbei method ; ( b ) ad method.the number of presence calls for a probe set in the 60 arrays for the six boxplots are : 09 , 1019 , 2029 , 3039 , 4049 , 5060 . the title of each boxplot is the number of probe sets used for the boxplot . after obtaining expression indexes using ad or mbei , fold changes can be calculated between two arrays for every gene and used to identify differentially expressed genes . usually , low or negative expressions are truncated to a small number before calculating fold changes , and genechip also cautions against using fold changes when the baseline expression is absent . the availability of ses for the model - based expression indexes allows us to obtain confidence intervals for fold changes . suppose where 1 and 2 are the real expression levels in the sample , and 1 and 2 are the model - based estimates of expression levels . we substitute the model - based ses for 1 and 2 letting r = 1/2 be the real fold change , then inference on r can be based on the quantity it can be shown that q has a distribution with 1 degree of freedom irrespective of the values of 1 and 2 . thus q is a pivotal quantity involving r. we can use q to construct fixed - level tests and to invert them to obtain confidence intervals ( ci ) for fold changes . table 1 presents the estimated expression indexes ( with ses ) in two arrays and the 90% confidence intervals of the fold changes for 14 genes . although all genes have similar estimated fold changes , the confidence intervals are very different . for example , gene 1 has fold change 2.47 and a tight confidence interval ( 2.06 , 3.02 ) . in contrast , gene 11 has a similar fold change of 2.48 but a much wider confidence interval ( 0.96 , 18.18 ) . thus the fold change around 2.5 for gene 11 is not as trustworthy as that for gene 1 . further examination reveals that this is due to the large ses relative to the expression indexes for gene 11 . this agrees with the intuition that when one or both expression levels are close to zero for one gene , the fold change can not be estimated with much accuracy . in addition , when image contamination results in unreliable expression values with large ses , the fold changes calculated using these expression value are attached with wide cis . in this manner , the measurement accuracy of expression values propagates to the estimation of fold changes . using expression levels and associated ses to determine confidence intervals of fold changes in practice , we find it useful to sort genes by the lower confidence bound ( ' lower cb ' in table 1 ) , which is a conservative estimate of the fold change . when an expression index is negative ( as a result of taking pm / mm differences ) , we do not calculate the confidence intervals . in such a case , it is more helpful to filter genes by presence calls . cluster analysis is a popular method for analyzing the data of a series of microarrays [ 6 , 7 ] . if two genes are co - regulated at the transcription level , their expression values across samples are likely to be correlated . clustering algorithms use these correlations ( or monotone transformation of correlations ) to cluster co - regulated genes together . the correlation based on the estimated expression levels may , however , be different from that based on the real but unobserved expression levels . also , the commonly used hierarchical clustering algorithm is an irreversible process : once two genes or nodes are merged , they will stay together , even if later on there is good reason to adjust previous clustering . thus there is a need to assess the reliability of clusters . a global way of using se in hierarchical clustering is to resample or bootstrap the whole ' gene by sample ' data matrix and redo the clustering in bittner et al . , the data matrix coming from cdna microarray experiments is resampled using the estimated variation derived from the median sd of log ratios for a gene across samples . as we now have ses for all data points , we can resample each expression value from a normal distribution with mean equal to the estimated expression value and sd equal to the attached se . figure 8a shows a hierarchical clustering tree of 225 selected genes with presence proportion > 0.5 and coefficient of variation ( sd / mean ) > 0.7 across the 20 samples in array set 2 . in trying to interpret this tree , we may be interested in the gene cluster colored in blue and the reliability of the gene members belonging to this cluster . the whole data matrix is resampled , and the clustering is performed again ( figure 8b ) . we notice that some blue genes ( genes in the original cluster are colored blue ) are clustered with other non - blue genes , and some non - blue genes are mixed into the main body of the blue genes . after each resampling , we identify a cluster that contains more than 80% of all the blue genes , but as few non - blue genes as possible ( measured as a percentage of all genes in this cluster ) . this cluster is considered to be the cluster that corresponds to the original one in figure 8a . in figure 8b the root node of the ' corresponding cluster ' is marked with small horizontal line intersecting the vertical line ( representing the range of the cluster ) on the right of the clustering picture . then , for each of all the 225 genes , if it belongs to this ' corresponding cluster ' , we increase its ' in - cluster ' count by 1 . after resampling 30 times , the in - cluster counts are indicated in gray - scale on the left side of the original clustering ( figure 8c ) , with black representing 30 and white representing zero . a high ' in - cluster ' count indicates a gene ' remains ' in the original cluster in most of the resampled clustering trees . gene clustering ( a ) 225 filtered genes are clustered based on their expression profiles across 20 samples . each gene 's expression values are standardized to have mean 0 and sd 1 across 20 samples . although the original ' blue ' genes are scattered to various places , we can still determine where the original cluster is , using the criteria described in the text . ( c ) after resampling 30 times , the reliability of the genes belonging to the original cluster is indicated by the vertical gray - scale bar on the left of the blue - red picture . we can see from figure 8c that most genes in the original cluster are reliable members , whereas a few genes at the bottom of the cluster are not ( in fact they are merged into the original cluster last ) . interestingly , some genes originally not in the original cluster group with the ' corresponding clusters ' during resampling many times and have gray ' in - cluster ' marks . these genes may be related to the original cluster in some way . in summary , this method can help us to distinguish reliable and unreliable gene members of a cluster , as well as draw our attention to related genes originally clustered somewhere else because of the accidental nature of hierarchical clustering . we have developed a software package dna - chip analyzer ( dchip ) to perform invariant - set normalization ( see below ) , calculation of mbei , computation of confidence intervals of fold changes , and hierarchical clustering with resampling . our experience is that more than 10 arrays are appropriate for model training , outlier detection and mbei calculation . researchers with fewer than 10 arrays may seek arrays of the same chip type and hybridizing to similar tissue samples , and combine them in a single dchip analysis session . we are exploring model - based meta - analysis of many arrays of the same chip type but hybridizing to a heterogeneous set of tissues samples , and will present such analysis in future work . as array images usually have different overall image brightness ( figure 9a ) , especially when they are generated at different times and places , proper normalization is required before comparing the expression levels of genes between arrays . model - based expression computation requires normalized probe - level data ( from affymetrix 's dat or cel files ) . for a group of arrays , we normalize all arrays ( except the baseline array ) to a common baseline array having the median overall brightness ( as measured by the median cel intensity in an array ) . ( a ) the cel intensities ( see text ) of a pair of replicate arrays ( array 11 and 12 in array set 5 ) are plotted against each other . the baseline array 11 ( shown on the y - axis ) is not as bright as array 12 ( shown on the x - axis ) . the smoothing spline ( green curve ) deviates from the diagonal line y = x ( blue curve ) , indicating the need for normalization . ( b ) the same plot as ( a ) with superimposed circles representing the invariant set , on the basis of which a piecewise linear normalization relationship is determined ( black dotted line , whose y - coordinate is the normalized value of array 12 ) . the normalization curve is close to the smoothing spline curve in ( a ) as the two arrays are replicated arrays and all probes should be invariant . ( c ) after normalization ( y - axis is the baseline array 11 , and x - axis the normalized value of array 12 ) , the scatterplot centers around the diagonal line and the array 12 is adjusted to have the similar overall brightness as array 11 . ( d ) the q - q plot of probe intensities of array 11 and normalized array 12 shows the probes in the two sets have almost the same distribution . a normalization relation can be understood as a curve in the scatterplot of two arrays with the baseline array drawn on the y - axis and the array to be normalized on the x - axis . a straight line running through the origin is a multiplicative normalization method ( genechip 's scaling method ) , and a smoothing spline through the scatterplot can also be used ( figure 9a , also see ) . we should base the normalization only on probe values that belong to non - differentially expressed genes , but generally we do not know which genes are non - differentially expressed ( control or housekeeping genes may also be variable across arrays ) . nevertheless , we expect that a probe of a non - differentially expressed gene in two arrays to have similar intensity ranks ( ranks are calculated in two arrays separately ) . we use an iterative procedure to identify a set of probes ( called the invariant set ) , which presumably consists of points from non - differentially expressed genes ( figure 9b ) . specifically , we start with points of all pm probes ( about 140,000 for hu6800 array ) . if a point 's proportion rank difference ( prd , absolute rank difference in two arrays divided by n = 140,000 ) is small enough , it is kept for the new set . here the threshold of being small is prd < 0.003 when a points 's average intensity ranks in the two arrays is small and prd < 0.007 when it is large , accounting for fewer points at high - intensity range ; and the threshold is interpolated in between . we chose these parameters empirically to make the selected points in the invariant set thin enough to naturally determine a normalization relation . in this way we may obtain a new set of 10,000 points , and the same procedure is applied to the new set iteratively , until the number of points in the new set does not decrease anymore . a piecewise linear running median line figure 10 shows another pair of arrays where the normalization relationship is non - linear . similar plots as in figure 9 for arrays hybridized to two different samples ( array 24 and 36 of array set 5 ) . ( a ) cel intensities ; ( b ) same plot as in ( a ) with superimposed circles representing the invariant set ; ( c ) after renormalization ; ( d ) q - q plot of normalized probe intensities . note that the smoothing spline in ( a ) is affected by several points at the lower - right corner , which might belong to differentially expressed genes . the invariant set , on the other hand , does not include these points when determining the normalization curve , leading to a different normalization relationship at the high end . we have developed a software package dna - chip analyzer ( dchip ) to perform invariant - set normalization ( see below ) , calculation of mbei , computation of confidence intervals of fold changes , and hierarchical clustering with resampling . our experience is that more than 10 arrays are appropriate for model training , outlier detection and mbei calculation . researchers with fewer than 10 arrays may seek arrays of the same chip type and hybridizing to similar tissue samples , and combine them in a single dchip analysis session . we are exploring model - based meta - analysis of many arrays of the same chip type but hybridizing to a heterogeneous set of tissues samples , and will present such analysis in future work . as array images usually have different overall image brightness ( figure 9a ) , especially when they are generated at different times and places , proper normalization is required before comparing the expression levels of genes between arrays . model - based expression computation requires normalized probe - level data ( from affymetrix 's dat or cel files ) . for a group of arrays , we normalize all arrays ( except the baseline array ) to a common baseline array having the median overall brightness ( as measured by the median cel intensity in an array ) . ( a ) the cel intensities ( see text ) of a pair of replicate arrays ( array 11 and 12 in array set 5 ) are plotted against each other . the baseline array 11 ( shown on the y - axis ) is not as bright as array 12 ( shown on the x - axis ) . the smoothing spline ( green curve ) deviates from the diagonal line y = x ( blue curve ) , indicating the need for normalization . ( b ) the same plot as ( a ) with superimposed circles representing the invariant set , on the basis of which a piecewise linear normalization relationship is determined ( black dotted line , whose y - coordinate is the normalized value of array 12 ) . the normalization curve is close to the smoothing spline curve in ( a ) as the two arrays are replicated arrays and all probes should be invariant . ( c ) after normalization ( y - axis is the baseline array 11 , and x - axis the normalized value of array 12 ) , the scatterplot centers around the diagonal line and the array 12 is adjusted to have the similar overall brightness as array 11 . ( d ) the q - q plot of probe intensities of array 11 and normalized array 12 shows the probes in the two sets have almost the same distribution . a normalization relation can be understood as a curve in the scatterplot of two arrays with the baseline array drawn on the y - axis and the array to be normalized on the x - axis . a straight line running through the origin is a multiplicative normalization method ( genechip 's scaling method ) , and a smoothing spline through the scatterplot can also be used ( figure 9a , also see ) . we should base the normalization only on probe values that belong to non - differentially expressed genes , but generally we do not know which genes are non - differentially expressed ( control or housekeeping genes may also be variable across arrays ) . nevertheless , we expect that a probe of a non - differentially expressed gene in two arrays to have similar intensity ranks ( ranks are calculated in two arrays separately ) . we use an iterative procedure to identify a set of probes ( called the invariant set ) , which presumably consists of points from non - differentially expressed genes ( figure 9b ) . specifically , we start with points of all pm probes ( about 140,000 for hu6800 array ) . if a point 's proportion rank difference ( prd , absolute rank difference in two arrays divided by n = 140,000 ) is small enough , it is kept for the new set . here the threshold of being small is prd < 0.003 when a points 's average intensity ranks in the two arrays is small and prd < 0.007 when it is large , accounting for fewer points at high - intensity range ; and the threshold is interpolated in between . we chose these parameters empirically to make the selected points in the invariant set thin enough to naturally determine a normalization relation . in this way we may obtain a new set of 10,000 points , and the same procedure is applied to the new set iteratively , until the number of points in the new set does not decrease anymore . a piecewise linear running median line figure 10 shows another pair of arrays where the normalization relationship is non - linear . similar plots as in figure 9 for arrays hybridized to two different samples ( array 24 and 36 of array set 5 ) . ( a ) cel intensities ; ( b ) same plot as in ( a ) with superimposed circles representing the invariant set ; ( c ) after renormalization ; ( d ) q - q plot of normalized probe intensities . note that the smoothing spline in ( a ) is affected by several points at the lower - right corner , which might belong to differentially expressed genes . the invariant set , on the other hand , does not include these points when determining the normalization curve , leading to a different normalization relationship at the high end . we thank sven de vos , dan tang , nik brown , stan nelson , jae k. lee , yaron hakak , john walker and arindam bhattacharjee for providing data , and the editor and referees who provided valuable suggestions . this work is supported in part by nih grant 1 ro1 hg02341 - 01 and nsf grant dbi-9904701 .

backgrounda model - based analysis of oligonucleotide expression arrays we developed previously uses a probe - sensitivity index to capture the response characteristic of a specific probe pair and calculates model - based expression indexes ( mbei ) . mbei has standard error attached to it as a measure of accuracy . here we investigate the stability of the probe - sensitivity index across different tissue types , the reproducibility of results in replicate experiments , and the use of mbei in perfect match ( pm)-only arrays.resultsprobe-sensitivity indexes are stable across tissue types . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . we extended the model to obtain expression values for pm - only arrays , and found that the 20-probe pm - only model is comparable to the 10-probe pm / mm difference model , in terms of the expression correlations with the original 20-probe pm / mm difference model . mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . the standard errors of mbei can be used to construct confidence intervals of fold changes , and the lower confidence bound of fold change is a better ranking statistic for filtering genes . we can assign reliability indexes for genes in a specific cluster of interest in hierarchical clustering by resampling clustering trees . a software dchip implementing many of these analysis methods is made available.conclusionsthe model - based approach reduces the variability of low expression estimates , and provides a natural method of calculating expression values for pm - only arrays . the standard errors attached to expression values can be used to assess the reliability of downstream analysis .

Background Results and discussion Probe-sensitivity indexes are stable across tissue types Model-based analysis for PM-only arrays MBEI reduces variability for low expression estimates Confidence interval for fold change Standard errors help to assess clustering results Methods and materials Software Normalization of arrays based on an 'invariant set' Acknowledgements

the statistical model proposed in for one probe set in multiple oligonucleotide arrays has the form it states that the perfect match ( pm)/mismatch ( mm ) difference in array i , probe j of this probe set is the product of model - based expression index ( mbei ) in array i ( i ) and probe - sensitivity index of probe j ( j ) plus random error . the image of outliers ( array and single outliers ) identified through model - fitting can be used to assess the quality of an experiment and to identify unexpected problems such as a misaligned corner of a dat file . we have investigated several important properties of the model , including the reliability and stability of the fitted parameters mbei ( ) and probe sensitivity indexes ( ) , the performance of mbei compared to the commonly used average difference ( ad ) , and how the availability of se facilitates downstream comparative and clustering analysis . ideally , the probe - sensitivity index ( ) should be independent of the tissue type . nevertheless , assuming that a non - target gene cross - hybridizes only to a few probes of a probe set , and its expression levels across arrays do not correlate with the target gene , the iterative probe - excluding procedure in may be able to exclude cross - hybridizing probes , regardless of the tissue type hybridized . in addition , the relative probe - sensitivity indexes of the good probes called by the model are likely to be similar across sets of arrays hybridizing to different tissue samples . the patterns resemble each other greatly , showing that the probe - sensitivity index is an inherent property of these non - cross - hybridizing probes and can be consistently identified from different sets of arrays . it is noteworthy that the probe 11 in array set 5 is likely to be cross - hybridizing , making its relative strength ( here mm is consistently larger than pm and this leads to a negative ) dissimilar to the probe 11 in other array sets . they are stratified by average lower presence proportion in two array sets ( the presence proportion of a probe set is the proportion of arrays in an array set where the target gene is called ' present ' by genechip 's algorithm ) . here , we can not obtain correct probe - sensitivity indexes because of the absence of the target gene . if the target gene becomes available for a future array set , the correct probe - sensitivity indexes will be recovered and these probes will be used for expression calculation . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . from figure 1 of , one can see that some mm probes may respond poorly to the changes in the expression level of the target gene . to investigate the relative performance of pm / mm versus pm - only designs , we exploited the model to estimate gene expression levels using only pm probes , and compared it to the mbei using both pm and mm probes . the full intensity model ( equation 1 of ) specifies the relationship of pm probe responses and expression level : where vj is the baseline response of probe pair j due to nonspecific hybridization , and ' j is the sensitivity of pm probe of the probe pair j. the parameter estimates can be obtained by iteratively fitting i and vj,'j , regarding the other set as known . we fit a pm - only and an mm - only model to obtain expression values of all 20-probe probe sets using array set 1 . for comparison , we also used half of the probe pairs ( by alternatively picking one out of every two probes ) in a 20-probe probe set to fit to the difference model ( equation 1 ) . for each probe set , these three sets of expression values were compared with the expression values of the original difference model using 20 probes , in terms of correlation of s obtained by two methods across the 21 arrays . if , for a probe set , a simplified model ( pm - only , mm - only or 10-probe difference model ) performs reasonably well , we expect its estimates to correlate with that from the 20-probe difference model . figure 3 shows the histogram and figure 4 the boxplot of correlations of s estimated from the 20-probe difference model and s estimated from the 10-probe difference model ( a ) , the 20-probe pm - only model ( b ) and the 20-probe mm - only model ( c ) . for probe sets with high presence proportion , both the 10-probe difference model and the pm - only model correlate well with the 20-probe difference model . we note that this comparison is intrinsically biased in favor of the 10-probe difference model because the ' truth ' is constructed from pm - mm differences . histogram of correlations between model - based expression values estimated using the 20-probe difference model and those estimated using different models . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; ( c ) 20-probe mm - only model . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; and ( c ) 20-probe mm - only model . we stress that , whereas the above analysis illustrates the applicability of model - based analysis to pm - only arrays , the assessment presented here is only tentative because of the limited information provided by the hu6800 arrays on the comparisons . definitive comparisons of the efficiency of the designs must await the availability of data from pm - only arrays . the differences between the expression values of the two replicate arrays in a pair are due to the variation introduced in experimental steps after the split , the array manufacturing difference and analytical methods such as normalization and expression calculation . this difference provides a lower bound of biological variation that can be detected between two independently amplified samples , and serves as a good statistic for comparing different analytical methods . also note that genechip software excludes probes whose pm / mm difference is outside three standard deviations ( sds ) of all probe differences in either of the two arrays in the comparison ; here , as we are comparing multiple arrays at the same time , when calculating ads a probe is excluded if its difference is an outlier in the above sense in any of the arrays , until a minimum of five probes is reached , where all five probes will be used ) . both the mbei and the ad method yielded some expression values differing by more than a factor of two , especially for genes at low expression level . between duplicate arrays , we expect these log ratios of expression values based on a good expression index ( ad or mbei ) to be close to zero . the average absolute log ratio distribution of the mbei method is significantly lower than that of the ad method when expression level is low ( and thus probe sets have a low proportion of detections of the target gene across arrays ) . as expression level becomes higher ( when the target gene of a probe set is detected in more arrays ) , the ad method shows a rapid improvement in performance , approaching the level of the mbei method . these results suggest that the mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . log ratios are not calculated for negative expression values or expression values identified as ' array - outliers ' by the mbei method in either array of a replicate pair , and are not used to calculate the average . after obtaining expression indexes using ad or mbei , fold changes can be calculated between two arrays for every gene and used to identify differentially expressed genes . the availability of ses for the model - based expression indexes allows us to obtain confidence intervals for fold changes . suppose where 1 and 2 are the real expression levels in the sample , and 1 and 2 are the model - based estimates of expression levels . we substitute the model - based ses for 1 and 2 letting r = 1/2 be the real fold change , then inference on r can be based on the quantity it can be shown that q has a distribution with 1 degree of freedom irrespective of the values of 1 and 2 . thus q is a pivotal quantity involving r. we can use q to construct fixed - level tests and to invert them to obtain confidence intervals ( ci ) for fold changes . table 1 presents the estimated expression indexes ( with ses ) in two arrays and the 90% confidence intervals of the fold changes for 14 genes . although all genes have similar estimated fold changes , the confidence intervals are very different . this agrees with the intuition that when one or both expression levels are close to zero for one gene , the fold change can not be estimated with much accuracy . in this manner , the measurement accuracy of expression values propagates to the estimation of fold changes . using expression levels and associated ses to determine confidence intervals of fold changes in practice , we find it useful to sort genes by the lower confidence bound ( ' lower cb ' in table 1 ) , which is a conservative estimate of the fold change . when an expression index is negative ( as a result of taking pm / mm differences ) , we do not calculate the confidence intervals . a global way of using se in hierarchical clustering is to resample or bootstrap the whole ' gene by sample ' data matrix and redo the clustering , then investigate the overall properties emerging from this repertoire of clustering trees . in trying to interpret this tree , we may be interested in the gene cluster colored in blue and the reliability of the gene members belonging to this cluster . ( c ) after resampling 30 times , the reliability of the genes belonging to the original cluster is indicated by the vertical gray - scale bar on the left of the blue - red picture . ideally , the probe - sensitivity index ( ) should be independent of the tissue type . nevertheless , assuming that a non - target gene cross - hybridizes only to a few probes of a probe set , and its expression levels across arrays do not correlate with the target gene , the iterative probe - excluding procedure in may be able to exclude cross - hybridizing probes , regardless of the tissue type hybridized . in addition , the relative probe - sensitivity indexes of the good probes called by the model are likely to be similar across sets of arrays hybridizing to different tissue samples . the patterns resemble each other greatly , showing that the probe - sensitivity index is an inherent property of these non - cross - hybridizing probes and can be consistently identified from different sets of arrays . it is noteworthy that the probe 11 in array set 5 is likely to be cross - hybridizing , making its relative strength ( here mm is consistently larger than pm and this leads to a negative ) dissimilar to the probe 11 in other array sets . the model identifies this probe as a ' probe - outlier ' only for array set 5 and excludes it when calculating mbei ( 0 ) for array set 5 . they are stratified by average lower presence proportion in two array sets ( the presence proportion of a probe set is the proportion of arrays in an array set where the target gene is called ' present ' by genechip 's algorithm ) . here , we can not obtain correct probe - sensitivity indexes because of the absence of the target gene . nevertheless , the pm - mm values for these probes are random fluctuations around zero , leading to a correct expression index close to zero . if the target gene becomes available for a future array set , the correct probe - sensitivity indexes will be recovered and these probes will be used for expression calculation . the target gene 's presence in many arrays of an array set allows the probe - sensitivity index to be estimated accurately . from figure 1 of , one can see that some mm probes may respond poorly to the changes in the expression level of the target gene . to investigate the relative performance of pm / mm versus pm - only designs , we exploited the model to estimate gene expression levels using only pm probes , and compared it to the mbei using both pm and mm probes . the full intensity model ( equation 1 of ) specifies the relationship of pm probe responses and expression level : where vj is the baseline response of probe pair j due to nonspecific hybridization , and ' j is the sensitivity of pm probe of the probe pair j. the parameter estimates can be obtained by iteratively fitting i and vj,'j , regarding the other set as known . we fit a pm - only and an mm - only model to obtain expression values of all 20-probe probe sets using array set 1 . for comparison , we also used half of the probe pairs ( by alternatively picking one out of every two probes ) in a 20-probe probe set to fit to the difference model ( equation 1 ) . for each probe set , these three sets of expression values were compared with the expression values of the original difference model using 20 probes , in terms of correlation of s obtained by two methods across the 21 arrays . if , for a probe set , a simplified model ( pm - only , mm - only or 10-probe difference model ) performs reasonably well , we expect its estimates to correlate with that from the 20-probe difference model . figure 3 shows the histogram and figure 4 the boxplot of correlations of s estimated from the 20-probe difference model and s estimated from the 10-probe difference model ( a ) , the 20-probe pm - only model ( b ) and the 20-probe mm - only model ( c ) . for probe sets with high presence proportion , both the 10-probe difference model and the pm - only model correlate well with the 20-probe difference model . we note that this comparison is intrinsically biased in favor of the 10-probe difference model because the ' truth ' is constructed from pm - mm differences . histogram of correlations between model - based expression values estimated using the 20-probe difference model and those estimated using different models . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; ( c ) 20-probe mm - only model . ( a ) 10-probe difference model ; ( b ) 20-probe pm - only model ; and ( c ) 20-probe mm - only model . we stress that , whereas the above analysis illustrates the applicability of model - based analysis to pm - only arrays , the assessment presented here is only tentative because of the limited information provided by the hu6800 arrays on the comparisons . definitive comparisons of the efficiency of the designs must await the availability of data from pm - only arrays . the differences between the expression values of the two replicate arrays in a pair are due to the variation introduced in experimental steps after the split , the array manufacturing difference and analytical methods such as normalization and expression calculation . this difference provides a lower bound of biological variation that can be detected between two independently amplified samples , and serves as a good statistic for comparing different analytical methods . also note that genechip software excludes probes whose pm / mm difference is outside three standard deviations ( sds ) of all probe differences in either of the two arrays in the comparison ; here , as we are comparing multiple arrays at the same time , when calculating ads a probe is excluded if its difference is an outlier in the above sense in any of the arrays , until a minimum of five probes is reached , where all five probes will be used ) . both the mbei and the ad method yielded some expression values differing by more than a factor of two , especially for genes at low expression level . between duplicate arrays , we expect these log ratios of expression values based on a good expression index ( ad or mbei ) to be close to zero . the average absolute log ratio distribution of the mbei method is significantly lower than that of the ad method when expression level is low ( and thus probe sets have a low proportion of detections of the target gene across arrays ) . as expression level becomes higher ( when the target gene of a probe set is detected in more arrays ) , the ad method shows a rapid improvement in performance , approaching the level of the mbei method . these results suggest that the mbei method is able to extend the reliable detection limit of expression to a lower mrna concentration . log ratios are not calculated for negative expression values or expression values identified as ' array - outliers ' by the mbei method in either array of a replicate pair , and are not used to calculate the average . the availability of ses for the model - based expression indexes allows us to obtain confidence intervals for fold changes . suppose where 1 and 2 are the real expression levels in the sample , and 1 and 2 are the model - based estimates of expression levels . we substitute the model - based ses for 1 and 2 letting r = 1/2 be the real fold change , then inference on r can be based on the quantity it can be shown that q has a distribution with 1 degree of freedom irrespective of the values of 1 and 2 . thus q is a pivotal quantity involving r. we can use q to construct fixed - level tests and to invert them to obtain confidence intervals ( ci ) for fold changes . table 1 presents the estimated expression indexes ( with ses ) in two arrays and the 90% confidence intervals of the fold changes for 14 genes . although all genes have similar estimated fold changes , the confidence intervals are very different . this agrees with the intuition that when one or both expression levels are close to zero for one gene , the fold change can not be estimated with much accuracy . in this manner , the measurement accuracy of expression values propagates to the estimation of fold changes . using expression levels and associated ses to determine confidence intervals of fold changes in practice , we find it useful to sort genes by the lower confidence bound ( ' lower cb ' in table 1 ) , which is a conservative estimate of the fold change . when an expression index is negative ( as a result of taking pm / mm differences ) , we do not calculate the confidence intervals . thus there is a need to assess the reliability of clusters . we notice that some blue genes ( genes in the original cluster are colored blue ) are clustered with other non - blue genes , and some non - blue genes are mixed into the main body of the blue genes . ( c ) after resampling 30 times , the reliability of the genes belonging to the original cluster is indicated by the vertical gray - scale bar on the left of the blue - red picture . we have developed a software package dna - chip analyzer ( dchip ) to perform invariant - set normalization ( see below ) , calculation of mbei , computation of confidence intervals of fold changes , and hierarchical clustering with resampling . researchers with fewer than 10 arrays may seek arrays of the same chip type and hybridizing to similar tissue samples , and combine them in a single dchip analysis session . we are exploring model - based meta - analysis of many arrays of the same chip type but hybridizing to a heterogeneous set of tissues samples , and will present such analysis in future work . model - based expression computation requires normalized probe - level data ( from affymetrix 's dat or cel files ) . a normalization relation can be understood as a curve in the scatterplot of two arrays with the baseline array drawn on the y - axis and the array to be normalized on the x - axis . we have developed a software package dna - chip analyzer ( dchip ) to perform invariant - set normalization ( see below ) , calculation of mbei , computation of confidence intervals of fold changes , and hierarchical clustering with resampling . researchers with fewer than 10 arrays may seek arrays of the same chip type and hybridizing to similar tissue samples , and combine them in a single dchip analysis session . we are exploring model - based meta - analysis of many arrays of the same chip type but hybridizing to a heterogeneous set of tissues samples , and will present such analysis in future work . a normalization relation can be understood as a curve in the scatterplot of two arrays with the baseline array drawn on the y - axis and the array to be normalized on the x - axis .

diabetic nephropathy ( dn ) is the single major cause of renal failure in many countries . in the past decades , a variety of rodent models these studies have given many new insights into the pathogenic pathways of dn and facilitated the development of therapeutic agents for this disease . however , most of the models displayed early nephropathic changes , including mild albuminuria , hyperfiltration and hypertrophy , mesangial expansion , and thickening of glomerular basement membrane ( gbm ) , and the advanced nephropathic changes such as pronounced albuminuria , decline of gfr , and glomerular mesangiolysis and nodular glomerulosclerosis were rarely observed in these models . thus , the lack of robust animal models of dn made it difficult to investigate the pathological mechanisms underlying advanced dn and to evaluate the effects of pharmacologic agents for progressive dn . although multiple cell types are involved , dn is in essence a microvascular disease that develops as a result of a confluence of hemodynamic and metabolic perturbations . there is compelling evidence that endothelial dysfunction serves as a key event in the development and progression of diabetic vascular complications , including nephropathy [ 24 ] . endothelial cells maintain vascular function and homeostasis by generating paracrine factors that regulate vascular tone , preventing coagulation and platelet aggregation , inhibiting adhesion of leukocytes , and limiting proliferation of vascular smooth muscle cells as well as by constituting a selective barrier to the diffusion of macromolecules into the interstitial space . further , it was shown that nitric oxide ( no ) produced by endothelial cells through the endothelial nitric oxide synthase ( enos ) plays a major role for many of these endothelial functions and that decreased no production and bioavailability largely contribute to endothelial dysfunction in diabetes [ 2 , 3 ] . in the past decades , a large body of experimental studies has suggested that development and/or progression of dn is associated with alterations in enos expression and activity . enos was shown to be the major nos enzyme in renal vasculature [ 68 ] , and enos expression was shown to be upregulated in early ( 16 weeks ) diabetic kidneys , especially in afferent and glomerular endothelium , concomitant with increases in diameter of afferent arterioles , glomerular volume and filtration rate , and urinary no metabolites ( nox ) [ 911 ] . further , nadph diaphorase staining suggested that enos activity is increased in afferent artery and glomerular endothelium in diabetic kidney . on the other hand , the studies assessing no production or responses in renal vasculature and glomerulus demonstrated decreased enos and no activity in dn , even when enos expression is upregulated [ 1215 ] . the enos uncoupling caused by reactive oxygen species has been suggested to be a mechanism underlying this paradox . further , a triphasic response of increased , unaltered , and impaired endothelial no - dependent vascular relaxation within the same diabetic animal and the downregulation of glomerular or renal enos expression in progressive models of dn ( ove26 mouse , zsf1 rat ) [ 17 , 18 ] suggested that enos - mediated no production is decreased during the progression of dn . in this context , it is noteworthy that a nonselective nos inhibitor attenuates the early renal vasodilatation and hyperfiltration in diabetic animals [ 19 , 20 ] , while its chronic treatment remarkably aggravates diabetic glomerular injury . in aggregate , these findings suggest that enos serves as a key mediator in the development and progression of dn . in humans , upregulated enos expression in glomerular endothelium was demonstrated in nephropathy patients with type 2 diabetes [ 22 , 23 ] . further , urine or serum no metabolites ( no2 + no3 ) were shown to be increased in normo- or microalbuminuric diabetes patients , associated with increases in glomerular filtration rate ( gfr ) [ 22 , 2426 ] , while preferential changes of plasma no3 in macroalbuminuric patients suggested decreased no bioavailability in these patients . it is of interest that african and asian type ii diabetic patients , who are susceptible to end - stage renal failure , exhibited lower no production compared to a comparable group of caucasian diabetic patients . last , genetic association studies have shown that enos polymorphisms that potentially impair enos gene transcription and activity are associated with an increased risk of advanced dn [ 29 , 30 ] . collectively , these experimental and clinical evidences suggest that renal enos expression and activity are increased early after the onset of diabetes , possibly mediating vasodilatation and hyperfiltration ; however , they are decreased with prolonged diabetes and the resulting vascular no deficiency may facilitate the progression of dn . multiple factors and mechanisms have been suggested for the enos upregulation or activation ( e.g. , high glucose , vegf , igf-1 , and shear stress ) , for the enos dysfunction , inactivation , and downregulation ( e.g. , ros , angiotensin ii , amda , pkc , age , and tnf ) , and for reduction of no bioavailability ( e.g. , excessive superoxide production ) in dn [ 2 , 31 ] . thus , there is substantial evidence that enos may serve as a key player in dn . however , the precise role of enos in this disease remained unknown . in the past decade , multiple groups have created the enos - deficient diabetic mice to investigate its role in dn and shown that these diabetic animals exhibit advanced nephropathic changes with the features similar to human dn . these studies clearly defined a pivotal role of enos in dn and developed a robust animal model of this disease . further , recent studies with this animal model have explored the novel mechanisms by which enos deficiency causes advanced dn and provided many new insights into the pathogenesis of dn . since this animal model is now widely used for the study of dn , herein we summarize the findings obtained with this animal model and discuss unresolved issues and future investigations . ( a ) nonobese models . we and nakagawa et al . induced diabetes in enos / male mice ( c57bl/6j strain ) using either low- ( 50 mg / kg , 5 days ) or high- ( 100 mg / kg , 2 days ) dose streptozotocin ( stz ) injections . also generated diabetic enos / mice ( c57bl/6j strain ) using low - dose stz injections and fed the mice either with a normal or high - fat diet . in this study , stz diabetes was induced at 46 months of age , while the former studies induced diabetes at 68 weeks of age . further , wang et al . created a spontaneous model of diabetic enos / and enos + / mice ( c57bl/6j:129s6/svevtac f1 hybrid ) by introducing the dominant akita diabetogenic mutation ( ins2 ) to enos + / and enos / mice . we and mohan et al . generated the enos - deficient db / db ( lepr ) mice by backcrossing enos / mice ( c57bl/6j strain ) onto db / db strain ( c57blks / j background ) . the blood glucose levels in diabetic enos / or enos + / mice were comparable to those in diabetic enos + /+ mice . in db / db model , db / db enos / mice developed obesity and diabetes similar to db / db enos + /+ mice . there was no difference in the timing of development of diabetes between these two groups . interestingly , db / db enos / mice showed significantly increased body weight , lower blood glucose levels , and prominent ( ~5 fold increase ) hyperinsulinemia as compared with db / db enos + /+ mice [ 36 , 37 ] . in addition , prominent islet enlargement and macrovesicular fat droplets were noted in the pancreas or liver in db / db enos / mice . in all studies , diabetic enos / or enos + / mice showed relatively lower survival rate ( 50% at 5 - 6 months after diabetes ) compared with diabetic enos + /+ mice ( 100% survival ) [ 33 , 35 ] . the low - dose stz enos / mice in which diabetes was induced at 46 months of age also showed lower survival rate ( 70% at 6 months after diabetes ) . in this study , it was also shown that the survival of stz enos / , but not stz enos + /+ , mice is significantly decreased by high - fat diet . in contrast , in our low - dose stz study , all stz enos / mice survived until 5 months after stz injections . thus , it seems that the survival rate of diabetic enos / mice differs among the models , perhaps due to the dose of stz or the age of onset of diabetes . nondiabetic enos / mice survived well during the study period [ 33 , 35 ] . for db / db enos / model , mohan et al . described that the average lifespan of db / db enos / mice is 9 months to 1 year without insulin treatment , while our db / db enos / mice only survive for 2630 weeks . high - dose stz enos / mice exhibited severe body weight loss compared with stz enos + /+ mice , while this phenotype was not observed in low - dose stz enos / and akita enos / ( 3-month old ) models . interestingly , 7-month old akita enos / or enos + / mice showed significantly higher body weight than akita enos + /+ mice . in db / db model , db / db enos / mice showed significantly higher body weight than db / db enos + /+ mice . difference was not observed in body weight between nondiabetic enos + /+ and enos / mice in all the models . diabetes further increased blood pressure ( bp ) of enos / mice in stz ( high- and low - dose ) and akita models [ 32 , 33 , 35 ] , while this effect was not observed in db / db model [ 36 , 37 ] and some low - dose stz studies [ 34 , 39 ] . of importance , in all studies , diabetic enos / mice showed significantly higher bp ( 1048 mmhg ) levels than diabetic enos + /+ mice ( table 1 ) . ( e ) hyperlipidemia . in low - dose stz study , diabetes or enos genotype did not affect plasma triglyceride and total cholesterol levels . it is of note that high - fat diet significantly increased plasma cholesterol levels in low - dose stz enos / mice , while its increase was limited in stz enos + /+ mice . plasma lipid levels are not described in high - dose stz and akita enos / studies . in db / db model , db / db enos / mice showed significantly higher plasma cholesterol levels compared with db / db enos + /+ and nondiabetic controls , while no significant difference was observed in plasma triglyceride levels between db / db enos / and other groups . diabetic enos / mice exhibited pronounced albuminuria compared with diabetic enos + /+ mice and the albuminuria further increased as the disease progressed . the severity of albuminuria appears to differ among the models and prominent albuminuria was observed in high - dose stz and db / db enos / mice ( table 2 ) . nondiabetic enos / mice also showed significant albuminuria in some [ 33 , 35 , 36 ] , but not all , studies , yet its levels were much lower than diabetic enos / mice , indicating that diabetes is required to cause pronounced albuminuria in enos / mice . it is of note that overt albuminuria was observed in low - dose stz enos / mice as early as at 14 days after stz injections , suggesting that hyperglycemia rapidly induces albuminuria in enos / mice . we also observed an increase in albuminuria in low - dose stz enos / mice as early as at 6 weeks after stz injections ; however , in other stz studies , the increases in albuminuria were not observed in stz enos / mice at 1.53 months after diabetes induction [ 33 , 34 ] . it should also be noted that akita enos + / mice developed albuminuria at levels comparable to those in akita enos / mice . given the fact that heterozygous deletion of enos gene reduces renal enos protein levels by ~65% , this finding indicates that not only deficiency but also reduction of enos are sufficient to accelerate albuminuria in diabetic mice . ( 1 ) light microscopy : compared with diabetic enos + /+ mice , diabetic enos / mice showed advanced glomerular lesions that are similar to what is seen with human dn . these include glomerular mesangiolysis and microaneurysms , advanced mesangial expansion occasionally forming nodular or kimmelstiel - wilson like lesion , nodular and global glomerulosclerosis , arteriolar hyalinosis , subendothelial hyaline deposition resembling fibrin caps , glomerular and arteriolar fibrin deposition , and tubulointerstitial fibrosis . mesangiolysis was occasionally accompanied by arteriolar lesions and intraluminal fibrin deposition [ 33 , 37 ] . adhesions to bowman 's capsule were also noted in db / db enos / mice . it is noteworthy that akita enos + / as well as enos / mice exhibited advanced glomerular lesions as compared with akita enos + /+ and nondiabetic enos / mice , although the lesions in akita enos + / were milder than akita enos / mice . this finding indicates that reduction of enos is sufficient to exacerbate histopathological changes of dn . mesangiolysis , glomerular fibrin deposition , and globally sclerotic glomeruli were also observed in nondiabetic enos / mice ; however , its frequency was much lower than in diabetic enos / mice [ 32 , 33 , 37 ] . importantly , the percentage of totally sclerotic glomeruli was 2.8-fold increased in db / db enos / mice from 16 to 40 weeks of age , indicating progressive glomerular injury in this model . it is of note that renal histopathology of diabetic enos / mice in low - dose stz and akita models appears to be milder than those in high - dose stz and db / db models [ 32 , 39 ] . also , glomeruloslerosis and tubulointerstitial fibrosis seem to be more severe in the stz enos / mice in which low - dose stz was injected at older ages . these findings suggest that the dose of stz , type of diabetes , or age may significantly affect renal injury in diabetic enos / mice . ( 2 ) electron microscopy : consistent with light microscopic findings , electron microscopy of diabetic enos / or enos + / glomeruli showed advanced glomerular lesions , including mesangiolysis that is characterized by accumulation of electron - lucent material in the mesangium and dissociation of the mesangial matrix , microaneurysm formation due to disruption of anchoring of the gbm to the mesangium , advanced mesangial expansion occasionally forming lobular or nodular mesangial architecture , and gbm thickening [ 3237 ] . the mesangiolysis accompanied destructive endothelial morphology similar to thrombotic microangiopathy . these include detachment of endothelial cells from the gbm , subendothelial accumulation of electron - lucent material , and intracapillary fibrin deposition and platelet accumulation [ 32 , 37 ] , suggesting advanced glomerular endothelial injury in diabetic enos / mice . it is of interest that enos / or enos + / genotype caused striking gbm thickening in akita model as compared with other models . ( 3 ) immunohistochemistry : diabetic enos / glomeruli showed increased macrophage infiltrate ( cd68 , f4/80 , or moma-2 immunostain ) [ 34 , 35 , 37 , 42 , 43 ] , focal loss of endothelial staining ( cd31 or cd34 immunostain ) [ 33 , 37 ] , fibrin deposition [ 3335 , 37 ] , and prominent fibronectin and collagen accumulation , whereas these changes were highly limited or not observed in diabetic enos + /+ mice and nondiabetic controls . in addition , tunel , pcna , or ki67 staining demonstrated endothelial apoptosis and proliferation in diabetic enos / glomeruli and peritubular capillaries [ 33 , 37 ] . electron dense deposits or immunoglobulin staining were not observed in diabetic enos / glomeruli , indicating that glomerular injury is not associated with immune - complex deposition . ( a ) glomerular filtration rate ( gfr ) . in a high - dose stz model , gfr was remarkably ( ~65% ) decreased in diabetic enos / mice compared with nondiabetic control , while in low - dose stz models diabetes increased gfr in enos / mice , although it did not reach the levels in diabetic enos + /+ mice [ 32 , 39 ] . interestingly , akita enos / and enos + / mice showed more pronounced diabetic hyperfiltration than akita enos + /+ mice , and the gfr declined in akita enos / mice as the disease progressed , showing significantly lower gfr than akita enos + /+ mice at 7 months of age , yet it was still higher than nondiabetic controls . gfr was progressively increased in akita enos + / mice and its decline was not observed at 7 months of age . thus , enos deficiency showed the opposite effects for diabetic hyperfiltration between stz and akita models . it should be noted that akita enos / study clearly demonstrated that prolonged enos deficiency leads to the decline in glomerular filtration in diabetic mice . db / db enos / mice showed significantly decreased gfr as compared with db / db or nondiabetic controls at 26 weeks of age . thus , db / db enos / mice showed lower gfr than those of nondiabetic control mice , suggesting more severe glomerular injury than in low - dose stz or akita enos / mice . stz enos / mice exhibited significant tubulointerstitial lesions compared with stz enos + /+ or nondiabetic enos / mice [ 34 , 44 , 45 ] . tubular tgf expression and interstitial macrophage infiltrate ( f4/80 staining ) and collagen deposition were also demonstrated in stz enos / mice [ 40 , 41 , 45 ] . however , surprisingly , akita enos / or enos + / mice showed significantly decreased tubulointerstitial fibrosis compared with akita enos + /+ mice . db / db enos / mice showed advanced tubulointerstitial injury as evidenced by destructive tubular morphology and interstitial collagen accumulation . further , kim-1 , tunel , pcna , and cd68 staining showed increased tubular injury , apoptosis , or proliferation , and interstitial macrophage infiltration in db / db enos / kidney [ 37 , 41 ] . thus , significant tubulointerstitial lesions were observed in stz and db / db enos / mice , while akita enos / mice had opposite results . enos deficiency did not alter renal oxidative stress in low - dose stz diabetic mice , whereas it was markedly increased in db / db enos / mice . surprisingly , akita enos / or enos + / kidneys showed less renal oxidative stress than akita enos + /+ or nondiabetic controls . nephropathy in low - dose stz or db / db enos / mice was remarkably suppressed by angiotensin - converting enzyme inhibitor ( acei ) or angiotensin receptor blocker ( arb ) [ 39 , 41 ] . however , these agents were ineffective for high - dose stz enos / mice . in summary , as compared with diabetic enos + /+ mice , diabetic enos / or enos + / mice showed advanced nephropathic changes that overlap to human dn , although some differences were observed between the models . given the fact that a much less severe renal phenotype was observed in nondiabetic enos / mice , these findings define a critical role of enos in the development and progression of dn . the renal phenotype in db / db enos / mice was more severe than in low - dose stz or akita enos / models . although the precise mechanism of this is currently unknown , this may be because ( 1 ) obesity - related factors , including insulin resistance , hyperinsulinemia , and hyperlipidemia , accelerate the renal injury in diabetic enos / mice . indeed , a high - fat diet significantly advanced the renal phenotype in low - dose stz enos / mice . also , hyperinsulinemia was shown to promote vascular inflammation and pai-1 expression [ 4749 ] . in addition , enos deficiency and hyperinsulinemia may induce abnormal endothelial insulin signaling and cause vascular disorders . the insulin receptor transduces two distinct signals in endothelium , ( i ) promoting no production through the pi3 k / akt / enos pathway and ( ii ) stimulating endothelial release of vasoconstrictor et-1 and expression of vcam-1/e - selection through the map kinase cascade . however , in the setting of enos deficiency , hyperinsulinemia may largely stimulate the latter endothelial insulin signaling . this imbalanced insulin signaling may promote vasoconstriction ( et-1 release ) and vascular inflammation , advancing renal pathology in db / db enos / mice . ( 2 ) the genetic background of db / db enos / mice causes more advanced dn . the db / db enos / mice were created with the c57blks / j strain , while stz or akita enos / mice studies were carried out with the c57bl/6j strain or c57bl/6j 129s6/svevtac f1 cross . the c57bl/6j strain is known to be relatively resistant to dn comparing with c57blks / j strain [ 5153 ] . the c57bl/6j 129s6/svevtac f1 cross also seems to be resistant to dn as albuminuria in akita enos + /+ mice is relatively mild ( < 100 g / day ) . hence , the more severe renal phenotype in db / db enos / mice may be associated with its genetic background . indeed , we have recently noted that c57blks / j - strain stz enos / mice develop more advanced nephropathy than c57bl/6j - strain stz enos / mice . however , the genetic background may not sufficiently explain the severity of dn in db / db enos / mice as the nephropathy in c57blks / j - strain stz enos / mice was milder than that in db / db enos / mice . ( 3 ) hyperleptinemia induced by the db / db mutation in obrb leptin receptor may modify the renal phenotype in db / db enos / mice as leptin has been shown to exhibit deleterious renal effects through other leptin receptor isoforms ( e.g. , obra receptor ) that is present in glomerular cells , promoting tgf and tgf type ii receptor expression and increasing collagen synthesis in glomerular cells . it should also be noted that high - dose stz enos / mice showed a much more severe phenotype than low - dose stz or akita models ( table 2 ) , although there was no difference in the severity of hyperglycemia among these models . stz is known to also be taken up by extraislet cells via glut2 and cause tissue injury , including liver ( hepatocytes ) and kidney ( proximal tubular cells and possibly podocytes ) [ 5558 ] . in kidney , it causes tubular injury and also advances albuminuria , especially when it is used at a high dose [ 1 , 58 , 59 ] . as shown in figure 1 , we have assessed tubular function in low - dose stz enos / mice using tc - mag3 renal scintigraphy . enos + /+ or nondiabetic control ( not shown ) , mice showed obvious tubular injury at 6 weeks after stz injections , as evidenced by delayed tc - mag3 excretion . however , surprisingly , the tubular injury was largely restored in stz enos / mice at 14 weeks after stz injections and the mice showed nearly normal tc - mag3 renograms up to 35 weeks after stz injections , suggesting mild or focal tubular injury in this model . this finding suggests that enos / mice may be more susceptible to stz tubular toxicity than enos + /+ mice , resulting in acute tubular injury , and that this tubular damage is mostly recovered by 14 weeks after stz injections ( when stz is used at a low dose ) . the renal phenotype in high - dose stz enos / mice was shown to be improved by insulin treatment ; however , it may be difficult to exclude stz renal toxicity with these data as insulin transduces a variety of biological signals in many cell types in addition to lowering blood glucose levels . also , it is possible that hyperglycemia largely enhances renal toxicity of stz in enos / mice ; accordingly , normalizing blood glucose levels reduces the stz renal toxicity as well as diabetic renal injury . together with the fact that tubular injury is severe in diabetic enos / mice in the order high - dose stz > low - dose stz > akita model , tubular pathology in stz enos / models may in part result from stz toxicity , even with low dose . the following points should also be noted with the renal phenotype of diabetic enos / mice : ( 1 ) overt dn was developed by enos deficiency in c57bl/6 strain mice that is known to be resistant to dn [ 51 , 52 ] . this finding indicates that enos deficiency is sufficient to develop as well as to progress dn . ( 2 ) despite evident glomerular capillary injury , the aorta was normal in db / db enos / and low - dose stz enos / ( kanetsuna y , unpublished data ) mice . early or established atherogenic lesions were not observed in these mice . ( 3 ) the increases in kidney / body weight ratio or kidney weight in diabetic enos / mice were comparable to those in diabetic enos + /+ mice , except for a high - dose stz model . the pronounced renal hypertrophy in high - dose stz enos / mice may be due to prominent body weight loss of these mice . the enos - derived no is known to mediate various biological functions and responses in vasculature and play a major role in vascular homeostasis . these include vasodilatation ( antihypertensive ) , inhibition of platelet aggregation and coagulation ( anti - thrombotic ) , and suppression of inflammation ( anti - inflammatory ) . further , no has been shown to prevent endothelial apoptosis induced by high glucose through inhibition of nf-b pathway ( antiapoptotic ) [ 62 , 63 ] , suppress cytokine - induced endothelial activation as well as production of cytokines in endothelial cells , and inhibit tgf , collagen , and fibronectin production in mesangial or endothelial cells ( antifibrotic ) [ 66 , 67 ] . thus , multiple mechanisms could be involved in the acceleration of glomerular injury in diabetic enos / mice and the renal phenotype of diabetic enos / mice seems to be well explained by the disruption of these reported vasoprotective actions of no . the following pathways or mechanisms have been investigated in more detail . hypertension and renin - angiotensin system . no negatively regulates blood pressure ( bp ) and counteracts angiotensin ii ( ang ii ) , which are key pathogenic mediators in dn . therefore , advanced nephropathy in diabetic enos / mice may be caused by hypertension and/or enhanced action of ang ii . have shown that lowering bp with hydralazine largely prevents glomerular injury in high - dose stz enos / mice , yet hydralazine treatment was ineffective for attenuating tubulointerstitial lesions . further , they also showed that acei ( enalapril ) or arb ( telmisartan ) is less effective in reducing bp and glomerular injury in stz enos / mice , whereas they are effective for stz enos + /+ mice . interestingly , enalapril or telmisartan increased serum aldosterone levels in stz enos / mice , while these agents decreased serum aldosterone in stz enos + /+ mice . furthermore , the authors demonstrated that aldosterone receptor antagonist ( spironolactone ) effectively reduces bp and glomerular and tubulointerstitial injury in stz enos / mice . these findings suggest that hypertension plays a critical role in the development of advanced glomerular lesions in diabetic enos / mice and that enos deficiency induces aldosterone breakthrough , reducing the efficacy of ras blockades for dn . on the other hand , yuen et al . have shown that acei ( captopril ) remarkably suppresses albuminuria in low - dose stz enos / mice , concomitant with reduction of bp . they also demonstrated that albuminuria in this model is prominently reduced by arb ( losartan ) at a dose that does not lower bp and that spironolactone is less effective than captopril or losartan . thus , low - dose stz enos / mice showed distinct responses to ras blockade . further , we have shown that antihypertensive triple therapy ( hydralazine + hydrochlorothiazide + reserpine ) or captopril normalizes bp and remarkably reduces glomerular and tubulointerstitial injury in db / db enos / mice . interestingly , captopril exhibited more profound effects in db / db enos / mice than antihypertensive therapy , whereas reduction of bp was comparable in both therapies , suggesting additive enos - independent renoprotective effects of acei in dn . collectively , these studies indicate that hypertension has a strong impact on renal injury in diabetic enos / mice and that the ras system may serve as a key mediator for this , yet acei or arb failed to reduce bp and renal injury in high - dose stz enos / mice ( this might be related to stz toxicity ) . diabetic enos / mice are hypertensive ; however , the elevation of bp is mild~moderate in these mice , especially in stz models ( table 1 ) . why does antihypertensive or ras blockade treatment has such a strong impact on glomerular injury in diabetic enos / mice ? the enos - derived no was shown to alter intraglomerular pressure and perfusion by modulating autoregulation or by counteracting ang ii vasoconstriction in afferent and efferent arteries [ 68 , 69 ] . therefore , an underlying mechanism could be ( 1 ) barotrauma to the glomerulus due to disruption of autoregulation and/or dominant vasoconstriction of efferent arteries and ( 2 ) glomerular hypoperfusion due to vasoconstriction of afferent or renal arteries . in this context have reported that inner lumen size in afferent arteries is increased in stz enos / mice , particularly in the glomerulus with mesangiolysis . in addition , we noted that mesangiolysis frequently accompanies afferent arteriolar lesions ( figure 2(a ) ) . these findings suggest that enos deficiency may impair autoregulation concomitant with afferent arteriolar injury in diabetic mice , leading to hyperperfusion of glomeruli . in addition , it should be noted that the severity of glomerular injury largely differs among individual glomeruli in diabetic enos / mice ( figures 2(b ) and 2(c ) ) ; 510% of glomeruli exhibited prominent mesangiolysis , while others do not . although the mechanism of this large heterogeneity of glomerular injury is currently unknown , this finding may indicate that the degree or mode of arteriolar injury differs in individual glomeruli , possibly associated with the location in the renal circulation or sensitivity of the glomerulus to ang ii . indeed , prominent mesangiolysis was more frequently observed in outer region of renal cortex ( ~80% in outer half of cortex and ~20% in inner half of cortex ) in stz enos / mice ( kanetsuna y. unpublished observation ) . thus , further investigation would be required to determine the effects of enos deficiency in glomerular ( and renal ) hemodynamics and autoregulation in diabetes . renal as well as systemic renin - angiotensin system ( ras ) is known to play a key role in dn . interestingly , intrarenal ang ii ( protein ) levels were decreased or unaltered in enos / mice with stz diabetes , while diabetes increased renal ang ii in enos + /+ mice [ 39 , 46 ] . thus , the levels of renal ang ii were lower in stz enos / mice than in stz enos + /+ mice . this finding suggests that enos is required for the increase in renal ang ii in diabetes and that increased ang ii sensitivity or responses of the target cells may be a responsible mechanism of ras activation in this model . further , alteration of renal ras components was examined in more detail in high - dose stz enos / and db / db enos / models [ 41 , 46 ] . in high - dose stz model , renal expression of ras components was changed in a similar manner in stz enos / and enos + /+ mice compared with nondiabetic counterparts as follows : ( upregulated ) angiotensinogen mrna , ace2 , at1 receptor , and mcr proteins , ( downregulated ) renin mrna , and ( unaltered ) ace protein . ( note that , in this study , the levels of ras components were not compared between stz enos / and enos + /+ mice . ) in db / db enos / and db / db kidneys , ras components were changed in the following manner compared with nondiabetic mice : ( upregulated ) angiotensinogen mrna , ( downregulated ) renin and ace1 mrna , and ( unaltered ) ace2 and ang ii receptors ( at1a , at1b , at2 , and mas ) . the db / db enos / kidney showed significantly higher angiotensinogen and lower renin mrna expression compared with db / db enos + /+ kidney . similar results were obtained with the glomeruli of these mice , yet pronounced downregulation of renin mrna was not observed in db / db enos / glomeruli and ace1 mrna was unaltered in db / db enos / and enos + /+ glomeruli . also , mas mrna was comparably downregulated in db / db enos + /+ and enos / glomeruli . thus , diabetes alters renal ras components in a similar manner in diabetic enos / and enos + /+ mice and the pronounced changes in renal angiotensinogen and renin mrna were noted in db / db enos / mice . however , it is still unclear how enos deficiency alters renal ras activity during the course of dn . endothelial no is also known to reduce endothelin-1 production and its action , a key pathogenic pathway in dn . abnormal angiogenesis has been implicated in the pathogenesis of dn , coupled with increased production of angiogenic factors such as vegf and angiopoietin-2 [ 70 , 71 ] . interestingly , nakagawa et al . have demonstrated that inhibition of no synthesis enhances vegf - induced endothelial cell proliferation through vegfr2-erk1/2 pathway in normal and high glucose culture conditions . further , the increased endothelial proliferation ( in glomerulus and peritubular capillaries ) and enhanced vegfr2 phosphorylation were noted in diabetic enos / kidney , compared with diabetic enos + /+ kidney , yet significant differences were not observed in mrna levels of renal vegf and vegfr2 between these two groups [ 33 , 37 , 39 ] . increased endothelial cell proliferation and vegfr2 phosphorylation were also noted in nondiabetic enos / kidney [ 33 , 39 ] ; however , its extent was limited in these mice , suggesting that diabetes or diabetes - mediated vegf upregulation is required for this effect . given the fact that enos activity is increased by vegf in most circumstances , nakagawa et al . vegf - enos uncoupling may be a mechanism that causes excessive vegf signaling and angiogenesis in dn . further , more recently , veron et al . demonstrated that overexpression of vegf in podocytes causes nodular glomerulosclerosis , mesangiolysis , microaneurysms , and arteriolar hyalinosis concurrent with massive proteinuria and renal failure in enos / mice in the absence of diabetes , suggesting that increased vegf activity and enos deficiency are sufficient to develop advanced dn - like lesions in mice . first , the detailed mechanisms by which enos deficiency enhances vegfr2 signaling and endothelial proliferation have not been determined yet . in addition , a body of studies has shown that enos deficiency inhibits vegf - mediated angiogenesis in various conditions including cancer [ 67 , 75 , 76 ] , indicating that enos is a critical downstream mediator of vegf angiogenesis . why does enos deficiency show a distinct effect in renal microvasculature , especially in the setting of diabetes ? second , it is also uncertain whether the enhanced vegf signaling and angiogenesis is a responsible mechanism of advanced dn in diabetic enos / mice . yuen et al . have recently shown that a vegfr inhibitor ( vatalanib ) is ineffective in reducing albuminuria in stz enos / mice , while it effectively decreases albuminuria in stz enos + /+ mice . this study also demonstrated that vegfr inhibition is ineffective in reducing mesangial expansion and gbm thickening in stz enos / mice , though it effectively suppressed glomerular capillary growth in this model . these findings suggest that ( 1 ) the enhanced vegfr activity accounts for a part of , but not all , the glomerular phenotype in stz enos / mice . ( 2 ) unlike other diabetic mouse models , diabetic albuminuria is caused by a vegf - independent mechanism in enos / mice . ( 3 ) vegf - mediated glomerular capillary growth occurs in diabetic mice in the absence of enos . in this context , it should be noted that enos deficiency was shown to alter morphogenesis of capillary growth and results in less sprouting and enlarged vessels ; therefore , the morphogenesis of glomerular capillary growth in diabetic enos / mice may differ from those in dn where enos activity is partially decreased . thus , further investigation would be required to determine whether vegf - enos deficiency induces the pathological signals that are analogous to progressive dn . ( note that no inhibition or enos knockdown was shown to upregulate vegf or vegfr2 expression in endothelial cells [ 73 , 78 ] . this may be another mechanism by which enos deficiency enhances vegf signaling , although there was no difference in total vegf and vegfr2 mrna levels between diabetic enos / and enos + /+ kidneys [ 33 , 39 ] . ) have recently shown that diabetic enos / mice ( stz and db / db models ) develop podocytopathy soon after the onset of diabetes , associated with the development of albuminuria , while podocyte injury is limited in diabetic enos + /+ or nondiabetic enos / mice . the podocytopathy was observed in stz enos / mice as early as 2 weeks after stz injections and in db / db enos / mice as early as 812 weeks of age . further , this study showed that ( 1 ) the podocytopathy and albuminuria in stz enos / mice are largely suppressed by captopril or losartan at a dose that does not lower bp . ( 2 ) the soluble factors , which are produced in diabetic enos / mice or in cultured enos / glomerular endothelial cells exposed to high glucose and ang ii , remarkably alter the cell shape and cytoskeleton arrangement in cultured podocytes , concomitant with an increase in rhoa activity . these findings suggest that enos deficiency confers ras - dependent acute podocytopathy to diabetic mice and that this podocytopathy may be caused by endocrine or paracrine mechanisms . for this topic , it was also shown that nicorandil reduces the podocyte oxidative stress and loss in stz enos / mice through atp - dependent k channel activation . a body of studies has shown that endothelial surface glycocalyx , a negatively charged surface layer of membrane - associated proteoglycans and glycosaminoglycans , serves as an important charge barrier in glomerular filtration , and its disruption may cause albuminuria . using cationic ferritin , we have shown that the anionic surface barrier in glomerular endothelium is damaged in stz enos / mice . this mechanism is also supported by the following evidence : ( 1 ) chronic no inhibition impairs glomerular endothelial charge barrier concomitant with albuminuria and gbm thickening . ( 2 ) the disrupted glomerular charge barrier in enos / mice was demonstrated by a recent mri study using cationic ferritin as a contrast agent . further , we noted that heparanase expression is upregulated in the glomerulus ( endothelial and mesangial cells ) of stz or db / db enos / mice ( takahashi t , unpublished observation ) . given the fact that heparanase impairs surface glycocalyx in glomerular endothelial cells , these findings suggest that no deficiency may upregulate heparanase expression in glomerulus in the setting of diabetes , disrupting the endothelial surface glycocalyx , and lead to albuminuria . since glomerular heparanase expression was shown to be associated with the development of dn in humans as well as in mice [ 83 , 84 ] , it would be of interest to examine the role of enos in the expression of heparanase and other glycocalyx disrupting enzymes in diabetic glomeruli . disruption of this barrier causes the influx of serum proteins to the subendothelial space , inducing vascular cellular responses and facilitating the formation of advanced vascular lesions . we have noted that immunoreactivity of ve - cadherin is decreased in the glomerular endothelium of low - dose stz enos / mice . although the precise mechanism of this is currently unknown , this finding suggests that enos deficiency may impair glomerular endothelial junctions in diabetes , causing insudation to subendothelial space and mesangium , and advance diabetic glomerular lesions . inflammatory gene expression . consistent with this finding , enhanced inflammatory gene expression was observed in diabetic enos / or enos + / kidneys , compared with diabetic enos + /+ kidney [ 34 , 35 , 41 ] . in addition , it was shown that enos deficiency and high - fat diet synergistically increases renal expression of il-1 and il-6 in stz - induced diabetic mice . given the fact that enos deficiency itself had no effects on renal inflammatory gene expression , these findings suggest that enos deficiency accelerates renal inflammatory gene expression in the setting of diabetes . we have recently demonstrated that renal expression of heparin - binding epidermal growth factor - like growth factor ( hb - egf ) is upregulated in both db / db enos / and nondiabetic enos / mice as early as at 8 weeks of age , including arterial and glomerular endothelium , podocytes , and tubular cells . egf expression was higher in db / db enos / mice and its levels were further increased during the progression of nephropathy , accompanied by egfr phosphorylation and urinary hb - egf excretion . further , we also demonstrated that ( 1 ) l - name treatment dramatically increases renal hb - egf expression and its urinary excretion in db / db enos + /+ mice . ( 2 ) replenishing no with sodium nitrate reduces renal hb - egf expression and its urinary excretion in db / db enos / mice and inhibits the progression of nephropathy . ( 3 ) genetic deletion of endothelial hb - egf expression attenuates glomerular injury in stz enos / mice . these findings suggest that no deficiency induces hb - egf expression in diabetic kidney and this may serve as an important mediator of progressive dn . in addition , we have recently shown that the renal phenotype in stz enos / mice is remarkably suppressed by an egfr inhibitor ( erlotinib ) . of interest , this was accompanied by activation of ampk pathway , inhibition of mtor pathway , decreased renal er stress , and increased autophagy . taken together , these findings indicate a pivotal role of egf pathway in this pathological condition . further investigation of this pathway should provide a new insight into the pathogenesis of dn . toll - like receptor tlr4 . lin et al . have recently shown that stz enos / kidney exhibits enhanced expression of toll - like receptor ( tlr ) tlr4 and its ligand hmgb1 , predominantly in tubular cells , and that synthetic tlr4 antagonist , crx-526 , significantly attenuates renal injury in this model without altering blood glucose and systolic blood pressure , including albuminuria , renal hypertrophy , and glomerular and tubulointerstitial injury . further , the authors demonstrated that crx-526 treatment decreases induction of chemokine ( c - c motif ) ligand ( ccl)-2 , osteopontin , ccl-5 , tgf- , and nf-b activation in stz enos / kidney and reduces macrophage infiltration and collagen deposition in glomerulus and interstitium . these results suggest that enos deficiency accelerates renal tlr4 pathway in diabetes and promotes renal inflammation and fibrosis . consistent with this finding , diabetic enos / or enos + / glomeruli showed increased thrombus formation that was accompanied by fibrin deposition and platelet accumulation [ 3235 , 37 ] and this was further enhanced by a high - fat diet . glomerular fibrin deposition was also observed in nondiabetic enos / mice ; however , its extent was highly limited in these mice [ 33 , 37 ] , indicating that enos deficiency promotes glomerular thrombus formation in the setting of diabetes . . demonstrated that renal tissue factor ( tf ) mrna levels and activity are increased in diabetic enos / and enos + / mice compared with diabetic enos + /+ mice in akita or stz models , and these mice exhibited prominent tf immunoreactivity in the mesangial area [ 34 , 35 ] . further , tf and moma-2 coimmunostaining indicated that all tf - positive cells are monocyte / macrophages . the authors also demonstrated that tf levels correlate with albuminuria , gbm thickening , and tubulointerstitial fibrosis ; tf expression is increased before the development of dn ; tf neutralizing antibody prominently decreases renal expression of inflammatory and fibrogenic genes in diabetic mice regardless of enos genotype ; high - fat diet additively increases renal tf levels and activities . an increase in renal tf expression was also observed in nondiabetic enos / mice ; however , it was relatively limited . in aggregate , these findings suggest that enos deficiency promotes tf expression in diabetic glomeruli through monocyte / macrophage infiltration and this activates the coagulation cascade in the mesangial area , promoting inflammatory and fibrogenic gene expression and accelerating glomerular injury . in addition , a recent study has demonstrated that enos / glomeruli show increased von willebrand factor ( vwf ) deposition both in glomerular capillaries and the mesangial area . given the fact that serum p - selectin levels are increased in enos / mice , suggesting exocytosis of weibel - palade body by the endothelium , the authors proposed a model that enos deficiency causes endothelial release of vwf , resulting in mesangial as well as intraluminal deposition of vwf and facilitating glomerular thrombus formation and mesangial expansion . although diabetic enos / mice were not investigated in this study , the finding suggests that a similar event may occur in diabetic enos / glomeruli . a large body of study has shown that increased oxidative stress plays a central role in the pathogenesis of dn . hence the effects of enos deficiency on renal oxidative stress have been investigated in some studies . in the low - dose stz model , oxidative stress ( plasma and renal cml and urinary 8-ohdg ) in diabetic enos / mice was comparable to those in diabetic enos + /+ mice , yet enos deficiency and high - fat diet synergistically increased oxidative stress ( plasma cml and urinary 8-ohdg ) in stz diabetic mice . similar results were also observed in our stz study ( kanetsuna y , unpublished observation ) ; both stz enos + /+ and enos / mice exhibited increased renal oxidative stress ( nitrotyrosine and 8-ohdg staining ) as compared with nondiabetic counterparts . surprisingly , renal oxidative stress was significantly reduced in akita enos / and enos + / mice compared with akita enos + /+ or nondiabetic enos / mice . in contrast , db / db enos / kidney showed pronounced nitrotyrosine and 4-hne staining . although a comparison between db / db enos + /+ and db / db enos / kidneys has not been performed , the finding suggests increased renal oxidative stress in this model . thus , the effects of enos deficiency on diabetic renal oxidative stress seem to be different among the models and oxidative stress may not be a key mechanism in low - dose stz and akita enos / models . other potential mechanisms . ( 1 ) a recent study showed that blockade of the ca2 + -activated k+ channel kca3.1 , which is expressed in proximal tubules , reduces albuminuria , renal hypertrophy , and the expression of inflammatory and fibrotic markers in tubulointerstitium in stz enos / mice without affecting blood pressure levels , suggesting involvement of kca3.1 pathway in this model . ( 2 ) no was shown to regulate the expression and activity of hypoxia - inducible factor ( hif1 ) , a key mediator in kidney disease , through multiple mechanisms in physiological and pathological conditions [ 87 , 88 ] . also , enos deficiency may lead to vasoconstriction , reducing renal blood flow , and cause renal hypoxia . although difference was not observed in renal hif-1 mrna levels between stz enos / and enos + /+ mice , further investigations should be performed on this subject , including hif1 protein level , s - nitrosylation , and its activity . ( 3 ) lipotoxic disruption of na+/h+ exchanger nhe1 interaction with pi(4,5)p2 was recently implicated in proximal tubule apoptosis in db / db enos / mice . in summary , multiple mechanisms are involved in the development of advanced dn in diabetic enos / mice ( figure 3 ) . among them , hypertension and ras system seem to play a major role in this model as in human dn . first , this model , especially db / db enos / mice , develops glomerular lesions of progressive dn . second , the nephropathy in this model progresses as the mice age , as indicated by declining gfr and increasing glomerulosclerosis . third , the pathophysiology ( decreased enos activity ) underlying this model is relevant to human dn . in addition , the glomerular gene expression profile in db / db enos / mice was recently shown to overlap to those in human dn . thus , this model provides an invaluable tool for us to study the enos - mediated pathogenic mechanisms underlying progressive dn . on the other hand , we may also need to realize that there are some differences between this animal model and advanced human dn . first , vegf and nephrin expression were shown to be downregulated in advanced human dn , concurrent with podocyte detachment and reduced glomerular capillary endothelial fenestration [ 9193 ] . however , glomerular vegf expression is upregulated in this model and endothelial fenestration and nephrin expression are still well preserved [ 32 , 39 ] ( advani a , unpublished observation ) . although the changes of glomerular vegf and nephrin expression have not been evaluated in db / db enos / model , given the fact that glomerular gene expression in db / db enos / mice overlaps to those in early human dn , this model seems to still display early dn or a transition phase to advanced dn . second , it is well known that diabetes causes enos uncoupling and the uncoupled enos serves as a major source of superoxide overproduction in diabetic endothelium . in addition , no was shown to promote superoxide generation by reducing oxygen consumption in mitochondria [ 94 , 95 ] . because these superoxide generations do not occur in enos deficient mice , the endothelial oxidative stress in enos - deficient diabetic mice may be lower than that in actual diabetic endothelium . further , enos deficiency may decrease the production of reactive nitrogen species ( e.g. , peroxynitrite ) that is harmful to the cells . these might be a reason why renal oxidative stress and tubulointerstitial lesion are decreased in akita enos / mice . last , the mesangiolysis seen in diabetic enos / mice is more severe than is often seen in human dn . its morphological features are similar to thrombotic microangiopathy rather than dn , indicating severe glomerular endothelial injury . thus , there are some differences in pathophysiology of renal injury between diabetic enos / mice ( enos deficiency - mediated ) and actual dn ( enos dysfunction ) . therefore , it would be very important to validate the findings obtained from this model with human subjects . in this context , heterozygous model may serve better than homozygous model and the creation of a diabetic mouse model in which enos is uncoupled would be of great interest . the following efforts and investigation would be required to better understand the role of enos in dn . first , the reported stz enos / studies have been done on the nephropathy - resistant genetic background ( c57bl/6j strain ) . in addition , the genetic background ( c57bl/6j 129 svev f1 ) of akita enos / mice seems to also be nephropathy - resistant as the urinary albumin excretion in akita enos + /+ mice was less than 100 g / day even at 7 months of age . therefore , it would be required to conduct those studies with nephropathy - prone strain , such as dba2/j , c57bl6 dba2 f1 hybrid , or 129svev [ 51 , 96 ] , to determine the precise effects of enos deficiency in dn that was caused by hypoinsulinemic diabetes . since stz is toxic to enos / mice , it would be desirable to conduct these studies with akita model . high - dose stz model should not be used for future works . also , the findings obtained with low - dose stz model should be confirmed with akita model . second , there are many unresolved issues with the reported models : ( 1 ) the cause of death in diabetic enos / mice is not defined , yet occasional bleeding in the chest and abdominal cavity and clots in the mesenteric arteries and necrosis of small intestine [ 34 , 35 ] suggest that hypercoagulability may in part contribute to premature death of these mice . ( 2 ) akita enos / study has emerged a role for enos to negatively regulate diabetic hyperfiltration , whereas previous studies proposed a role of enos as a positive regulator of diabetic hyperfiltration based on the effects of nonselective nos inhibitors . also , to date , it is unclear whether db / db enos / mice exhibit pronounced ( or reduced ) diabetic hyperfiltration at early age . db / db enos / mice developed prominent hyperinsulinemia ; however , the mechanism of this is currently unknown . since enos deficiency was shown to reduce insulin sensitivity [ 97 , 98 ] , this may be due to the enhanced insulin resistance in these mice . third , enos / mice have been shown to exhibit various congenital vascular anomalies with high frequency , including congenital septal defects and postnatal heart failure , abnormal aortic valve development , and defects in pulmonary vascular development . further , enos / mice were shown to exhibit congenital renal defects including renal scars containing crowded small glomeruli and progressive renal disease . in addition , we have recently noted that the number of perfusable renal vessels is remarkably decreased in enos / mice compared with wild type mice ( figure 4 ) . therefore , it is still unclear whether enos deficiency accelerates dn or diabetes accelerates the renal injury in enos / mice . also , enos is expressed in various cell types including cardiomyocytes , hematopoietic cells such as erythrocytes and platelets , adipocytes , and renal tubular epithelial cells [ 103 , 104 ] . consequently , the precise role of enos in diabetic endothelium or vasculature is not defined clearly . the postnatal conditional knockout study to target endothelial or vascular ( endothelial and hematopoietic ) enos in diabetic mice last , a body of studies has shown a critical role of enos - derived no in regulation of renal hemodynamics and oxygen consumption . further , the fact that antihypertensive or ras inhibitor treatment remarkably reduces glomerular injury in diabetic enos / mice suggests that hemodynamic events may play a key role in the pathology of this model . however , it is still unclear how enos deficiency alters renal or glomerular hemodynamics and oxygenation in diabetic mice and what factors are involved in these changes . in addition , cardiac phenotype is not investigated in detail , yet significant changes were not observed in the pulse levels between db / db enos / and enos + /+ mice . in recent decade , several groups have generated diabetic enos / models , either with hypoinsulinemic or type 2 diabetes , and defined the critical role of enos in the development of advanced diabetic renal lesions . further , the efforts to investigate the underlying mechanisms in this model explored unreported enos - associated pathogenic mechanisms in this disease . the complete enos deficiency does not occur in actual dn ; therefore , diabetic enos / model may include some artificial events that are irrelevant to dn . however , given the facts that this animal model was developed through a mechanism that is relevant to human dn and exhibits the most advanced diabetic renal lesion among the reported mouse models , further investigations of this model should largely advance our understanding of the pathogenesis of progressive dn and these studies enable a more efficient interrogation of novel therapeutics for this disease .

diabetic nephropathy ( dn ) is the leading cause of end - stage renal disease in many countries . the animal models that recapitulate human dn undoubtedly facilitate our understanding of this disease and promote the development of new diagnostic markers and therapeutic interventions . based on the clinical evidence showing the association of enos dysfunction with advanced dn , we and others have created diabetic mice that lack enos expression and shown that enos - deficient diabetic mice exhibit advanced nephropathic changes with distinct features of progressive dn , including pronounced albuminuria , nodular glomerulosclerosis , mesangiolysis , and arteriolar hyalinosis . these studies clearly defined a critical role of enos in dn and developed a robust animal model of this disease , which enables us to study the pathogenic mechanisms of progressive dn . further , recent studies with this animal model have explored the novel mechanisms by which enos deficiency causes advanced dn and provided many new insights into the pathogenesis of dn . therefore , here we summarize the findings obtained with this animal model and discuss the roles of enos in dn , unresolved issues , and future investigations of this animal model study .

1. Introduction 2. Diabetic eNOS Knockout Study 3. Proposed Mechanisms of Advanced DN in Diabetic eNOS / Mice 4. Future Directions 5. Conclusions

diabetic nephropathy ( dn ) is the single major cause of renal failure in many countries . in the past decades , a variety of rodent models these studies have given many new insights into the pathogenic pathways of dn and facilitated the development of therapeutic agents for this disease . however , most of the models displayed early nephropathic changes , including mild albuminuria , hyperfiltration and hypertrophy , mesangial expansion , and thickening of glomerular basement membrane ( gbm ) , and the advanced nephropathic changes such as pronounced albuminuria , decline of gfr , and glomerular mesangiolysis and nodular glomerulosclerosis were rarely observed in these models . thus , the lack of robust animal models of dn made it difficult to investigate the pathological mechanisms underlying advanced dn and to evaluate the effects of pharmacologic agents for progressive dn . endothelial cells maintain vascular function and homeostasis by generating paracrine factors that regulate vascular tone , preventing coagulation and platelet aggregation , inhibiting adhesion of leukocytes , and limiting proliferation of vascular smooth muscle cells as well as by constituting a selective barrier to the diffusion of macromolecules into the interstitial space . further , it was shown that nitric oxide ( no ) produced by endothelial cells through the endothelial nitric oxide synthase ( enos ) plays a major role for many of these endothelial functions and that decreased no production and bioavailability largely contribute to endothelial dysfunction in diabetes [ 2 , 3 ] . in the past decades , a large body of experimental studies has suggested that development and/or progression of dn is associated with alterations in enos expression and activity . enos was shown to be the major nos enzyme in renal vasculature [ 68 ] , and enos expression was shown to be upregulated in early ( 16 weeks ) diabetic kidneys , especially in afferent and glomerular endothelium , concomitant with increases in diameter of afferent arterioles , glomerular volume and filtration rate , and urinary no metabolites ( nox ) [ 911 ] . on the other hand , the studies assessing no production or responses in renal vasculature and glomerulus demonstrated decreased enos and no activity in dn , even when enos expression is upregulated [ 1215 ] . further , a triphasic response of increased , unaltered , and impaired endothelial no - dependent vascular relaxation within the same diabetic animal and the downregulation of glomerular or renal enos expression in progressive models of dn ( ove26 mouse , zsf1 rat ) [ 17 , 18 ] suggested that enos - mediated no production is decreased during the progression of dn . in aggregate , these findings suggest that enos serves as a key mediator in the development and progression of dn . it is of interest that african and asian type ii diabetic patients , who are susceptible to end - stage renal failure , exhibited lower no production compared to a comparable group of caucasian diabetic patients . last , genetic association studies have shown that enos polymorphisms that potentially impair enos gene transcription and activity are associated with an increased risk of advanced dn [ 29 , 30 ] . collectively , these experimental and clinical evidences suggest that renal enos expression and activity are increased early after the onset of diabetes , possibly mediating vasodilatation and hyperfiltration ; however , they are decreased with prolonged diabetes and the resulting vascular no deficiency may facilitate the progression of dn . , high glucose , vegf , igf-1 , and shear stress ) , for the enos dysfunction , inactivation , and downregulation ( e.g. however , the precise role of enos in this disease remained unknown . in the past decade , multiple groups have created the enos - deficient diabetic mice to investigate its role in dn and shown that these diabetic animals exhibit advanced nephropathic changes with the features similar to human dn . these studies clearly defined a pivotal role of enos in dn and developed a robust animal model of this disease . further , recent studies with this animal model have explored the novel mechanisms by which enos deficiency causes advanced dn and provided many new insights into the pathogenesis of dn . since this animal model is now widely used for the study of dn , herein we summarize the findings obtained with this animal model and discuss unresolved issues and future investigations . generated the enos - deficient db / db ( lepr ) mice by backcrossing enos / mice ( c57bl/6j strain ) onto db / db strain ( c57blks / j background ) . given the fact that heterozygous deletion of enos gene reduces renal enos protein levels by ~65% , this finding indicates that not only deficiency but also reduction of enos are sufficient to accelerate albuminuria in diabetic mice . these include glomerular mesangiolysis and microaneurysms , advanced mesangial expansion occasionally forming nodular or kimmelstiel - wilson like lesion , nodular and global glomerulosclerosis , arteriolar hyalinosis , subendothelial hyaline deposition resembling fibrin caps , glomerular and arteriolar fibrin deposition , and tubulointerstitial fibrosis . this finding indicates that reduction of enos is sufficient to exacerbate histopathological changes of dn . mesangiolysis , glomerular fibrin deposition , and globally sclerotic glomeruli were also observed in nondiabetic enos / mice ; however , its frequency was much lower than in diabetic enos / mice [ 32 , 33 , 37 ] . ( 2 ) electron microscopy : consistent with light microscopic findings , electron microscopy of diabetic enos / or enos + / glomeruli showed advanced glomerular lesions , including mesangiolysis that is characterized by accumulation of electron - lucent material in the mesangium and dissociation of the mesangial matrix , microaneurysm formation due to disruption of anchoring of the gbm to the mesangium , advanced mesangial expansion occasionally forming lobular or nodular mesangial architecture , and gbm thickening [ 3237 ] . in summary , as compared with diabetic enos + /+ mice , diabetic enos / or enos + / mice showed advanced nephropathic changes that overlap to human dn , although some differences were observed between the models . given the fact that a much less severe renal phenotype was observed in nondiabetic enos / mice , these findings define a critical role of enos in the development and progression of dn . although the precise mechanism of this is currently unknown , this may be because ( 1 ) obesity - related factors , including insulin resistance , hyperinsulinemia , and hyperlipidemia , accelerate the renal injury in diabetic enos / mice . however , in the setting of enos deficiency , hyperinsulinemia may largely stimulate the latter endothelial insulin signaling . no negatively regulates blood pressure ( bp ) and counteracts angiotensin ii ( ang ii ) , which are key pathogenic mediators in dn . these findings suggest that hypertension plays a critical role in the development of advanced glomerular lesions in diabetic enos / mice and that enos deficiency induces aldosterone breakthrough , reducing the efficacy of ras blockades for dn . further , we have shown that antihypertensive triple therapy ( hydralazine + hydrochlorothiazide + reserpine ) or captopril normalizes bp and remarkably reduces glomerular and tubulointerstitial injury in db / db enos / mice . these findings suggest that enos deficiency may impair autoregulation concomitant with afferent arteriolar injury in diabetic mice , leading to hyperperfusion of glomeruli . thus , further investigation would be required to determine the effects of enos deficiency in glomerular ( and renal ) hemodynamics and autoregulation in diabetes . renal as well as systemic renin - angiotensin system ( ras ) is known to play a key role in dn . abnormal angiogenesis has been implicated in the pathogenesis of dn , coupled with increased production of angiogenic factors such as vegf and angiopoietin-2 [ 70 , 71 ] . demonstrated that overexpression of vegf in podocytes causes nodular glomerulosclerosis , mesangiolysis , microaneurysms , and arteriolar hyalinosis concurrent with massive proteinuria and renal failure in enos / mice in the absence of diabetes , suggesting that increased vegf activity and enos deficiency are sufficient to develop advanced dn - like lesions in mice . first , the detailed mechanisms by which enos deficiency enhances vegfr2 signaling and endothelial proliferation have not been determined yet . in addition , a body of studies has shown that enos deficiency inhibits vegf - mediated angiogenesis in various conditions including cancer [ 67 , 75 , 76 ] , indicating that enos is a critical downstream mediator of vegf angiogenesis . ( 3 ) vegf - mediated glomerular capillary growth occurs in diabetic mice in the absence of enos . in this context , it should be noted that enos deficiency was shown to alter morphogenesis of capillary growth and results in less sprouting and enlarged vessels ; therefore , the morphogenesis of glomerular capillary growth in diabetic enos / mice may differ from those in dn where enos activity is partially decreased . thus , further investigation would be required to determine whether vegf - enos deficiency induces the pathological signals that are analogous to progressive dn . this may be another mechanism by which enos deficiency enhances vegf signaling , although there was no difference in total vegf and vegfr2 mrna levels between diabetic enos / and enos + /+ kidneys [ 33 , 39 ] . ) have recently shown that diabetic enos / mice ( stz and db / db models ) develop podocytopathy soon after the onset of diabetes , associated with the development of albuminuria , while podocyte injury is limited in diabetic enos + /+ or nondiabetic enos / mice . these findings suggest that enos deficiency confers ras - dependent acute podocytopathy to diabetic mice and that this podocytopathy may be caused by endocrine or paracrine mechanisms . further , we noted that heparanase expression is upregulated in the glomerulus ( endothelial and mesangial cells ) of stz or db / db enos / mice ( takahashi t , unpublished observation ) . since glomerular heparanase expression was shown to be associated with the development of dn in humans as well as in mice [ 83 , 84 ] , it would be of interest to examine the role of enos in the expression of heparanase and other glycocalyx disrupting enzymes in diabetic glomeruli . although the precise mechanism of this is currently unknown , this finding suggests that enos deficiency may impair glomerular endothelial junctions in diabetes , causing insudation to subendothelial space and mesangium , and advance diabetic glomerular lesions . in addition , it was shown that enos deficiency and high - fat diet synergistically increases renal expression of il-1 and il-6 in stz - induced diabetic mice . given the fact that enos deficiency itself had no effects on renal inflammatory gene expression , these findings suggest that enos deficiency accelerates renal inflammatory gene expression in the setting of diabetes . we have recently demonstrated that renal expression of heparin - binding epidermal growth factor - like growth factor ( hb - egf ) is upregulated in both db / db enos / and nondiabetic enos / mice as early as at 8 weeks of age , including arterial and glomerular endothelium , podocytes , and tubular cells . further , we also demonstrated that ( 1 ) l - name treatment dramatically increases renal hb - egf expression and its urinary excretion in db / db enos + /+ mice . in addition , we have recently shown that the renal phenotype in stz enos / mice is remarkably suppressed by an egfr inhibitor ( erlotinib ) . further investigation of this pathway should provide a new insight into the pathogenesis of dn . have recently shown that stz enos / kidney exhibits enhanced expression of toll - like receptor ( tlr ) tlr4 and its ligand hmgb1 , predominantly in tubular cells , and that synthetic tlr4 antagonist , crx-526 , significantly attenuates renal injury in this model without altering blood glucose and systolic blood pressure , including albuminuria , renal hypertrophy , and glomerular and tubulointerstitial injury . further , the authors demonstrated that crx-526 treatment decreases induction of chemokine ( c - c motif ) ligand ( ccl)-2 , osteopontin , ccl-5 , tgf- , and nf-b activation in stz enos / kidney and reduces macrophage infiltration and collagen deposition in glomerulus and interstitium . glomerular fibrin deposition was also observed in nondiabetic enos / mice ; however , its extent was highly limited in these mice [ 33 , 37 ] , indicating that enos deficiency promotes glomerular thrombus formation in the setting of diabetes . the authors also demonstrated that tf levels correlate with albuminuria , gbm thickening , and tubulointerstitial fibrosis ; tf expression is increased before the development of dn ; tf neutralizing antibody prominently decreases renal expression of inflammatory and fibrogenic genes in diabetic mice regardless of enos genotype ; high - fat diet additively increases renal tf levels and activities . in aggregate , these findings suggest that enos deficiency promotes tf expression in diabetic glomeruli through monocyte / macrophage infiltration and this activates the coagulation cascade in the mesangial area , promoting inflammatory and fibrogenic gene expression and accelerating glomerular injury . given the fact that serum p - selectin levels are increased in enos / mice , suggesting exocytosis of weibel - palade body by the endothelium , the authors proposed a model that enos deficiency causes endothelial release of vwf , resulting in mesangial as well as intraluminal deposition of vwf and facilitating glomerular thrombus formation and mesangial expansion . a large body of study has shown that increased oxidative stress plays a central role in the pathogenesis of dn . ( 1 ) a recent study showed that blockade of the ca2 + -activated k+ channel kca3.1 , which is expressed in proximal tubules , reduces albuminuria , renal hypertrophy , and the expression of inflammatory and fibrotic markers in tubulointerstitium in stz enos / mice without affecting blood pressure levels , suggesting involvement of kca3.1 pathway in this model . also , enos deficiency may lead to vasoconstriction , reducing renal blood flow , and cause renal hypoxia . in summary , multiple mechanisms are involved in the development of advanced dn in diabetic enos / mice ( figure 3 ) . first , this model , especially db / db enos / mice , develops glomerular lesions of progressive dn . thus , this model provides an invaluable tool for us to study the enos - mediated pathogenic mechanisms underlying progressive dn . on the other hand , we may also need to realize that there are some differences between this animal model and advanced human dn . although the changes of glomerular vegf and nephrin expression have not been evaluated in db / db enos / model , given the fact that glomerular gene expression in db / db enos / mice overlaps to those in early human dn , this model seems to still display early dn or a transition phase to advanced dn . because these superoxide generations do not occur in enos deficient mice , the endothelial oxidative stress in enos - deficient diabetic mice may be lower than that in actual diabetic endothelium . further , enos deficiency may decrease the production of reactive nitrogen species ( e.g. thus , there are some differences in pathophysiology of renal injury between diabetic enos / mice ( enos deficiency - mediated ) and actual dn ( enos dysfunction ) . therefore , it would be very important to validate the findings obtained from this model with human subjects . in this context , heterozygous model may serve better than homozygous model and the creation of a diabetic mouse model in which enos is uncoupled would be of great interest . the following efforts and investigation would be required to better understand the role of enos in dn . therefore , it would be required to conduct those studies with nephropathy - prone strain , such as dba2/j , c57bl6 dba2 f1 hybrid , or 129svev [ 51 , 96 ] , to determine the precise effects of enos deficiency in dn that was caused by hypoinsulinemic diabetes . also , the findings obtained with low - dose stz model should be confirmed with akita model . second , there are many unresolved issues with the reported models : ( 1 ) the cause of death in diabetic enos / mice is not defined , yet occasional bleeding in the chest and abdominal cavity and clots in the mesenteric arteries and necrosis of small intestine [ 34 , 35 ] suggest that hypercoagulability may in part contribute to premature death of these mice . ( 2 ) akita enos / study has emerged a role for enos to negatively regulate diabetic hyperfiltration , whereas previous studies proposed a role of enos as a positive regulator of diabetic hyperfiltration based on the effects of nonselective nos inhibitors . further , enos / mice were shown to exhibit congenital renal defects including renal scars containing crowded small glomeruli and progressive renal disease . therefore , it is still unclear whether enos deficiency accelerates dn or diabetes accelerates the renal injury in enos / mice . consequently , the precise role of enos in diabetic endothelium or vasculature is not defined clearly . the postnatal conditional knockout study to target endothelial or vascular ( endothelial and hematopoietic ) enos in diabetic mice last , a body of studies has shown a critical role of enos - derived no in regulation of renal hemodynamics and oxygen consumption . further , the fact that antihypertensive or ras inhibitor treatment remarkably reduces glomerular injury in diabetic enos / mice suggests that hemodynamic events may play a key role in the pathology of this model . in recent decade , several groups have generated diabetic enos / models , either with hypoinsulinemic or type 2 diabetes , and defined the critical role of enos in the development of advanced diabetic renal lesions . further , the efforts to investigate the underlying mechanisms in this model explored unreported enos - associated pathogenic mechanisms in this disease . the complete enos deficiency does not occur in actual dn ; therefore , diabetic enos / model may include some artificial events that are irrelevant to dn . however , given the facts that this animal model was developed through a mechanism that is relevant to human dn and exhibits the most advanced diabetic renal lesion among the reported mouse models , further investigations of this model should largely advance our understanding of the pathogenesis of progressive dn and these studies enable a more efficient interrogation of novel therapeutics for this disease .

the global initiative for chronic obstructive lung disease defines chronic obstructive pulmonary disease ( copd ) as a common preventable and treatable disease characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases . exacerbations and comorbidities contribute to the overall severity in individual patients.1 exacerbations are associated with increased airway and systemic inflammation , which lead to airway wall edema , sputum plugging , and bronchoconstriction . copd remains a major global health and economic burden that is expected to be the third leading cause of death , and the fifth leading cause of disability by 2020.1 in 2010 , copd accounted for $ 49.9 billion in health care expenditures in the united states alone ( $ 29.5 billion in direct health care expenditures , $ 8.0 billion in indirect morbidity costs , and $ 12.4 billion in indirect mortality costs).2 in europe , copd accounts for 10.3 billion euros in health care spending a year.3 pharmacological therapy is used to control symptoms , as well as to reduce exacerbations , and to improve exercise tolerance . ambulatory copd patients are currently treated with long - acting bronchodilators and inhaled corticosteroids , along with systemic corticosteroids during exacerbations . there is a pressing need to develop novel approaches for the treatment of copd and the prevention or reduction of acute exacerbations of copd . existing therapies give partial benefits either by incompletely improving airflow limitation or reducing acute exacerbations , hence the need for newer , more effective therapies . evidence that existing medications reduce lung function decline in the long term has been inconclusive . the torch study4 investigated the effects of combined salmeterol plus fluticasone , either component alone , and placebo , on the rate of postbronchodilator forced expiratory volume in 1 second ( fev1 ) decline . the investigators found that salmeterol plus fluticasone reduced the rate of fev1 decline by 16 ml / year compared with placebo . despite this large study , a meta - analysis conducted by soriano et al5 concluded that inhaled corticosteroids showed a significant improvement in lung function decline compared with placebo at 3 months , however , after 6 months there was no significant difference between placebo and inhaled corticosteroid treatment . one of the main challenges in developing new therapeutic agents for the treatment or prevention of acute exacerbations of copd is that their potential success can not be entirely known until the investigational therapies enter relatively large phase ii studies , assessing clinical outcome over a 3- to 6-month period or longer.6,7 this article reviews the experimental challenges that can be performed relatively early in drug development for the treatment of copd in order to obtain preliminary signals of safety and efficacy in humans . these challenge models are representative of the local inflammatory response caused by an exacerbation of copd ; however it is important to note that these models do not reflect the actual exacerbation milieu . the models chosen are those models that have successfully been used to date in copd drug development . depending on the dose given during inhalation , these challenge models may also cause local and systemic inflammation , thus making them ideal for assessing the inflammatory processes in the lungs during an exacerbation and the potential therapeutic benefit of novel agents , as they mimic the local inflammatory response in the lung during an exacerbation . lipopolysaccharide ( lps ) is a macromolecular cell wall surface antigen of gram - negative bacteria . it is made up of three components : the o antigen ( or o polysaccharide ) side chain , the core oligosaccharide , and the lipid a moiety.8 lps is an extremely biologically active substance and has been used for many years in preclinical and clinical research , due to its role in activating many transcription factors . in the serum , lps binds to a lipid - binding protein , which facilitates the association between lps and cd14 on the cell membranes . this in turn facilitates the transfer of lps to the trl4/md2 complex ( figure 1).9 this triggers a signaling cascade in macrophage lineage and endothelial cells , resulting in the secretion of proinflammatory cytokines and nitric oxide , and the activation of complement and the coagulation systems that contribute to characteristic features of inflammation , and with excessive stimulation , endotoxic shock . in monocytes and macrophages , lps triggers the production of powerful inflammatory mediators including cytokines ( eg , interleukin [ il]-1 , il-6 , il-8 , tumor necrosis factor [ tnf]- and platelet - activating factor ) , which stimulate production of prostaglandins and leukotrienes . activation of alternative complement pathway factors c3a and c5a induce histamine release , and affect neutrophil chemotaxis and accumulation . the release of these mediators and subsequent systemic response make lps a powerful research tool in evaluating inflammatory pathways . healthy subjects inhaling endotoxin show a systemic and pulmonary inflammatory response , recruiting neutrophils and macrophages to the lung tissue . inhalation of nebulized doses ( up to 50 g ) of lps via a dosimeter in healthy volunteers , leads to an increase in temperature , blood c - reactive protein ( crp ) , blood and sputum neutrophils , blood monocytes and lymphocytes , and blood and sputum proinflammatory mediators , including : il-8 , tnf , myeloperoxidase ( mpo ) , matrix metalloproteinase-9 , il-6 , il-1 , monocyte chemotactic protein-1 , and macrophage inflammatory protein-1.10,11 bronchial segmental instillation induces an early phase response ( 024 hours ) , resulting in a statistically significant increase in neutrophils , tnf , il-1 , il-1r antagonist , il-6 , and granulocyte colony - stimulating factor . neutrophils , macrophages , and monocytes increase 2448 hours post instillation.11,12 intranasal lps challenge may be the least invasive and best tolerated model . clinical symptoms are minimal ; however , very little or no data have been published to date using this model . further validation of the intranasal model needs to be performed to compare it to the lps inhalation challenge model , to better understand the relevance of the lps - triggered nasal inflammation to the phenomena that occur in the central and distal airways in copd . many of the effects of exogenous lps can be blocked by medications . in one study , pretreatment with oral prednisolone or cilomilast had no effect on the local lps - induced inflammatory response in the lung;13 pretreatment with prednisolone alone significantly inhibited the lps - induced crp response . similarly , in another study , subjects who received simvastatin 4080 mg demonstrated a reduction in neutrophils , mpo , tnf , matrix metalloproteinase-7 , -8 , and -9 in the bronchoalveolar lavage fluid ( balf ) , as well as a reduction in plasma crp , versus placebo.14 hohlfeld et al15 used lps to show that roflumilast reduced the influx of total cells , neutrophils , and eosinophils into the airways of healthy subjects after segmental challenge with endotoxin , with statistical significance . it is important to remember that inhaled lps challenge is a model of acute neutrophilic inflammation and not a model of copd.16 the model can be used to understand the biological effects of compounds that inhibit the lps pathway ( table 1 ) . it is formed in the troposphere from primary precursor pollutants . in the presence of light , no2 is cleaved by sunlight to no + o , allowing the formation of o3 ( o2 + o ) ( figure 2 ) . in epidemiological studies , o3 levels have been associated with exacerbations of asthma , copd , and pneumonia.1719 experimental o3 exposure in healthy human subjects is known to elicit a reversible impairment in lung function , as well as acute proximal airways neutrophilic inflammation , and an increase in the concentration of several cytokines and mediators of inflammation within the airways.20 in the first reported study of the inflammatory effects of low - level o3 exposure ( 80 ppb o3 for 6.6 hours ) in healthy volunteers,21 there were statistically significant increases in polymorphononuclear neutrophils , prostaglandin e2 , lactate dehydrogenase , il-6 , 1-antitrypsin , and decreased phagocytosis via the complement receptor . this is similar to a more recent study with low - level exposure to o3 at 80 ppb for 6.6 hours,22 in which there were increased airway neutrophils , monocytes , and dendritic cells , as well as modifications of the expression of cd14 , hla - dr , cd80 , and cd86 on monocytes . in another study examining whether circulating cd11b plays a role in the inflammatory response following inhaled o3 exposure , 22 volunteers underwent controlled exposure to o3 ( 400 ppb for 2 hours ) and to clean air on two separate occasions.23 induced sputum collected from subjects exposed to o3 revealed marked neutrophilia , and increased expression of mcd14 on airway macrophages and monocytes . circulating cd11b levels also predicted the magnitude of the airway neutrophil response a number of different classes of therapeutic agents have been studied in the o3 challenge model in healthy volunteers . therapeutic classes include corticosteroids ( administered orally and by inhalation ) and nonsteroidal anti - inflammatory drugs . more recently , studies investigating the effects of cxc chemokine receptor ( cxcr ) 1 , 2 antagonists have been reported.2428 holz et al24 conducted a double - blind , double - dummy , placebo - controlled , three - period crossover study . eighteen healthy subjects , who had been shown at screening to produce more than a 10% increase in sputum neutrophils in response to exposure to 250 ppb o3 , were randomly assigned to receive alternating single orally inhaled doses of fluticasone 2 mg , 50 mg of prednisolone orally , and placebo at least 2 weeks apart . compared with placebo , pretreatment with inhaled or oral corticosteroids resulted in a significant reduction of sputum neutrophils , by 62% and 64% , respectively . this was associated with statistically significant reductions in sputum mpo , by 55% for inhaled corticosteroids and 42% for oral steroids . compared with placebo , there was a mean reduction in sputum il-8 levels , by 49% after inhaled corticosteroids and 34% after oral corticosteroids . similar results were obtained in a study conducted by alexis et al.25 in subjects receiving fluticasone 0.5 mg and 2 mg , sputum neutrophilia was significantly reduced by 18% and 35% , respectively . the following inflammatory markers were also significantly reduced in a dose - dependent manner in subjects receiving fluticasone : cd11b , mcd14 , cd64 , cd16 , hla - dr , and cd86 on sputum monocytes . serum clara cell protein 16 levels ( a marker of pulmonary damage ) were significantly increased post - o3 challenge . schelegle et al26 pretreated healthy volunteers with indomethacin , which was shown to significantly reduce o3-induced decrements in fev1 and forced vital capacity , when compared to no drug or placebo . this was associated with reductions in subjective symptoms of cough , shortness of breath , and throat tickle on indomethacin treatment , suggesting that cyclooxygenase products play a partial role in subjective symptoms associated with o3 exposure . hazucha et al29 demonstrated a similar reduction of o3-induced decrements in fev1 and forced vital capacity following single - dose treatment with either 200 mg or 800 mg of ibuprofen compared to placebo , which was associated with reduced post - o3 balf levels of prostaglandin e2 , thromboxane b2 , and il-6 . sch527123 is a novel , selective , oral cxc chemokine receptor 2 antagonist that inhibits neutrophil activation and modulates neutrophil trafficking in animal models . eighteen healthy o3 responders ( > 20% increase in sputum neutrophils ) underwent o3 challenge tests ( 250 ppb , 3 hours intermittent exercise ) 1 hour after the last treatment dose , and sputum was induced at 3 hours postchallenge27 following sch527123 treatment . the o3 challenge resulted in statistically significantly lower sputum neutrophil counts ( 0.136 10 ml ) compared with prednisolone ( 0.846 10 ml ; p = 0.001 ) or placebo ( 2.986 10 ml ; p = 0.001 ) . comparable results were obtained for total cell count , percentage of sputum neutrophils , and for il-8 and myeloperoxidase in sputum supernatant . post challenge , sch527123 inhibited neutrophilia in peripheral blood , but significantly less than in sputum . gaga et al30 investigated the use of sch527123i in subjects with severe neutrophilic asthma . when averaged over the 4-week treatment period , sputum neutrophils were significantly reduced in the active group by 57% compared to placebo ( p = 0.01 ) . there were fewer mild exacerbations ( 1.3 vs 2.25 ; p = 0.05 ) , and there was a trend towards fewer severe exacerbations in the sch527123 group . the asthma control questionnaire score was improved by 0.42 points in the active group compared with the placebo - treated group ( p = 0.053 ) , although the difference did not reach clinical significance , and no changes were observed in fev1 . this shows that in asthma , the o3 challenge model has proved useful in demonstrating inhibition of neutrophil numbers , associated with a clinical meaningful effect . more recently , in a randomized , double - blind , placebo controlled three - way crossover trial of the cxcr2 antagonist sb-656933 , 24 healthy nonsmoking male subjects were randomly assigned to receive a single dose of 50 mg , 150 mg , or placebo , 1 hour prior to o3 challenge.31 single doses of sb-656933 reduced o3-induced airway inflammation in a dose - dependent manner . there were corresponding reductions in myeloperoxidase levels in the sputum supernatant , by 32.8% ( confidence interval : 9.2 , 50.3 ) and 50.5% ( confidence interval : 33.3 , 63.3 ) . studies in copd have been conducted using o3 concentrations in the range of 120250 ppb with 7.515 minutes of exercise every 30 minutes , aiming to maintain a ventilatory rate of between 2030 l / min.3234 in a study of nine subjects with copd and ten age - matched controls , gong et al35 found an increase in specific airway resistance and a statistically significant decrease in fev1 in the copd subjects versus the age - matched controls . in summary , o3 challenge has been well tolerated in healthy volunteers and in older subjects , as well as subjects with asthma or copd . the o3-challenge model potentially provides critical decision - making data in understanding whether new compounds have the desired biological effect in healthy volunteers and patients with copd ; hence it can de - risk decisions to move forwards into large phase ii safety and efficacy trials . rhinovirus is responsible for the common cold and is spread through infected respiratory secretions from one person to another . human rhinovirus ( hrv ) replicates at 33c35c and thus has been linked to upper airway infections , where mucosal surfaces are cooler . gern et al36 detected hrv using reverse transcription polymerase chain reaction in both nasal secretions and balf , in healthy subjects after experimental infection with rhinovirus 16 . hrv binds to intracellular adhesion molecule ( icam)-1 , the major hrv receptor.37 the low - density - lipoprotein receptor38 binds to a minor group of hrv ( hrv2 ) . the gene for icam-1 maps to human chromosome 19 , as do the genes for a number of other picornavirus receptors . several studies have shown induction of proinflammatory genes implicated in neutrophil activation following rhinovirus induction of bronchial epithelial cells ( eg , il-8 regulated by nf- signaling pathways and gro).3941 rhinovirus infection of epithelial cells leads to the release of proinflammatory cytokines and chemokines including il-6 and il-8 . these cells release toxic products , stimulating mucus production and leading to tissue damage , with possible long - term loss of lung function . some mediators , such as endothelin-1 , have a direct effect in causing bronchoconstriction and vasoconstriction , resulting in airflow obstruction and impaired gas exchange . healthy subjects , subjects with asthma , and subjects with allergic asthma have been intensively studied in clinical trials inoculating them with rhinovirus 16 or other rhinovirus serotypes.4244 these studies demonstrated that rhinovirus infection of the lower airways is common after experimental inoculation . several studies looking at causes of exacerbations in copd have shown that viruses account for up to 60% of exacerbations , and that hrv is numerically the most important virus type.30,4550 figure 3 depicts the total viral and hrv exacerbation rate in seven exacerbation studies . other viruses associated with acute exacerbations of copd are coronavirus , influenza a and b , parainfluenza , adenovirus , and respiratory syncytial virus.45,5155 to develop a model of viral exacerbation in subjects with copd , mallia et al56 conducted a virus dose - escalating study infecting four copd subjects with rhinovirus . in this study , the median tissue culture infective dose ( tcid50 ) of rhinovirus was administered by the inhaled route using a nebulizer , to elicit a copd exacerbation . although there was a decrease in fev1 ( 16% ) and peak expiratory flow ( 12% ) maximal on day 9 , there was not a statistically significant increase in total sputum cell count or peripheral neutrophil count . symptoms of cold and lower respiratory tract symptoms , as well as lung function changes that are characteristic of viral - induced exacerbations of copd , were observed . there was an increase , although not statistically significant , in the proinflammatory cytokines il-6 and il-8 . in another study , there were significant increases in total respiratory scores in both copd subjects and healthy controls.57 peak expiratory flow fell by 23.5 ml in the controls ( p = not significant ) and by 50.5 ml ( p < 0.05 ) in the copd patients . more recently , both the upper and lower symptom scores were found to be significantly higher in the copd subjects.58 in this study , ten of the 11 infected copd subjects met the criteria defining an exacerbation of copd . subjects in the copd group demonstrated significant decreased peak expiratory flow from baseline , while those in the control group did not . the blood and sputum showed a significant increase in peripheral neutrophils in the copd subjects but not the controls . subjects in the copd group had significantly increased sputum neutrophil elastase levels over baseline on days 9 and 15 , as well as il-8 levels on day 9 . sputum neutrophil elastase levels were significantly higher in subjects with copd , compared with control subjects on days 915 . to date , only one laboratory has published on this model and as such , the data should be interpreted with caution . the main advantage of this model is that it will give a clear understanding and insight into the molecular and cellular inflammatory processes that take place during a viral - associated exacerbation of copd . there are no published data to date on the effect of pharmacological interventions in this model . the rhinovirus challenge model has the potential for use as a preclinical and clinical tool to identify and investigate novel drug targets and establish whether new therapeutic agents have potential clinical utility . these include ( but are not limited to ) targets against soluble icam-1 and thus inhibit interaction of hrv with icam-1,59,60 inhibitors of rhinovirus rna - dependent rna polymerases 3d,61 activators of retinoic acid - inducible gene 1,62 inhibitors of rhinovirus capsid protein vp-163 and inhibitors of different rhinovirus proteases ( eg , 2a , 3c).64,65 currently , this is the only model that reflects the underlying mechanism of viral exacerbations of copd . lipopolysaccharide ( lps ) is a macromolecular cell wall surface antigen of gram - negative bacteria . it is made up of three components : the o antigen ( or o polysaccharide ) side chain , the core oligosaccharide , and the lipid a moiety.8 lps is an extremely biologically active substance and has been used for many years in preclinical and clinical research , due to its role in activating many transcription factors . in the serum , lps binds to a lipid - binding protein , which facilitates the association between lps and cd14 on the cell membranes . this in turn facilitates the transfer of lps to the trl4/md2 complex ( figure 1).9 this triggers a signaling cascade in macrophage lineage and endothelial cells , resulting in the secretion of proinflammatory cytokines and nitric oxide , and the activation of complement and the coagulation systems that contribute to characteristic features of inflammation , and with excessive stimulation , endotoxic shock . in monocytes and macrophages , lps triggers the production of powerful inflammatory mediators including cytokines ( eg , interleukin [ il]-1 , il-6 , il-8 , tumor necrosis factor [ tnf]- and platelet - activating factor ) , which stimulate production of prostaglandins and leukotrienes . activation of alternative complement pathway factors c3a and c5a induce histamine release , and affect neutrophil chemotaxis and accumulation . the release of these mediators and subsequent systemic response make lps a powerful research tool in evaluating inflammatory pathways . healthy subjects inhaling endotoxin show a systemic and pulmonary inflammatory response , recruiting neutrophils and macrophages to the lung tissue . inhalation of nebulized doses ( up to 50 g ) of lps via a dosimeter in healthy volunteers , leads to an increase in temperature , blood c - reactive protein ( crp ) , blood and sputum neutrophils , blood monocytes and lymphocytes , and blood and sputum proinflammatory mediators , including : il-8 , tnf , myeloperoxidase ( mpo ) , matrix metalloproteinase-9 , il-6 , il-1 , monocyte chemotactic protein-1 , and macrophage inflammatory protein-1.10,11 bronchial segmental instillation induces an early phase response ( 024 hours ) , resulting in a statistically significant increase in neutrophils , tnf , il-1 , il-1r antagonist , il-6 , and granulocyte colony - stimulating factor . neutrophils , macrophages , and monocytes increase 2448 hours post instillation.11,12 intranasal lps challenge may be the least invasive and best tolerated model . clinical symptoms are minimal ; however , very little or no data have been published to date using this model . further validation of the intranasal model needs to be performed to compare it to the lps inhalation challenge model , to better understand the relevance of the lps - triggered nasal inflammation to the phenomena that occur in the central and distal airways in copd . many of the effects of exogenous lps can be blocked by medications . in one study , pretreatment with oral prednisolone or cilomilast had no effect on the local lps - induced inflammatory response in the lung;13 pretreatment with prednisolone alone significantly inhibited the lps - induced crp response . similarly , in another study , subjects who received simvastatin 4080 mg demonstrated a reduction in neutrophils , mpo , tnf , matrix metalloproteinase-7 , -8 , and -9 in the bronchoalveolar lavage fluid ( balf ) , as well as a reduction in plasma crp , versus placebo.14 hohlfeld et al15 used lps to show that roflumilast reduced the influx of total cells , neutrophils , and eosinophils into the airways of healthy subjects after segmental challenge with endotoxin , with statistical significance . it is important to remember that inhaled lps challenge is a model of acute neutrophilic inflammation and not a model of copd.16 the model can be used to understand the biological effects of compounds that inhibit the lps pathway ( table 1 ) . it is formed in the troposphere from primary precursor pollutants . in the presence of light , no2 is cleaved by sunlight to no + o , allowing the formation of o3 ( o2 + o ) ( figure 2 ) . in epidemiological studies , o3 levels have been associated with exacerbations of asthma , copd , and pneumonia.1719 experimental o3 exposure in healthy human subjects is known to elicit a reversible impairment in lung function , as well as acute proximal airways neutrophilic inflammation , and an increase in the concentration of several cytokines and mediators of inflammation within the airways.20 in the first reported study of the inflammatory effects of low - level o3 exposure ( 80 ppb o3 for 6.6 hours ) in healthy volunteers,21 there were statistically significant increases in polymorphononuclear neutrophils , prostaglandin e2 , lactate dehydrogenase , il-6 , 1-antitrypsin , and decreased phagocytosis via the complement receptor . this is similar to a more recent study with low - level exposure to o3 at 80 ppb for 6.6 hours,22 in which there were increased airway neutrophils , monocytes , and dendritic cells , as well as modifications of the expression of cd14 , hla - dr , cd80 , and cd86 on monocytes . in another study examining whether circulating cd11b plays a role in the inflammatory response following inhaled o3 exposure , 22 volunteers underwent controlled exposure to o3 ( 400 ppb for 2 hours ) and to clean air on two separate occasions.23 induced sputum collected from subjects exposed to o3 revealed marked neutrophilia , and increased expression of mcd14 on airway macrophages and monocytes . circulating cd11b levels also predicted the magnitude of the airway neutrophil response following inhaled o3 exposure . a number of different classes of therapeutic agents have been studied in the o3 challenge model in healthy volunteers . therapeutic classes include corticosteroids ( administered orally and by inhalation ) and nonsteroidal anti - inflammatory drugs . more recently , studies investigating the effects of cxc chemokine receptor ( cxcr ) 1 , 2 antagonists have been reported.2428 holz et al24 conducted a double - blind , double - dummy , placebo - controlled , three - period crossover study . eighteen healthy subjects , who had been shown at screening to produce more than a 10% increase in sputum neutrophils in response to exposure to 250 ppb o3 , were randomly assigned to receive alternating single orally inhaled doses of fluticasone 2 mg , 50 mg of prednisolone orally , and placebo at least 2 weeks apart . compared with placebo , pretreatment with inhaled or oral corticosteroids resulted in a significant reduction of sputum neutrophils , by 62% and 64% , respectively . this was associated with statistically significant reductions in sputum mpo , by 55% for inhaled corticosteroids and 42% for oral steroids . compared with placebo , there was a mean reduction in sputum il-8 levels , by 49% after inhaled corticosteroids and 34% after oral corticosteroids . similar results were obtained in a study conducted by alexis et al.25 in subjects receiving fluticasone 0.5 mg and 2 mg , sputum neutrophilia was significantly reduced by 18% and 35% , respectively . the following inflammatory markers were also significantly reduced in a dose - dependent manner in subjects receiving fluticasone : cd11b , mcd14 , cd64 , cd16 , hla - dr , and cd86 on sputum monocytes . serum clara cell protein 16 levels ( a marker of pulmonary damage ) were significantly increased post - o3 challenge . schelegle et al26 pretreated healthy volunteers with indomethacin , which was shown to significantly reduce o3-induced decrements in fev1 and forced vital capacity , when compared to no drug or placebo . this was associated with reductions in subjective symptoms of cough , shortness of breath , and throat tickle on indomethacin treatment , suggesting that cyclooxygenase products play a partial role in subjective symptoms associated with o3 exposure . hazucha et al29 demonstrated a similar reduction of o3-induced decrements in fev1 and forced vital capacity following single - dose treatment with either 200 mg or 800 mg of ibuprofen compared to placebo , which was associated with reduced post - o3 balf levels of prostaglandin e2 , thromboxane b2 , and il-6 . sch527123 is a novel , selective , oral cxc chemokine receptor 2 antagonist that inhibits neutrophil activation and modulates neutrophil trafficking in animal models . eighteen healthy o3 responders ( > 20% increase in sputum neutrophils ) underwent o3 challenge tests ( 250 ppb , 3 hours intermittent exercise ) 1 hour after the last treatment dose , and sputum was induced at 3 hours postchallenge27 following sch527123 treatment . the o3 challenge resulted in statistically significantly lower sputum neutrophil counts ( 0.136 10 ml ) compared with prednisolone ( 0.846 10 ml ; p = 0.001 ) or placebo ( 2.986 10 ml ; p = 0.001 ) . comparable results were obtained for total cell count , percentage of sputum neutrophils , and for il-8 and myeloperoxidase in sputum supernatant . post challenge , sch527123 inhibited neutrophilia in peripheral blood , but significantly less than in sputum . gaga et al30 investigated the use of sch527123i in subjects with severe neutrophilic asthma . when averaged over the 4-week treatment period , sputum neutrophils were significantly reduced in the active group by 57% compared to placebo ( p = 0.01 ) . there were fewer mild exacerbations ( 1.3 vs 2.25 ; p = 0.05 ) , and there was a trend towards fewer severe exacerbations in the sch527123 group . the asthma control questionnaire score was improved by 0.42 points in the active group compared with the placebo - treated group ( p = 0.053 ) , although the difference did not reach clinical significance , and no changes were observed in fev1 . this shows that in asthma , the o3 challenge model has proved useful in demonstrating inhibition of neutrophil numbers , associated with a clinical meaningful effect . more recently , in a randomized , double - blind , placebo controlled three - way crossover trial of the cxcr2 antagonist sb-656933 , 24 healthy nonsmoking male subjects were randomly assigned to receive a single dose of 50 mg , 150 mg , or placebo , 1 hour prior to o3 challenge.31 single doses of sb-656933 reduced o3-induced airway inflammation in a dose - dependent manner . there were corresponding reductions in myeloperoxidase levels in the sputum supernatant , by 32.8% ( confidence interval : 9.2 , 50.3 ) and 50.5% ( confidence interval : 33.3 , 63.3 ) . studies in copd have been conducted using o3 concentrations in the range of 120250 ppb with 7.515 minutes of exercise every 30 minutes , aiming to maintain a ventilatory rate of between 2030 l / min.3234 in a study of nine subjects with copd and ten age - matched controls , gong et al35 found an increase in specific airway resistance and a statistically significant decrease in fev1 in the copd subjects versus the age - matched controls . in summary , o3 challenge has been well tolerated in healthy volunteers and in older subjects , as well as subjects with asthma or copd . the o3-challenge model potentially provides critical decision - making data in understanding whether new compounds have the desired biological effect in healthy volunteers and patients with copd ; hence it can de - risk decisions to move forwards into large phase ii safety and efficacy trials . rhinovirus is responsible for the common cold and is spread through infected respiratory secretions from one person to another . human rhinovirus ( hrv ) replicates at 33c35c and thus has been linked to upper airway infections , where mucosal surfaces are cooler . gern et al36 detected hrv using reverse transcription polymerase chain reaction in both nasal secretions and balf , in healthy subjects after experimental infection with rhinovirus 16 . hrv binds to intracellular adhesion molecule ( icam)-1 , the major hrv receptor.37 the low - density - lipoprotein receptor38 binds to a minor group of hrv ( hrv2 ) . the gene for icam-1 maps to human chromosome 19 , as do the genes for a number of other picornavirus receptors . several studies have shown induction of proinflammatory genes implicated in neutrophil activation following rhinovirus induction of bronchial epithelial cells ( eg , il-8 regulated by nf- signaling pathways and gro).3941 rhinovirus infection of epithelial cells leads to the release of proinflammatory cytokines and chemokines including il-6 and il-8 . these cells release toxic products , stimulating mucus production and leading to tissue damage , with possible long - term loss of lung function . some mediators , such as endothelin-1 , have a direct effect in causing bronchoconstriction and vasoconstriction , resulting in airflow obstruction and impaired gas exchange . healthy subjects , subjects with asthma , and subjects with allergic asthma have been intensively studied in clinical trials inoculating them with rhinovirus 16 or other rhinovirus serotypes.4244 these studies demonstrated that rhinovirus infection of the lower airways is common after experimental inoculation . several studies looking at causes of exacerbations in copd have shown that viruses account for up to 60% of exacerbations , and that hrv is numerically the most important virus type.30,4550 figure 3 depicts the total viral and hrv exacerbation rate in seven exacerbation studies . other viruses associated with acute exacerbations of copd are coronavirus , influenza a and b , parainfluenza , adenovirus , and respiratory syncytial virus.45,5155 to develop a model of viral exacerbation in subjects with copd , mallia et al56 conducted a virus dose - escalating study infecting four copd subjects with rhinovirus . in this study , the median tissue culture infective dose ( tcid50 ) of rhinovirus was administered by the inhaled route using a nebulizer , to elicit a copd exacerbation . although there was a decrease in fev1 ( 16% ) and peak expiratory flow ( 12% ) maximal on day 9 , there was not a statistically significant increase in total sputum cell count or peripheral neutrophil count . symptoms of cold and lower respiratory tract symptoms , as well as lung function changes that are characteristic of viral - induced exacerbations of copd , were observed . there was an increase , although not statistically significant , in the proinflammatory cytokines il-6 and il-8 . in another study , there were significant increases in total respiratory scores in both copd subjects and healthy controls.57 peak expiratory flow fell by 23.5 ml in the controls ( p = not significant ) and by 50.5 ml ( p < 0.05 ) in the copd patients . more recently , both the upper and lower symptom scores were found to be significantly higher in the copd subjects.58 in this study , ten of the 11 infected copd subjects met the criteria defining an exacerbation of copd . subjects in the copd group demonstrated significant decreased peak expiratory flow from baseline , while those in the control group did not . the blood and sputum showed a significant increase in peripheral neutrophils in the copd subjects but not the controls . subjects in the copd group had significantly increased sputum neutrophil elastase levels over baseline on days 9 and 15 , as well as il-8 levels on day 9 . sputum neutrophil elastase levels were significantly higher in subjects with copd , compared with control subjects on days 915 . to date , only one laboratory has published on this model and as such , the data should be interpreted with caution . the main advantage of this model is that it will give a clear understanding and insight into the molecular and cellular inflammatory processes that take place during a viral - associated exacerbation of copd . there are no published data to date on the effect of pharmacological interventions in this model . the rhinovirus challenge model has the potential for use as a preclinical and clinical tool to identify and investigate novel drug targets and establish whether new therapeutic agents have potential clinical utility . these include ( but are not limited to ) targets against soluble icam-1 and thus inhibit interaction of hrv with icam-1,59,60 inhibitors of rhinovirus rna - dependent rna polymerases 3d,61 activators of retinoic acid - inducible gene 1,62 inhibitors of rhinovirus capsid protein vp-163 and inhibitors of different rhinovirus proteases ( eg , 2a , 3c).64,65 currently , this is the only model that reflects the underlying mechanism of viral exacerbations of copd . the use of challenge models has the potential to significantly inform early decision making , before embarking on long - term phase ii and iii clinical trials designed to test interventions that may treat or avert exacerbations of copd . although challenge models are good predictive models of acute exacerbations of copd , there are ethical considerations associated with inducing exacerbations in subjects with copd . therefore , safety boards may be advised to only consider subjects with mild copd for inclusion in these studies . healthy subjects could be used as an alternative , when determining the effects of a developmental drug s mechanism of action on lung inflammation . the lps and o3 models have been used successfully in healthy subjects.1315,22,24,2628 as these models represent the local inflammation in the lung during an exacerbation , and test the mechanism of action of potential novel drugs , these data may be used for future decision making . pharmaceutical companies have used lps models as a means to establish proof of principle early during the clinical development process , because they are relatively simple to perform and have few adverse events . this model is cost effective because it can be conducted in healthy subjects , who are easy to recruit . lps challenge data , whether positive or negative , can provide valuable information to aid investment decision making . one disadvantage of the lps model is that it is a model of lung inflammation , but not of the disease state , thus preclinical validation of the developmental drug s effects on lps pathways is essential . for anti - inflammatory targets that are involved in the toll - like receptor 4 , nf- pathway , the lps challenge model is the model of choice . despite the longstanding knowledge and understanding of the adverse effects of o3 on pulmonary biology , the use of o3 as a challenge model to assess the potential of new drugs for the prevention of acute exacerbations in copd , is relatively new . the model has been shown to be safe and to have few side effects in healthy volunteers , and in patients with asthma and copd.24,25,28,66 additionally , it is reliable and reproducible . it has been used successfully to generate biological effect and systemic effect for fluticasone and the cxcr2 antagonists , sch527123 and sb-656933.27,31 a limitation of the o3 model is that it has yet to be determined whether inhibition of neutrophilia translates into clinical benefits for patients with copd . preliminary data indicate that inhibition of the neutrophil response following o3 challenge may be associated with beneficial changes in system scores obtained in subjects with copd . challenge with rhinovirus 16 to elicit mild exacerbations in subjects with copd appears to be safe and well tolerated , but only a few copd subjects have been exposed to this model . the observation that fev1 does not always return to baseline after inducing an exacerbation in copd subjects may call to question the feasibility of using the challenge in a broader population of patients with copd , in addition to raising ethical considerations . this remains an exciting model with a great deal of potential , as rhinovirus models are good predictive models of viral - induced acute exacerbations of copd . in order for pharmaceutical companies to succeed in the copd arena , innovative approaches to clinical trial design and conduct are required that will generate critical , high - quality proof of efficacy and biologic target engagement data to support early investment decision making , early drug termination , and facilitation of better - informed decisions regarding those drugs in which proof of effect has been clearly demonstrated . challenge models in copd , which expose fewer individuals for short periods of time to eliciting agents , may serve as surrogate of potential efficacy and thus may help early decision making and reduction in clinical development timelines .

chronic obstructive pulmonary disease ( copd ) is a major cause of morbidity and mortality . current therapies confer partial benefits either by incompletely improving airflow limitation or by reducing acute exacerbations , hence new therapies are desirable . in the absence of robust early predictors of clinical efficacy , the potential success of novel therapeutic agents in copd will not entirely be known until the drugs enter relatively large and costly clinical trials . new predictive models in humans , and new study designs are being sought to allow for confirmation of pharmacodynamic and potentially clinically meaningful effects in early development . this review focuses on human challenge models with lipopolysaccharide endotoxin , ozone , and rhinovirus , in the early clinical development phases of novel therapeutic agents for the treatment and reduction of exacerbations in copd .

Introduction Lipopolysaccharide challenge Ozone challenge Rhinovirus challenge Discussion Conclusion

the global initiative for chronic obstructive lung disease defines chronic obstructive pulmonary disease ( copd ) as a common preventable and treatable disease characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases . exacerbations and comorbidities contribute to the overall severity in individual patients.1 exacerbations are associated with increased airway and systemic inflammation , which lead to airway wall edema , sputum plugging , and bronchoconstriction . copd remains a major global health and economic burden that is expected to be the third leading cause of death , and the fifth leading cause of disability by 2020.1 in 2010 , copd accounted for $ 49.9 billion in health care expenditures in the united states alone ( $ 29.5 billion in direct health care expenditures , $ 8.0 billion in indirect morbidity costs , and $ 12.4 billion in indirect mortality costs).2 in europe , copd accounts for 10.3 billion euros in health care spending a year.3 pharmacological therapy is used to control symptoms , as well as to reduce exacerbations , and to improve exercise tolerance . there is a pressing need to develop novel approaches for the treatment of copd and the prevention or reduction of acute exacerbations of copd . existing therapies give partial benefits either by incompletely improving airflow limitation or reducing acute exacerbations , hence the need for newer , more effective therapies . the torch study4 investigated the effects of combined salmeterol plus fluticasone , either component alone , and placebo , on the rate of postbronchodilator forced expiratory volume in 1 second ( fev1 ) decline . one of the main challenges in developing new therapeutic agents for the treatment or prevention of acute exacerbations of copd is that their potential success can not be entirely known until the investigational therapies enter relatively large phase ii studies , assessing clinical outcome over a 3- to 6-month period or longer.6,7 this article reviews the experimental challenges that can be performed relatively early in drug development for the treatment of copd in order to obtain preliminary signals of safety and efficacy in humans . these challenge models are representative of the local inflammatory response caused by an exacerbation of copd ; however it is important to note that these models do not reflect the actual exacerbation milieu . the models chosen are those models that have successfully been used to date in copd drug development . depending on the dose given during inhalation , these challenge models may also cause local and systemic inflammation , thus making them ideal for assessing the inflammatory processes in the lungs during an exacerbation and the potential therapeutic benefit of novel agents , as they mimic the local inflammatory response in the lung during an exacerbation . lipopolysaccharide ( lps ) is a macromolecular cell wall surface antigen of gram - negative bacteria . it is made up of three components : the o antigen ( or o polysaccharide ) side chain , the core oligosaccharide , and the lipid a moiety.8 lps is an extremely biologically active substance and has been used for many years in preclinical and clinical research , due to its role in activating many transcription factors . in the serum , lps binds to a lipid - binding protein , which facilitates the association between lps and cd14 on the cell membranes . this in turn facilitates the transfer of lps to the trl4/md2 complex ( figure 1).9 this triggers a signaling cascade in macrophage lineage and endothelial cells , resulting in the secretion of proinflammatory cytokines and nitric oxide , and the activation of complement and the coagulation systems that contribute to characteristic features of inflammation , and with excessive stimulation , endotoxic shock . inhalation of nebulized doses ( up to 50 g ) of lps via a dosimeter in healthy volunteers , leads to an increase in temperature , blood c - reactive protein ( crp ) , blood and sputum neutrophils , blood monocytes and lymphocytes , and blood and sputum proinflammatory mediators , including : il-8 , tnf , myeloperoxidase ( mpo ) , matrix metalloproteinase-9 , il-6 , il-1 , monocyte chemotactic protein-1 , and macrophage inflammatory protein-1.10,11 bronchial segmental instillation induces an early phase response ( 024 hours ) , resulting in a statistically significant increase in neutrophils , tnf , il-1 , il-1r antagonist , il-6 , and granulocyte colony - stimulating factor . further validation of the intranasal model needs to be performed to compare it to the lps inhalation challenge model , to better understand the relevance of the lps - triggered nasal inflammation to the phenomena that occur in the central and distal airways in copd . similarly , in another study , subjects who received simvastatin 4080 mg demonstrated a reduction in neutrophils , mpo , tnf , matrix metalloproteinase-7 , -8 , and -9 in the bronchoalveolar lavage fluid ( balf ) , as well as a reduction in plasma crp , versus placebo.14 hohlfeld et al15 used lps to show that roflumilast reduced the influx of total cells , neutrophils , and eosinophils into the airways of healthy subjects after segmental challenge with endotoxin , with statistical significance . in the presence of light , no2 is cleaved by sunlight to no + o , allowing the formation of o3 ( o2 + o ) ( figure 2 ) . in epidemiological studies , o3 levels have been associated with exacerbations of asthma , copd , and pneumonia.1719 experimental o3 exposure in healthy human subjects is known to elicit a reversible impairment in lung function , as well as acute proximal airways neutrophilic inflammation , and an increase in the concentration of several cytokines and mediators of inflammation within the airways.20 in the first reported study of the inflammatory effects of low - level o3 exposure ( 80 ppb o3 for 6.6 hours ) in healthy volunteers,21 there were statistically significant increases in polymorphononuclear neutrophils , prostaglandin e2 , lactate dehydrogenase , il-6 , 1-antitrypsin , and decreased phagocytosis via the complement receptor . in another study examining whether circulating cd11b plays a role in the inflammatory response following inhaled o3 exposure , 22 volunteers underwent controlled exposure to o3 ( 400 ppb for 2 hours ) and to clean air on two separate occasions.23 induced sputum collected from subjects exposed to o3 revealed marked neutrophilia , and increased expression of mcd14 on airway macrophages and monocytes . circulating cd11b levels also predicted the magnitude of the airway neutrophil response a number of different classes of therapeutic agents have been studied in the o3 challenge model in healthy volunteers . eighteen healthy subjects , who had been shown at screening to produce more than a 10% increase in sputum neutrophils in response to exposure to 250 ppb o3 , were randomly assigned to receive alternating single orally inhaled doses of fluticasone 2 mg , 50 mg of prednisolone orally , and placebo at least 2 weeks apart . compared with placebo , pretreatment with inhaled or oral corticosteroids resulted in a significant reduction of sputum neutrophils , by 62% and 64% , respectively . hazucha et al29 demonstrated a similar reduction of o3-induced decrements in fev1 and forced vital capacity following single - dose treatment with either 200 mg or 800 mg of ibuprofen compared to placebo , which was associated with reduced post - o3 balf levels of prostaglandin e2 , thromboxane b2 , and il-6 . there were fewer mild exacerbations ( 1.3 vs 2.25 ; p = 0.05 ) , and there was a trend towards fewer severe exacerbations in the sch527123 group . the asthma control questionnaire score was improved by 0.42 points in the active group compared with the placebo - treated group ( p = 0.053 ) , although the difference did not reach clinical significance , and no changes were observed in fev1 . more recently , in a randomized , double - blind , placebo controlled three - way crossover trial of the cxcr2 antagonist sb-656933 , 24 healthy nonsmoking male subjects were randomly assigned to receive a single dose of 50 mg , 150 mg , or placebo , 1 hour prior to o3 challenge.31 single doses of sb-656933 reduced o3-induced airway inflammation in a dose - dependent manner . studies in copd have been conducted using o3 concentrations in the range of 120250 ppb with 7.515 minutes of exercise every 30 minutes , aiming to maintain a ventilatory rate of between 2030 l / min.3234 in a study of nine subjects with copd and ten age - matched controls , gong et al35 found an increase in specific airway resistance and a statistically significant decrease in fev1 in the copd subjects versus the age - matched controls . rhinovirus is responsible for the common cold and is spread through infected respiratory secretions from one person to another . healthy subjects , subjects with asthma , and subjects with allergic asthma have been intensively studied in clinical trials inoculating them with rhinovirus 16 or other rhinovirus serotypes.4244 these studies demonstrated that rhinovirus infection of the lower airways is common after experimental inoculation . several studies looking at causes of exacerbations in copd have shown that viruses account for up to 60% of exacerbations , and that hrv is numerically the most important virus type.30,4550 figure 3 depicts the total viral and hrv exacerbation rate in seven exacerbation studies . other viruses associated with acute exacerbations of copd are coronavirus , influenza a and b , parainfluenza , adenovirus , and respiratory syncytial virus.45,5155 to develop a model of viral exacerbation in subjects with copd , mallia et al56 conducted a virus dose - escalating study infecting four copd subjects with rhinovirus . there was an increase , although not statistically significant , in the proinflammatory cytokines il-6 and il-8 . the blood and sputum showed a significant increase in peripheral neutrophils in the copd subjects but not the controls . subjects in the copd group had significantly increased sputum neutrophil elastase levels over baseline on days 9 and 15 , as well as il-8 levels on day 9 . the rhinovirus challenge model has the potential for use as a preclinical and clinical tool to identify and investigate novel drug targets and establish whether new therapeutic agents have potential clinical utility . lipopolysaccharide ( lps ) is a macromolecular cell wall surface antigen of gram - negative bacteria . it is made up of three components : the o antigen ( or o polysaccharide ) side chain , the core oligosaccharide , and the lipid a moiety.8 lps is an extremely biologically active substance and has been used for many years in preclinical and clinical research , due to its role in activating many transcription factors . this in turn facilitates the transfer of lps to the trl4/md2 complex ( figure 1).9 this triggers a signaling cascade in macrophage lineage and endothelial cells , resulting in the secretion of proinflammatory cytokines and nitric oxide , and the activation of complement and the coagulation systems that contribute to characteristic features of inflammation , and with excessive stimulation , endotoxic shock . activation of alternative complement pathway factors c3a and c5a induce histamine release , and affect neutrophil chemotaxis and accumulation . further validation of the intranasal model needs to be performed to compare it to the lps inhalation challenge model , to better understand the relevance of the lps - triggered nasal inflammation to the phenomena that occur in the central and distal airways in copd . similarly , in another study , subjects who received simvastatin 4080 mg demonstrated a reduction in neutrophils , mpo , tnf , matrix metalloproteinase-7 , -8 , and -9 in the bronchoalveolar lavage fluid ( balf ) , as well as a reduction in plasma crp , versus placebo.14 hohlfeld et al15 used lps to show that roflumilast reduced the influx of total cells , neutrophils , and eosinophils into the airways of healthy subjects after segmental challenge with endotoxin , with statistical significance . in epidemiological studies , o3 levels have been associated with exacerbations of asthma , copd , and pneumonia.1719 experimental o3 exposure in healthy human subjects is known to elicit a reversible impairment in lung function , as well as acute proximal airways neutrophilic inflammation , and an increase in the concentration of several cytokines and mediators of inflammation within the airways.20 in the first reported study of the inflammatory effects of low - level o3 exposure ( 80 ppb o3 for 6.6 hours ) in healthy volunteers,21 there were statistically significant increases in polymorphononuclear neutrophils , prostaglandin e2 , lactate dehydrogenase , il-6 , 1-antitrypsin , and decreased phagocytosis via the complement receptor . this is similar to a more recent study with low - level exposure to o3 at 80 ppb for 6.6 hours,22 in which there were increased airway neutrophils , monocytes , and dendritic cells , as well as modifications of the expression of cd14 , hla - dr , cd80 , and cd86 on monocytes . in another study examining whether circulating cd11b plays a role in the inflammatory response following inhaled o3 exposure , 22 volunteers underwent controlled exposure to o3 ( 400 ppb for 2 hours ) and to clean air on two separate occasions.23 induced sputum collected from subjects exposed to o3 revealed marked neutrophilia , and increased expression of mcd14 on airway macrophages and monocytes . a number of different classes of therapeutic agents have been studied in the o3 challenge model in healthy volunteers . hazucha et al29 demonstrated a similar reduction of o3-induced decrements in fev1 and forced vital capacity following single - dose treatment with either 200 mg or 800 mg of ibuprofen compared to placebo , which was associated with reduced post - o3 balf levels of prostaglandin e2 , thromboxane b2 , and il-6 . comparable results were obtained for total cell count , percentage of sputum neutrophils , and for il-8 and myeloperoxidase in sputum supernatant . there were fewer mild exacerbations ( 1.3 vs 2.25 ; p = 0.05 ) , and there was a trend towards fewer severe exacerbations in the sch527123 group . the asthma control questionnaire score was improved by 0.42 points in the active group compared with the placebo - treated group ( p = 0.053 ) , although the difference did not reach clinical significance , and no changes were observed in fev1 . there were corresponding reductions in myeloperoxidase levels in the sputum supernatant , by 32.8% ( confidence interval : 9.2 , 50.3 ) and 50.5% ( confidence interval : 33.3 , 63.3 ) . studies in copd have been conducted using o3 concentrations in the range of 120250 ppb with 7.515 minutes of exercise every 30 minutes , aiming to maintain a ventilatory rate of between 2030 l / min.3234 in a study of nine subjects with copd and ten age - matched controls , gong et al35 found an increase in specific airway resistance and a statistically significant decrease in fev1 in the copd subjects versus the age - matched controls . rhinovirus is responsible for the common cold and is spread through infected respiratory secretions from one person to another . healthy subjects , subjects with asthma , and subjects with allergic asthma have been intensively studied in clinical trials inoculating them with rhinovirus 16 or other rhinovirus serotypes.4244 these studies demonstrated that rhinovirus infection of the lower airways is common after experimental inoculation . several studies looking at causes of exacerbations in copd have shown that viruses account for up to 60% of exacerbations , and that hrv is numerically the most important virus type.30,4550 figure 3 depicts the total viral and hrv exacerbation rate in seven exacerbation studies . other viruses associated with acute exacerbations of copd are coronavirus , influenza a and b , parainfluenza , adenovirus , and respiratory syncytial virus.45,5155 to develop a model of viral exacerbation in subjects with copd , mallia et al56 conducted a virus dose - escalating study infecting four copd subjects with rhinovirus . there was an increase , although not statistically significant , in the proinflammatory cytokines il-6 and il-8 . the rhinovirus challenge model has the potential for use as a preclinical and clinical tool to identify and investigate novel drug targets and establish whether new therapeutic agents have potential clinical utility . the use of challenge models has the potential to significantly inform early decision making , before embarking on long - term phase ii and iii clinical trials designed to test interventions that may treat or avert exacerbations of copd . although challenge models are good predictive models of acute exacerbations of copd , there are ethical considerations associated with inducing exacerbations in subjects with copd . the lps and o3 models have been used successfully in healthy subjects.1315,22,24,2628 as these models represent the local inflammation in the lung during an exacerbation , and test the mechanism of action of potential novel drugs , these data may be used for future decision making . pharmaceutical companies have used lps models as a means to establish proof of principle early during the clinical development process , because they are relatively simple to perform and have few adverse events . for anti - inflammatory targets that are involved in the toll - like receptor 4 , nf- pathway , the lps challenge model is the model of choice . despite the longstanding knowledge and understanding of the adverse effects of o3 on pulmonary biology , the use of o3 as a challenge model to assess the potential of new drugs for the prevention of acute exacerbations in copd , is relatively new . the model has been shown to be safe and to have few side effects in healthy volunteers , and in patients with asthma and copd.24,25,28,66 additionally , it is reliable and reproducible . the observation that fev1 does not always return to baseline after inducing an exacerbation in copd subjects may call to question the feasibility of using the challenge in a broader population of patients with copd , in addition to raising ethical considerations . this remains an exciting model with a great deal of potential , as rhinovirus models are good predictive models of viral - induced acute exacerbations of copd . in order for pharmaceutical companies to succeed in the copd arena , innovative approaches to clinical trial design and conduct are required that will generate critical , high - quality proof of efficacy and biologic target engagement data to support early investment decision making , early drug termination , and facilitation of better - informed decisions regarding those drugs in which proof of effect has been clearly demonstrated . challenge models in copd , which expose fewer individuals for short periods of time to eliciting agents , may serve as surrogate of potential efficacy and thus may help early decision making and reduction in clinical development timelines .

a meta - analysis of 35 studies between 1990 and 2005 indicated that medication errors occurred in a mean of 5.7% of all drug administration episodes while adverse drug events affected 6.1 patients per 100 hospitalized . many factors are involved in drug prescription errors including polypharmacy , lack of sufficient pharmacological knowledge , errors in patients charts or documentation by nurses , inadequate pharmacy service , being a female , age > 65 years , renal excretion of drugs , drugs with narrow therapeutic index and the use of anticoagulants or diuretics . furthermore , several studies in the united states have consistently reported adverse drug events ranging from 3% to 12% . these studies indicate that 1.5 - 3% of all adverse drug events occur in the emergency department ( ed ) . however , the eds had the highest proportion of prevalence of preventable ( 70 - 82% ) errors . patients come to the ed for evaluation of emergent or urgent conditions for after - hours medical care , or by referral from their primary physician . in the ed , doctors face urgent and sever cases that need to be treated quickly with high quality . this creates a challenge for physicians to initiate and select appropriate drugs for the patient . furthermore , the unique operating characteristics of ed make the ed vulnerable to medical errors including medication errors and adverse drug events . many factors , either intrinsic or extrinsic , influence the quality of health care in the ed . these include : high levels of activity , high cognitive load , high decision density , high levels of diagnostic uncertainty , inexperience of physicians and nurses , distractions , narrow time window and shift work . the world health organization ( who ) compiled a set of core drug use indicators that are useful for studying patterns of drug prescribing in health care facilities . the who also stated that : rational use of drugs requires that patients receive medications appropriate to their clinical needs , in doses that meet their own individual requirements for an adequate period of time and at the lowest cost to them and their community . the who core indicators for drug utilization include : average number of drugs per encounter , percentage of drugs prescribed by generic name , percentage of encounters with an antibiotic , percentage of encounters with an injection , percentage of drugs prescribed from the essential drug list . another tool used to control and contrast the rational use of drugs is the hospital 's pharmaco - therapeutic guide , which is used and studied by the pharmaceutical and therapeutics commission of any hospital . in contrast , irrational use of drugs refers to the distribution or consumption of drugs in ways that negate or reduce their efficacy or in situations where they are unlikely to have the desire effect . irrational prescription of drugs leads to unproductive and risky treatment and poses a major risk of present day medical practice . the problem of irrational drug prescription is not restricted to developing countries as there is evidence from many developed countries on the inappropriate use of drugs . appropriate medication use is of both clinical and economic significance to any health system and should be given adequate attention . drug utilization research is a component of medical audit that plays an important role in pharmaco - epidemiological studies . this is because it reports the extent , quality , determinants and outcome of drug exposure . in addition , it helps in assessing rational usage and cost control of various medications used in the hospital . pharmaco - epidemiological studies detailing prescribing patterns of physicians are very few from developing countries . currently , there is limited local data on the prescribing habits of doctors at the ed . the aim of this study was to assess drug prescribing trends , average number of drugs per prescription , the who core indicators for drug utilization and prescription cost during patients visits at the ed at sultan qaboos university hospital ( squh ) . this study was conducted at the ed of squh , a tertiary care hospital , in muscat , the sultanate of oman . this was a retrospective cross - sectional study of all patients ( n = 300 ) who attended the ed at squh in may of 2012 . the who prescribing indicators mentioned above were measured retrospectively from the hospital 's medical records . no information was collected about the signs and symptoms of diseases as this is not a requirement as per the who guidelines in this type of study . the cost of medications was tested for normal distribution using one - sample kolmogorov - smirnov test . the association between drugs cost and patient 's gender was conducted by mann - whitney test and between the drug cost and type of emergency and patient 's outcome . however , the correlation between drug cost and duration of stay at the ed and patient 's age was performed using spearman 's correlation coefficient . an a priori two - tailed level of significance was set at the 0.05 level . statistical analyses were conducted using stata version 12.1 ( stata corporation , college station , tx , usa ) . ethical approval for the study was obtained through the medical research and ethics committee at the college of medicine and health sciences . this study was conducted at the ed of squh , a tertiary care hospital , in muscat , the sultanate of oman . this was a retrospective cross - sectional study of all patients ( n = 300 ) who attended the ed at squh in may of 2012 . the who prescribing indicators mentioned above were measured retrospectively from the hospital 's medical records . no information was collected about the signs and symptoms of diseases as this is not a requirement as per the who guidelines in this type of study . descriptive statistics were used to describe the data . for categorical variables , frequencies and percentages were reported . for continuous variables , the cost of medications was tested for normal distribution using one - sample kolmogorov - smirnov test . the association between drugs cost and patient 's gender was conducted by mann - whitney test and between the drug cost and type of emergency and patient 's outcome . however , the correlation between drug cost and duration of stay at the ed and patient 's age was performed using spearman 's correlation coefficient . an a priori two - tailed level of significance was set at the 0.05 level . statistical analyses were conducted using stata version 12.1 ( stata corporation , college station , tx , usa ) . ethical approval for the study was obtained through the medical research and ethics committee at the college of medicine and health sciences . among the recruited patients , 155 ( 52% ) were males and 145 ( 48% ) were females . only 15% ( n = 45 ) of patients were referred to other departments for further management . summary of the characteristics of the 300 patients in the ed at sultan qaboos university hospital the total number of prescriptions for the 300 patients over the month was 939 . the distribution of drugs among patients included in this study was : 55 ( 18% ) patients received one drug ; 78 ( 26% ) patients received two drugs ; 60 ( 20% ) patients received three drugs ; 47 ( 16% ) patients received four drugs ; 30 ( 18% ) patients received five drugs ; and the rest received more than five drugs ( 10% ) . the majority of drugs were administered by the oral route ( n = 481 ; 51% ) followed by the parenteral route ( n = 357 ; 38% ) and then topical route ( n = 54 ; 6% ) the non - steroidal anti - inflammatory drugs ( nsaids ) were the most commonly prescribed class of drugs ( 38% ) followed by the gastro - intestinal tract ( git ) drugs ( 19% ) and central - nervous system ( cns ) drugs ( 13% ) . paracetamol was the most commonly prescribed drug ( n = 195 ; 21% ) followed by morphine ( n = 67 ; 7% ) and diclofenac ( n = 62 ; 7% ) . percentage of different classes of drugs prescribed to 300 patients ( n = 939 prescriptions ) in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system the top 10 drugs prescribed to 300 patients in the ed at sultan qaboos university hospital in muscat , oman table 3 shows the distribution of drug classes across various emergency types , indicating that nsaids and cns drugs were mostly prescribed for hematological patients ( 56 and 54 cases , respectively ) , whereas asthma drugs and steroids were mostly prescribed for respiratory patients ( 21 and 6 cases , respectively ) . distribution of drug classes across various emergency types among 300 patients in the ed at sultan qaboos university hospital in muscat , oman the who core prescribing indicators are shown in table 4 . who core drug prescribing indicators in the study population ( n=300 ) at the ed at sultan qaboos university hospital in muscat , oman as shown in table 5 , the average cost per prescription was 242 631 us$. table 6 shows the top 10 drugs that account for the highest cost among all drugs prescribed . morphine had the highest cost ( 1884 us$ ) followed by cefuroxime ( 1404 us$ ) and filgrastim ( 940 us$ ) . figure 2 represents the percentage of the cost of different drug classes showing that anti - infective drugs incurred the highest cost ( 2810 us$ ) followed by cns drugs ( 2004 us$ ) . drug indications , prescribing trends and prescription cost in the ed at sultan qaboos university hospital in muscat , oman the top 10 drugs cost of prescribing for 300 patients in the ed at sultan qaboos university hospital in muscat , oman the percentage of the cost of different drug classes of 300 patients in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system there was no significant association between the hospital cost and patient 's gender ( p = 0.730 ) and clinical outcome ( p = 0.958 ) . however , there was a significant correlation between the hospital cost and age ( older patients had higher medication cost ; p < 0.001 ) , duration of stay at the ed ( as the hospital stay increases so too was the cost ; p = 0.008 ) and emergency types ( higher cost was associated with infection ; p < 0.001 ) . among the recruited patients , 155 ( 52% ) were males and 145 ( 48% ) were females . only 15% ( n = 45 ) of patients were referred to other departments for further management . summary of the characteristics of the 300 patients in the ed at sultan qaboos university hospital the distribution of drugs among patients included in this study was : 55 ( 18% ) patients received one drug ; 78 ( 26% ) patients received two drugs ; 60 ( 20% ) patients received three drugs ; 47 ( 16% ) patients received four drugs ; 30 ( 18% ) patients received five drugs ; and the rest received more than five drugs ( 10% ) . the majority of drugs were administered by the oral route ( n = 481 ; 51% ) followed by the parenteral route ( n = 357 ; 38% ) and then topical route ( n = 54 ; 6% ) . a total of 103 drugs , belonging to seven categories , were prescribed . the non - steroidal anti - inflammatory drugs ( nsaids ) were the most commonly prescribed class of drugs ( 38% ) followed by the gastro - intestinal tract ( git ) drugs ( 19% ) and central - nervous system ( cns ) drugs ( 13% ) . paracetamol was the most commonly prescribed drug ( n = 195 ; 21% ) followed by morphine ( n = 67 ; 7% ) and diclofenac ( n = 62 ; 7% ) . percentage of different classes of drugs prescribed to 300 patients ( n = 939 prescriptions ) in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system the top 10 drugs prescribed to 300 patients in the ed at sultan qaboos university hospital in muscat , oman table 3 shows the distribution of drug classes across various emergency types , indicating that nsaids and cns drugs were mostly prescribed for hematological patients ( 56 and 54 cases , respectively ) , whereas asthma drugs and steroids were mostly prescribed for respiratory patients ( 21 and 6 cases , respectively ) . distribution of drug classes across various emergency types among 300 patients in the ed at sultan qaboos university hospital in muscat , oman the who core prescribing indicators are shown in table 4 . who core drug prescribing indicators in the study population ( n=300 ) at the ed at sultan qaboos university hospital in muscat , oman as shown in table 5 , the average cost per prescription was 242 631 us$. table 6 shows the top 10 drugs that account for the highest cost among all drugs prescribed . morphine had the highest cost ( 1884 us$ ) followed by cefuroxime ( 1404 us$ ) and filgrastim ( 940 us$ ) . figure 2 represents the percentage of the cost of different drug classes showing that anti - infective drugs incurred the highest cost ( 2810 us$ ) followed by cns drugs ( 2004 us$ ) . drug indications , prescribing trends and prescription cost in the ed at sultan qaboos university hospital in muscat , oman the top 10 drugs cost of prescribing for 300 patients in the ed at sultan qaboos university hospital in muscat , oman the percentage of the cost of different drug classes of 300 patients in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system there was no significant association between the hospital cost and patient 's gender ( p = 0.730 ) and clinical outcome ( p = 0.958 ) . however , there was a significant correlation between the hospital cost and age ( older patients had higher medication cost ; p < 0.001 ) , duration of stay at the ed ( as the hospital stay increases so too was the cost ; p = 0.008 ) and emergency types ( higher cost was associated with infection ; p < 0.001 ) . there was no significant association between the hospital cost and patient 's gender ( p = 0.730 ) and clinical outcome ( p = 0.958 ) . however , there was a significant correlation between the hospital cost and age ( older patients had higher medication cost ; p < 0.001 ) , duration of stay at the ed ( as the hospital stay increases so too was the cost ; p = 0.008 ) and emergency types ( higher cost was associated with infection ; p < 0.001 ) . studying drug utilization pattern in the ed provides a means of assessing drug prescribing trends , efficiency and cost - effectiveness of hospital formularies . to the best of our knowledge , this is the first study in sultanate of oman to analyze drug utilization patterns in the ed . the mean age of the patients was 34 19 years and 155 ( 52% ) were males . the average number of drugs per prescription , which is an important index of the standard of prescribing , was 3.16 1.89 , with a significant number of the patients in this study ( 26% ) receiving at least two drugs per episode . cardiovascular diseases had the highest average number of drugs per prescription ( 3.70 1.49 ) . the average number of drugs per prescription is higher than the who recommended average number of drugs per prescription of 2.0 . two studies conducted in india ( n = 200 and n = 259 ) showed an average number of drugs per prescription of 4.2 and 3.3 , respectively . in these two studies , cardiovascular disease and alcoholic liver disease had the highest average number of drugs per prescription ( 5.4 and 3.9 , respectively ) . another study conducted in spain ( n = 669 ) showed an average number of drugs per prescription of 1.7 . in addition , a study on elderly patients ( n = 124 ) presented to the ed in the usa found an average number per prescriptions of 8.6 ( range 0 - 21 ) . one reason for the higher average number of drugs per prescription in usa compared with the who standards is that physicians tend to administer polypharmacy during the initial contact with patient while the diagnosis is not yet confirmed and waiting for laboratory results . despite this , keeping the mean number of drugs per prescription to as low as possible is always preferable to reduce the risk of drug - drug interactions , development of drugs resistance and adverse drug events . this study demonstrated a high use of nsaids in the ed and this could be explained by the large therapeutic range as shown in table 3 of this class , especially their usage as analgesics . furthermore , the reason for high indication of morphine use could be explained by the fact that most patients that visited the ed during the study period were hematological cases ( 18% ; n = 54 ) having mainly sickle cell disease crisis which required morphine to manage their crisis [ table 3 ] . this study also showed git drugs as the second most commonly prescribed medications ( 19% ) with ranitidine being the most commonly prescribed drug of this class ( 5% ) [ table 2 ] . this could be reflected by the fact that git cases represent 17% ( n = 51 ) of all drug indications in the study [ table 5 ] . a study evaluated the use of drugs across the different indications in the ed in india ( n = 200 patients ; 629 prescriptions ) and found that antibiotics were the most commonly prescribed drugs ( 22% ) followed by nsaids ( 12% ) and a class of git drugs called proton pump inhibitors ( 12% ) . in contrast to our study , the most common indication for drug prescription in the indian study was infection ( 19% ) , while infection was the third most common indication for drug prescription in our study ( 15% ) . paracetamol was also the most commonly prescribed drug ( 10% ) in a study conducted at the ed in spain , which is similar to our study results , followed by ibuprofen - an nsaid-(9% ) and omeprazole ( 7% ) . however , in the spanish study , the most common indication for drug prescribing was the git and metabolism ( 22% ) followed by blood and hematopoietic system ( 2% ) , which is almost similar to our data . in our study , the highest average number of drugs per prescription was for cardiovascular disease ( 3.70 1.49 ) followed by respiratory disease ( 3.41 2.45 ) and gastrointestinal disease ( 3.39 2.32 ) . in the indian study , the average number of drugs per prescription was highest in the cardiovascular disease ( 5.4 1.2 ) followed by the central nervous system ( 4.5 1.0 ) and renal disease ( 4.2 0.9 ) . anti - infective drugs cost ( 2810 us$ ) was the highest among all drug classes prescribed followed by cns drugs ( 2004 us$ ) and nsaids ( 878 us$ ) [ figure 2 ] . this high cost of these classes was due to the high frequency of drugs prescription of these classes as shown in table 3 . moreover , most patients came to the ed with hematological , gastrointestinal and infectious diseases which require prescription of these classes of drugs . the total cost of all drug classes was 8690 us$ over the 1-month period and the average cost per prescription was 242 631 us$. in a study conducted in india , the mean cost per prescriptions was 784 134 inr ( 14 2.4 us$ ) . it should be kept in mind that in order to have a realist view on the total cost of ed visits , other aspects of health care such as investigations , stay in hospital and other intangible costs should be calculated . the who core indicators of prescribing practices measure the performance of health care providers in several key dimensions related to the appropriate use of drugs . in our study , the average number of drugs per prescription was 3.16 1.89 , the percentage of antibiotics use was 10% , the percentage of injectable drug use was 38% and about 58% of drugs were prescribed from the who essential drug list [ table 4 ] . several drug use studies using these standard drug - use indicators have been performed in many developing countries under the supervision of the who to provide ideal values for each indicator ( more details can be found in hogerzeil et al . 1993 and in the who manual ) . in these studies , the percentage of antibiotics use ranged from 25% to 63% while the percentage of injectable drug use ranged from 0.2% to 48% respectively . furthermore , some of these studies showed that about 85 - 88% of drugs prescribed were from the who essential drug list . our study showed that there was a significant association between the hospital cost and age . older patients had higher medication cost because as a person gets older he / she are more prone to have variants of diseases as well as chronic diseases with their complications that require further medication management . moreover , duration of stay in the ed had a significant association with the hospital cost . as the hospital stay increases so too was the cost because long stays mean further investigations and management that are required for the patient . furthermore , there was also a significant association between the hospital cost and emergency types . no power analysis was performed in this study and one could not be certain that our sample size ( n = 300 ) was a representative sample of the general omani population . however , the sample size was in accordance with who recommendation for practice assessment in individual facilities which requires that a minimum of 100 samples per facility should be collected for the purpose of evaluation . this sample size will give a 95% confidence interval of within 10% for the individual result . furthermore , since this study was performed in only 1 month , it could not have captured seasonal variations , which could have affected prescribing patterns . the average number of drugs prescribed per patients in the ed over the 1 month period was 3.16 1.89 . nsaids were the most frequently class of drugs administered to the patients followed by git drugs . the highest number of drugs was prescribed for cardiovascular system diseases followed by respiratory and gastrointestinal . anti - infective drug cost was the highest among all other classes followed by cns drugs . the results of this type of studies highlight the importance of strategies that have to be implemented to optimize medication use at the ed . these include ensuring that all persons involved in the medication process have good pharmacological knowledge , computerization of the entire medication process and engagement of clinical pharmacists in such process . the importance of these strategies should be emphasized in medical curricula and continuing medical education of health professionals .

objectives : the aim of this study was to assess the prescribing trends and costs of drugs in the emergency department ( ed ) at sultan qaboos university hospital ( squh ) , a tertiary care hospital , in muscat , the sultanate of oman.materials and methods : this was a retrospective cross - sectional study of all patients ( n = 300 ) who attended the ed at squh in may 2012 . analyses were performed using descriptive and univariate statistics.results:the average age of patients was 34 19 years . the average number of drugs prescribed per patients was 3.2 1.9 and the majority of the patients ( n = 78 ; 26% ) received two drugs . the most common route of drug administration was the oral route ( n = 481 ; 51% ) followed by parenterally ( n = 357 ; 38% ) . non - steroidal anti - inflammatory drugs ( nsaids ) were the most commonly prescribed class of drugs ( 38% ) followed by the gastro - intestinal tract drugs ( 19% ) and central nervous system drugs ( 13% ) . the average cost per prescription was 242 632 us$. morphine had the highest cost ( 1885 us$ ) followed by cefuroxime ( 1404 us$ ) and filgrastim ( 939 us$ ) over the 1-month period . there was a significant positive correlation between hospital cost and age ( p < 0.001 ) , duration of stay at the ed ( p = 0.008 ) and emergency types ( p < 0.001).conclusion : nsaids were the most frequent class of drugs administered to patients . highest number of drugs was prescribed for cardiovascular diseases followed by respiratory and gastrointestinal diseases . anti - infective drugs cost was the highest among all other classes . the results of the present study are attempts to highlight the importance of strategies that have to be implemented to optimize medication use at the ed .

Introduction Materials and Methods Study setting Study design and subjects WHO core drug prescribing indicators Statistical analysis Ethical approval Results General characteristics of the patients Drug utilization pattern among patients WHO core indicators of drug utilization pattern Drug prescribing cost at the ED at SQUH Correlation of drug utilization pattern and cost with different patients Parameters Discussion Conclusion

a meta - analysis of 35 studies between 1990 and 2005 indicated that medication errors occurred in a mean of 5.7% of all drug administration episodes while adverse drug events affected 6.1 patients per 100 hospitalized . many factors are involved in drug prescription errors including polypharmacy , lack of sufficient pharmacological knowledge , errors in patients charts or documentation by nurses , inadequate pharmacy service , being a female , age > 65 years , renal excretion of drugs , drugs with narrow therapeutic index and the use of anticoagulants or diuretics . these studies indicate that 1.5 - 3% of all adverse drug events occur in the emergency department ( ed ) . however , the eds had the highest proportion of prevalence of preventable ( 70 - 82% ) errors . in the ed , doctors face urgent and sever cases that need to be treated quickly with high quality . furthermore , the unique operating characteristics of ed make the ed vulnerable to medical errors including medication errors and adverse drug events . many factors , either intrinsic or extrinsic , influence the quality of health care in the ed . the who also stated that : rational use of drugs requires that patients receive medications appropriate to their clinical needs , in doses that meet their own individual requirements for an adequate period of time and at the lowest cost to them and their community . the who core indicators for drug utilization include : average number of drugs per encounter , percentage of drugs prescribed by generic name , percentage of encounters with an antibiotic , percentage of encounters with an injection , percentage of drugs prescribed from the essential drug list . another tool used to control and contrast the rational use of drugs is the hospital 's pharmaco - therapeutic guide , which is used and studied by the pharmaceutical and therapeutics commission of any hospital . in contrast , irrational use of drugs refers to the distribution or consumption of drugs in ways that negate or reduce their efficacy or in situations where they are unlikely to have the desire effect . the problem of irrational drug prescription is not restricted to developing countries as there is evidence from many developed countries on the inappropriate use of drugs . appropriate medication use is of both clinical and economic significance to any health system and should be given adequate attention . this is because it reports the extent , quality , determinants and outcome of drug exposure . in addition , it helps in assessing rational usage and cost control of various medications used in the hospital . currently , there is limited local data on the prescribing habits of doctors at the ed . the aim of this study was to assess drug prescribing trends , average number of drugs per prescription , the who core indicators for drug utilization and prescription cost during patients visits at the ed at sultan qaboos university hospital ( squh ) . this study was conducted at the ed of squh , a tertiary care hospital , in muscat , the sultanate of oman . this was a retrospective cross - sectional study of all patients ( n = 300 ) who attended the ed at squh in may of 2012 . the association between drugs cost and patient 's gender was conducted by mann - whitney test and between the drug cost and type of emergency and patient 's outcome . however , the correlation between drug cost and duration of stay at the ed and patient 's age was performed using spearman 's correlation coefficient . an a priori two - tailed level of significance was set at the 0.05 level . statistical analyses were conducted using stata version 12.1 ( stata corporation , college station , tx , usa ) . ethical approval for the study was obtained through the medical research and ethics committee at the college of medicine and health sciences . this study was conducted at the ed of squh , a tertiary care hospital , in muscat , the sultanate of oman . this was a retrospective cross - sectional study of all patients ( n = 300 ) who attended the ed at squh in may of 2012 . for continuous variables , the cost of medications was tested for normal distribution using one - sample kolmogorov - smirnov test . the association between drugs cost and patient 's gender was conducted by mann - whitney test and between the drug cost and type of emergency and patient 's outcome . however , the correlation between drug cost and duration of stay at the ed and patient 's age was performed using spearman 's correlation coefficient . ethical approval for the study was obtained through the medical research and ethics committee at the college of medicine and health sciences . among the recruited patients , 155 ( 52% ) were males and 145 ( 48% ) were females . only 15% ( n = 45 ) of patients were referred to other departments for further management . summary of the characteristics of the 300 patients in the ed at sultan qaboos university hospital the total number of prescriptions for the 300 patients over the month was 939 . the distribution of drugs among patients included in this study was : 55 ( 18% ) patients received one drug ; 78 ( 26% ) patients received two drugs ; 60 ( 20% ) patients received three drugs ; 47 ( 16% ) patients received four drugs ; 30 ( 18% ) patients received five drugs ; and the rest received more than five drugs ( 10% ) . the majority of drugs were administered by the oral route ( n = 481 ; 51% ) followed by the parenteral route ( n = 357 ; 38% ) and then topical route ( n = 54 ; 6% ) the non - steroidal anti - inflammatory drugs ( nsaids ) were the most commonly prescribed class of drugs ( 38% ) followed by the gastro - intestinal tract ( git ) drugs ( 19% ) and central - nervous system ( cns ) drugs ( 13% ) . paracetamol was the most commonly prescribed drug ( n = 195 ; 21% ) followed by morphine ( n = 67 ; 7% ) and diclofenac ( n = 62 ; 7% ) . percentage of different classes of drugs prescribed to 300 patients ( n = 939 prescriptions ) in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system the top 10 drugs prescribed to 300 patients in the ed at sultan qaboos university hospital in muscat , oman table 3 shows the distribution of drug classes across various emergency types , indicating that nsaids and cns drugs were mostly prescribed for hematological patients ( 56 and 54 cases , respectively ) , whereas asthma drugs and steroids were mostly prescribed for respiratory patients ( 21 and 6 cases , respectively ) . distribution of drug classes across various emergency types among 300 patients in the ed at sultan qaboos university hospital in muscat , oman the who core prescribing indicators are shown in table 4 . who core drug prescribing indicators in the study population ( n=300 ) at the ed at sultan qaboos university hospital in muscat , oman as shown in table 5 , the average cost per prescription was 242 631 us$. table 6 shows the top 10 drugs that account for the highest cost among all drugs prescribed . morphine had the highest cost ( 1884 us$ ) followed by cefuroxime ( 1404 us$ ) and filgrastim ( 940 us$ ) . figure 2 represents the percentage of the cost of different drug classes showing that anti - infective drugs incurred the highest cost ( 2810 us$ ) followed by cns drugs ( 2004 us$ ) . drug indications , prescribing trends and prescription cost in the ed at sultan qaboos university hospital in muscat , oman the top 10 drugs cost of prescribing for 300 patients in the ed at sultan qaboos university hospital in muscat , oman the percentage of the cost of different drug classes of 300 patients in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system there was no significant association between the hospital cost and patient 's gender ( p = 0.730 ) and clinical outcome ( p = 0.958 ) . however , there was a significant correlation between the hospital cost and age ( older patients had higher medication cost ; p < 0.001 ) , duration of stay at the ed ( as the hospital stay increases so too was the cost ; p = 0.008 ) and emergency types ( higher cost was associated with infection ; p < 0.001 ) . among the recruited patients , 155 ( 52% ) were males and 145 ( 48% ) were females . only 15% ( n = 45 ) of patients were referred to other departments for further management . summary of the characteristics of the 300 patients in the ed at sultan qaboos university hospital the distribution of drugs among patients included in this study was : 55 ( 18% ) patients received one drug ; 78 ( 26% ) patients received two drugs ; 60 ( 20% ) patients received three drugs ; 47 ( 16% ) patients received four drugs ; 30 ( 18% ) patients received five drugs ; and the rest received more than five drugs ( 10% ) . the majority of drugs were administered by the oral route ( n = 481 ; 51% ) followed by the parenteral route ( n = 357 ; 38% ) and then topical route ( n = 54 ; 6% ) . the non - steroidal anti - inflammatory drugs ( nsaids ) were the most commonly prescribed class of drugs ( 38% ) followed by the gastro - intestinal tract ( git ) drugs ( 19% ) and central - nervous system ( cns ) drugs ( 13% ) . paracetamol was the most commonly prescribed drug ( n = 195 ; 21% ) followed by morphine ( n = 67 ; 7% ) and diclofenac ( n = 62 ; 7% ) . percentage of different classes of drugs prescribed to 300 patients ( n = 939 prescriptions ) in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system the top 10 drugs prescribed to 300 patients in the ed at sultan qaboos university hospital in muscat , oman table 3 shows the distribution of drug classes across various emergency types , indicating that nsaids and cns drugs were mostly prescribed for hematological patients ( 56 and 54 cases , respectively ) , whereas asthma drugs and steroids were mostly prescribed for respiratory patients ( 21 and 6 cases , respectively ) . distribution of drug classes across various emergency types among 300 patients in the ed at sultan qaboos university hospital in muscat , oman the who core prescribing indicators are shown in table 4 . who core drug prescribing indicators in the study population ( n=300 ) at the ed at sultan qaboos university hospital in muscat , oman as shown in table 5 , the average cost per prescription was 242 631 us$. table 6 shows the top 10 drugs that account for the highest cost among all drugs prescribed . morphine had the highest cost ( 1884 us$ ) followed by cefuroxime ( 1404 us$ ) and filgrastim ( 940 us$ ) . figure 2 represents the percentage of the cost of different drug classes showing that anti - infective drugs incurred the highest cost ( 2810 us$ ) followed by cns drugs ( 2004 us$ ) . drug indications , prescribing trends and prescription cost in the ed at sultan qaboos university hospital in muscat , oman the top 10 drugs cost of prescribing for 300 patients in the ed at sultan qaboos university hospital in muscat , oman the percentage of the cost of different drug classes of 300 patients in the emergency department at sultan qaboos university hospital in muscat , oman , nsaid : non - steroidal anti - inflammatory drugs , git : gastrointestinal tract , cns : central nervous system , cvs : cardiovascular system there was no significant association between the hospital cost and patient 's gender ( p = 0.730 ) and clinical outcome ( p = 0.958 ) . however , there was a significant correlation between the hospital cost and age ( older patients had higher medication cost ; p < 0.001 ) , duration of stay at the ed ( as the hospital stay increases so too was the cost ; p = 0.008 ) and emergency types ( higher cost was associated with infection ; p < 0.001 ) . there was no significant association between the hospital cost and patient 's gender ( p = 0.730 ) and clinical outcome ( p = 0.958 ) . however , there was a significant correlation between the hospital cost and age ( older patients had higher medication cost ; p < 0.001 ) , duration of stay at the ed ( as the hospital stay increases so too was the cost ; p = 0.008 ) and emergency types ( higher cost was associated with infection ; p < 0.001 ) . studying drug utilization pattern in the ed provides a means of assessing drug prescribing trends , efficiency and cost - effectiveness of hospital formularies . to the best of our knowledge , this is the first study in sultanate of oman to analyze drug utilization patterns in the ed . the mean age of the patients was 34 19 years and 155 ( 52% ) were males . the average number of drugs per prescription , which is an important index of the standard of prescribing , was 3.16 1.89 , with a significant number of the patients in this study ( 26% ) receiving at least two drugs per episode . cardiovascular diseases had the highest average number of drugs per prescription ( 3.70 1.49 ) . the average number of drugs per prescription is higher than the who recommended average number of drugs per prescription of 2.0 . two studies conducted in india ( n = 200 and n = 259 ) showed an average number of drugs per prescription of 4.2 and 3.3 , respectively . in these two studies , cardiovascular disease and alcoholic liver disease had the highest average number of drugs per prescription ( 5.4 and 3.9 , respectively ) . another study conducted in spain ( n = 669 ) showed an average number of drugs per prescription of 1.7 . in addition , a study on elderly patients ( n = 124 ) presented to the ed in the usa found an average number per prescriptions of 8.6 ( range 0 - 21 ) . one reason for the higher average number of drugs per prescription in usa compared with the who standards is that physicians tend to administer polypharmacy during the initial contact with patient while the diagnosis is not yet confirmed and waiting for laboratory results . despite this , keeping the mean number of drugs per prescription to as low as possible is always preferable to reduce the risk of drug - drug interactions , development of drugs resistance and adverse drug events . this study demonstrated a high use of nsaids in the ed and this could be explained by the large therapeutic range as shown in table 3 of this class , especially their usage as analgesics . furthermore , the reason for high indication of morphine use could be explained by the fact that most patients that visited the ed during the study period were hematological cases ( 18% ; n = 54 ) having mainly sickle cell disease crisis which required morphine to manage their crisis [ table 3 ] . this study also showed git drugs as the second most commonly prescribed medications ( 19% ) with ranitidine being the most commonly prescribed drug of this class ( 5% ) [ table 2 ] . this could be reflected by the fact that git cases represent 17% ( n = 51 ) of all drug indications in the study [ table 5 ] . a study evaluated the use of drugs across the different indications in the ed in india ( n = 200 patients ; 629 prescriptions ) and found that antibiotics were the most commonly prescribed drugs ( 22% ) followed by nsaids ( 12% ) and a class of git drugs called proton pump inhibitors ( 12% ) . in contrast to our study , the most common indication for drug prescription in the indian study was infection ( 19% ) , while infection was the third most common indication for drug prescription in our study ( 15% ) . paracetamol was also the most commonly prescribed drug ( 10% ) in a study conducted at the ed in spain , which is similar to our study results , followed by ibuprofen - an nsaid-(9% ) and omeprazole ( 7% ) . however , in the spanish study , the most common indication for drug prescribing was the git and metabolism ( 22% ) followed by blood and hematopoietic system ( 2% ) , which is almost similar to our data . in our study , the highest average number of drugs per prescription was for cardiovascular disease ( 3.70 1.49 ) followed by respiratory disease ( 3.41 2.45 ) and gastrointestinal disease ( 3.39 2.32 ) . in the indian study , the average number of drugs per prescription was highest in the cardiovascular disease ( 5.4 1.2 ) followed by the central nervous system ( 4.5 1.0 ) and renal disease ( 4.2 0.9 ) . anti - infective drugs cost ( 2810 us$ ) was the highest among all drug classes prescribed followed by cns drugs ( 2004 us$ ) and nsaids ( 878 us$ ) [ figure 2 ] . moreover , most patients came to the ed with hematological , gastrointestinal and infectious diseases which require prescription of these classes of drugs . the total cost of all drug classes was 8690 us$ over the 1-month period and the average cost per prescription was 242 631 us$. in a study conducted in india , the mean cost per prescriptions was 784 134 inr ( 14 2.4 us$ ) . in our study , the average number of drugs per prescription was 3.16 1.89 , the percentage of antibiotics use was 10% , the percentage of injectable drug use was 38% and about 58% of drugs were prescribed from the who essential drug list [ table 4 ] . 1993 and in the who manual ) . furthermore , some of these studies showed that about 85 - 88% of drugs prescribed were from the who essential drug list . our study showed that there was a significant association between the hospital cost and age . moreover , duration of stay in the ed had a significant association with the hospital cost . furthermore , there was also a significant association between the hospital cost and emergency types . no power analysis was performed in this study and one could not be certain that our sample size ( n = 300 ) was a representative sample of the general omani population . however , the sample size was in accordance with who recommendation for practice assessment in individual facilities which requires that a minimum of 100 samples per facility should be collected for the purpose of evaluation . furthermore , since this study was performed in only 1 month , it could not have captured seasonal variations , which could have affected prescribing patterns . the average number of drugs prescribed per patients in the ed over the 1 month period was 3.16 1.89 . nsaids were the most frequently class of drugs administered to the patients followed by git drugs . the highest number of drugs was prescribed for cardiovascular system diseases followed by respiratory and gastrointestinal . anti - infective drug cost was the highest among all other classes followed by cns drugs . the results of this type of studies highlight the importance of strategies that have to be implemented to optimize medication use at the ed . these include ensuring that all persons involved in the medication process have good pharmacological knowledge , computerization of the entire medication process and engagement of clinical pharmacists in such process . the importance of these strategies should be emphasized in medical curricula and continuing medical education of health professionals .

exposure to internal emitters such as cesium-137 is an inevitable consequence of nuclear accidents , such as chernobyl and fukushima daiichi . in addition to such large - scale events , occupational and industrial exposure , as in the case of the goiania scrap metal incident or as in the case of a terrorist - plotted radiological dispersal device , raise serious concerns . because of its environmental persistence and ease of dispersal , cs poses a great health risk to the general public . thus , our team set out to develop early and robust markers for cs exposure in the easily obtainable biofluids , urine , and serum . such exposure markers can be used to screen and assess the risk of exposure in a given population , which , in turn , may help triage the affected population in a faster and more efficient manner . many of the previous ionizing radiation ( ir ) studies focused on the dna damage / repair machinery and its markers in biological samples . although such genomic and transcriptomic studies have helped bring to light many facts about ir - induced injury and inflammation responses , they are limited in scope and have failed to capture the molecular and structural effects of ir in cells . proteomics and more recently metabolomics have filled this gap and contributed to the field of biodosimetry by revealing the molecular targets of ir and their associated signaling networks . advances in technology , particularly in the field of mass spectrometry , have enabled our laboratory to make significant progress in determining even the slight and subtle changes in the molecular composition , namely , the metabolome , of biofluids as a result of exposure to external beam and internal emitters using mouse models . this study is complementary to our previous work on the metabolic perturbations in urine of c57/bl6 mice over a 30-day period after exposure to cs , through employing the sensitivity of time - of - flight mass spectrometry ( tofms ) coupled to high resolving power of ultra performance liquid chromatography ( uplc ) . herein we utilized the same platform to determine the unique and robust responses of serum metabolites to cs exposure but in a new approach entailing a fast and simple lipid profiling method with relative quantitation called ms . by using this method , we were able to not only identify several different classes of lipids along with their relative abundances but also gain structural information on these chemical species . furthermore , we utilized a comprehensive statistical analysis workflow we have specifically developed for metabolomics , called metabolyzer , to determine cs - specific perturbations in metabolites / lipids and their respective pathways . the statistically significant metabolites and lipids were then compared with known external - beam -irradiation markers to determine similarities and differences in terms of responses to these two types of exposure . to date , this is the first time that mass spectrometry has been utilized to study the effects of exposure to an internal emitter in serum of mice . the results of this study and other radiation - induced signaling studies in easily accessible biofluids may help uncover the mechanism behind ir - induced inflammation and injury , which will ultimately lead to the discovery of novel biomarkers of ir exposure . the following fatty acids and lipid standards were purchased from avanti polar lipids ( alabaster , al ) : sphingolipid mix ( sm ) ii , phosphatidylethanolamine pe ( 14:0/14:0 ) , phosphatidylcholine pc ( 14:0/14:0 ) , phosphatidic acid pa ( 14:0/14:0 ) , phosphatidylserine ps ( 14:0/14:0 ) , phosphatidylinositol pi ( 17:0/20:4 ) , and lysophosphatidylcholine lpc ( 17:1 ) . prostaglandin standard and leukotriene were purchased from cayman chemical ( ann arbor , mi ) , and fatty acid standard fa(17:1 ) was purchased from nu - chek prep ( elysian , mn ) . debrisoquine sulfate , 4-nitrobenzoic acid ( 4-nba ) , and uplc - grade solvents such as acetonitrile , water , and isopropanol were purchased from fisher scientific ( hanover park , il ) . glucose , riboflavin , arachidonic acid , linoleic acid , oleic acid , palmitic acid , hippuric acid , nicotinic acid , lactic acid , uridine , taurine , -ketobutyric acid , uric acid , hydroxyphenylpyruvic acid , acetylcarnitine , and carnitine were purchased from sigma - aldrich ( seelze , germany ) . the tandem ms spectrum of dityrosine provided by hanft et al . was used as a validation reference spectrum . in addition , the ms / ms spectra provided by scripps center for metabolomics , metlin , ( la jolla , ca ) were used as reference spectra for hydroquinone , inositol , gentisic acid , and dihydrolipoamide . metlin was also used to putatively identify the ion at m / z of 337.1048 as the sodium adduct of 7,8-dihydropteroic acid . all animal experiments were conducted in accordance with applicable federal and state guidelines and were approved by the institutional animal care and use committee of the lovelace biomedical and environmental research institute ( lberi ) . c57bl/6 mice ( approximately 1012 weeks old , 2530 g ) were received from charles river laboratories ( frederick , md ) and were quarantined for 14 days prior to group assignment by body - weight stratification for randomization into the study . animals were injected intraperitoneally with 8.0 0.3 mbq cscl solution in a volume of 50 l . nearly identical biokinetics have been found following inhalation , intraperitoneal , or intravenous administration of cs , which was confirmed in a pilot study comparison of the biokinetics between intravenous and intraperitoneal administration in c57bl/6 mice ( unpublished results ) . on the basis of these results , after dose administration , mice were housed individually in microisolator cages with lead shielding used to minimize cross - irradiation from adjacent mice . all animals had unlimited access to teklad certified global rodent diet 2016 ( harlan teklad , madison , wi ) and water except during dose administration and whole - body in vivo counting . mice injected with cscl initially lost weight , then resumed weight gain at approximately the same rate as the unexposed controls from day 3 after injection . no adverse effects were noted on the animals during the course of the study . serum was collected at necropsy by cardiac stick at 2 , 3 , 5 , 20 , and 30 days postinjection . each time point consists of eight mice per group with the exception of day 2 and day 30 control mice with seven mice per group ( table 1 ) . average body weight of mice per study group before treatment ( pre ) in kilograms . average body weight of mice per study group after treatment ( post ) in kilograms . serum samples were prepared by adding one part serum to four parts of a chilled chloroform and methanol mixture ( 2:1 v / v ) containing nonendogenous metabolite and lipid standards in a sterile siliconized tube . the list of nonendogenous internal standards along with their concentrations used in this experiment can be found in supplementary table 1 in the supporting information . each sample was then vortexed vigorously for 30 s at room temperature followed by centrifugation at 13 000 g for 5 min to separate the polar and nonpolar species into two phases . the upper aqueous phase containing primarily polar metabolites was collected , dried in a vacuum to < 10 l , and resuspended in 50 l of 50% acetonitrile . the lower phase was also collected , dried under a gentle stream of nitrogen , and resuspended in 50 l of a isopropanol and 50% acetonitrile mixture ( 1:1 v / v ) . to establish appropriate standard curves for the lipid internal standards , we carried out a series of two - fold dilutions at the initial concentration of 350 g / ml to the final concentration of 2 g / ml for phosphatidylcholine 14:0/14:0 ( pc 14:0/14:0 ) , phosphatidylethanolamine 14:0/14:0 ( pe 14:0/14:0 ) , phosphatidylglycerol d4pge2 ( pg d4pge2 ) , and sphingolipid mix ii ( sm mix ii ) . the concentration range for lysophosphatidylcholine 17:1 ( lpc 17:1 ) and fatty acid 17:1 ( fa 17:1 ) internal standards was at 128 to 1 the internal standards were also spiked into pooled control serum samples and processed via uplc these nonendogenous internal standards were quickly identified based on their unique mass , retention time , and fragmentation profiles . the calculated standard curve for each internal standard was then used to determine the relative abundance of different classes of lipids in each ionization mode . the metabolomic analysis was performed by injecting 2 l of aliquot of each sample into a reverse - phase 50 2.1 mm h - class uplc acquity 1.7-m beh c18 column ( waters corp , milford , ma ) coupled to a time - of - flight mass spectrometry ( tofms ) . the mobile phase consisted of water and 0.1% formic acid ( solvent a ) , 100% acetonitrile ( solvent b ) . the gradient for the metabolomic analysis switched from 98% aqueous solvent a to 40% solvent a and 60% solvent b after 4 min and to 98% solvent b at 8 min for 1 min and back to 98% solvent a for the last 2 min of the 11 min gradient at a flow rate of 0.5 ml / min . the xevo g2-s mass spectrometer ( waters ) was operated in both ms and ms modes scanning a 501200 m / z range with low collision energy of 10.0 ev for the precursor ions and collision energy ramp of 1050 ev for the product ions . the lipidomic samples were injected into a csh c18 column 150 m 100 mm ( waters ) with the h - class uplc acquity . the solvents used for the lipidomic analysis were 50% acetonitrile with 0.1% formic acid and 10 mm ammonium formate ( solvent c ) and isopropanol / acetonitrile ( 90:10 v : v ) with 10 mm ammonium formate ( solvent d).the gradient started with 60% solvent c at 0.45 ml / min for the initial 8 min , then switched to 100% solvent d for 1 min , and back to 60% solvent c for the remaining 2 min of the 11 min long gradient . the xevo g2-s qtof mass spectrometer was operated in positive ( esi ) and negative ( esi ) electrospray ionization ( esi ) modes over a mass range of 50 to 1200 da in two channels , ms and ms . the low energy ms channel was operated at 10.0 ev of collision energy while the ms channel included an energy ramp of 1050 ev . the lock - spray consisted of leucine enkephalin ( 556.2771 [ m + h ] and 554.2615 [ m - h ] ) . the ms data were acquired in centroid mode and processed using masslynx software ( waters ) as described later . as described previously markerlynx software ( waters ) was used to construct a data matrix consisting of the retention time , m / z , and abundance value ( via the normalized peak area ) for each ion using the raw ms chromatograms . to determine the peak areas of internal standards , quanlynx ( waters ) was used . for analyzing the ms data , the high energy scans ( fragments ) our in - house statistical analysis program , metabolyzer , was used to analyze the data and identify statistically significant ions . metabolyzer allowed for extraction of the ions with nonzero abundance values , which were detected in at least 70% of samples in each study group , called complete - presence ions . data were then log - transformed and analyzed for statistical significance via the nonparametric mann statistical significance testing for ions with nonzero abundance values in at least 70% of the samples in only one group ( partial - presence ions ) were analyzed as categorical variables for presence status ( i.e. , nonzero abundance ) via fisher s exact test ( p value < 0.05 ) . the log - transformed data for statistically significant complete - presence ions were then utilized for principal component analysis ( pca ) via singular value decomposition for the purpose of data visualization statistically significant ions were putatively identified via metabolyzer , which utilizes the human metabolome database ( hmdb ) , lipidmaps , and the kyoto encyclopedia of genes and genomes ( kegg ) database . the m / z values were used to putatively assign ids to the ions by neutral mass elucidation , which was accomplished by considering the possible adducts , h , na , or nh4 in the esi mode and h and cl in the esi mode . the masses were then compared with the exact mass of small molecules in the databases , from which putative metabolites were identified with a mass error of 20 ppm ( ppm ) or less . kegg - annotated pathways associated with these putative metabolites were also identified . to extract structural information on the putative identities of the metabolite , we explored ms data via quanlynx for alignment of the low energy scans with high energy scans . the fragmentation pattern of each metabolite and lipid of interest was compared against that of its pure chemical form either in online databases or the in - house database . in this study we utilized the sensitivity of mass spectrometry ( xevo - g2 , waters ) to detect changes in the sera of mice exposed to cs over the course of 30 days . the comprehensive analysis of serum metabolites and lipids along with their relative abundances were made possible by a feature of the xevo - g2 called ms , where many precursor , neutral loss , and product ion scans are acquired for every injection . these spectral features were used to determine the changes in the serum metabolome of mice after exposure to cs at different time points / doses ( table 1 ) . for example , the overall serum metabolomic profile of mice 2 days after cscl injection at a cumulative dose of 1.95 gy showed significant changes when compared with the serum metabolomic profile of matched control mice , as expected . this is evident from the clear separation in pca of figure 1a , where cs - exposed mice ( red circles ) are tightly clustered and clearly dichotomized from the control mice ( blue triangles ) . the heatmap in figure 1b shows a panel of putative metabolites on the vertical axis , whose serum abundances change significantly ( p < 0.05 ) 2 days post - cs - exposure and contributed the most to the separation seen in the pca of figure 1a . each circle in the volcano plot of panel c represents a putative metabolite , with the boxed red circles on the top half representing those , which were statistically significantly perturbed in the serum of cs - exposed mice 2 days after exposure . this initial statistical analysis using traditional tests was extended to other time points / doses with similar results . to see how the overall metabolomic profiles of all time points / doses compared the resulting proximity - matrix - based mds plot in figure 1d shows that the overall metabolomic profile of serum from control mice is clearly separated from that of cs - exposed mice at all time point / doses based on the top 100 ranked putative metabolite . these 100 putative metabolite can be used to assign with 82% accuracy any serum sample to its correct dose group based on its relative serum abundance ( data from esi mode ) . comparative analysis of serum metabolomic profiles of control mice and those exposed to cs at a cumulative dose of 1.95 gy at 2 days post - exposure . panel a is a principle component analysis ( pca ) plot showing clear separation of metabolomic signatures of sera from control ( blue triangles ) and cs - exposed mice ( red circles ) . panel b is a heatmap of metabolites whose serum levels change significantly 2 days post - cs - exposure . the top half of this heatmap displays metabolites whose levels in serum dropped post - exposure and those of metabolites on the bottom half increased post - exposure after 2 days . panel c is a volcano plot , which highlights many statistically significant metabolites post - exposure . panel d is an mds plot generated in random forests showing the spatial separation between the overall metabolomic profiles of serum samples from control mice and those of serum samples from cs - exposed mice at 2 , 3 , 5 , 20 , and 30 days post - exposure . panels a c were created in metabolyzer , while panel d was generated in random forests . after thorough analysis of the statistically significant metabolites from all of the time - points / doses we were able to group the metabolites into several key pathways , such as riboflavin metabolism and linoleic acid metabolism in esi mode ( supplemental figure 1 in the supporting information ) and glycolysis , tca cycle , tyrosine , and phenylalanine metabolism in esi mode . the results of this analysis suggested that exposure to cs caused an increase in the serum levels of metabolites from these pathways particularly at earlier time points ( day 3 and day 5 post - exposure ) . some metabolites levels dropped to their pre - exposure levels after the initial increase at days 3 and 5 , but most metabolites showed a persistent increase in their response to cs during the entire course of the study , as shown in figure 2 . for instance , two metabolites associated with riboflavin pathway , hydroquinone , and riboflavin ( reduced form ) , showed increased serum levels post - exposure , particularly after 3 and 5 days ( cumulative doses of 2.70 and 4.14 gy ) . the levels of these metabolites remained elevated until 30 days post - exposure ( the last time - point in the study ) . changes in the serum abundances of selected metabolites post cs exposure are represented as the ratio of their responses in cs - exposed mice to those in control mice . the responses were calculated as the area under the peak for each metabolite in cs - treated serum divided by that in control serum ( y axis ) . the identities of these ions were validated via ms / ms against pure standards or through ms / ms spectra published in online databases ( metlin and hmdb ) . tight clustering of fold - change values for glucose and dityrosine is shown at each time point . furthermore , the serum abundance of a few metabolites associated with energy metabolism was determined to be significantly attenuated as a result of exposure to cs . lactic acid is an important metabolite of energy metabolism , and changes in its levels may be indicative of a change in energy supply and glycolytic flux . the serum levels of this metabolite increased as a result of exposure to cs in the earlier time points and returned to its pre - exposure levels by the end of the experiment ( day 30 ) . increased level of this metabolite interestingly , we determined a similar increase in the levels of dityrosine , which is a cross - linked species formed upon protein modification due to ros - induced damage . the rise in dityrosine levels during earlier time points matched the increase in serum lactic acid during these time points , which may suggest a link between these two metabolites and internal emitter exposure - induced ros damage . additionally , glucose level was determined to be elevated by almost two - fold in mice exposed to cs starting as early as 2 days post - exposure . together with an increase in serum levels of nicotinic acid , members of the tca cycle such as citrate , malate , and metabolites associated with tyrosine and phenylalanine metabolism ( figure 2 and table 2 ) may suggest an increase in the rate of glycolysis . taurine and uric acid listed in table 2 have been reported in gamma irradiation studies as potential markers of external beam exposure . these metabolites were also found to be significantly perturbed in the urine of cs - exposed mice in this study . taurine shows a significant decrease in its serum concentration post - cs - exposure , while its urinary abundance was determined to increase in the same mice post - exposure . uric acid in the urine of these mice showed a late increased response , while its serum levels suggest an early increase in response . inositol is yet another metabolite reported in literature as a potential serum marker for gamma irradiation , whose increased response to radiation in a dose - specific manner may indicate a perturbation in hepatic lipid metabolism . we determined that the serum levels of this metabolite increased post cs - exposure ; however , this persistent increase was not dose - specific and may suggest a more systemic perturbation in lipid metabolism as a result of exposure to an internal emitter . fold change was calculated by dividing the relative concentration of a metabolite in post cs exposure serum samples by that in respective control samples . putative name was assigned based on accurate mass matched to metlin database ( mass error < 10 ppm ) . figure 3 demonstrates how retention time window and specific fragments of lipid head groups can be used to do just that . the use of lipid and fatty acid standards in both esi modes further facilitated their identification due to the differences in preferential ionization of polar head groups . for instance , in esi mode , the phosphatidylcholine ( pc ) and lysophosphatidylcholine ( lpc ) standards were used for identification and relative quantification of lipids belonging to these classes , while in esi mode ( phosphatidylethanolamine ( pe ) , lysophosphatidylethanolamine ( lpe ) , phosphatidylglycerol ( pg ) , and ( fatty acid ) fa were used . the two - fold serial dilutions established linearity between peak area and concentration for the mentioned standards ( supplemental table 1 in the supporting information ) . more than 800 spectral features were detected in both esi modes combined , from which 48 were determined to be pcs , 12 sms , 7 pes , 15 lpcs , 2 lpes , 3 fas , and 1 pg . we were able to detect more pcs than the other lipid species in this study . among the pcs , the ion at m / z of 758.5685 and retention time of 5.95 min was determined to be the most statistically significant pc . figure 4 shows the low and high energy scans for this pc with calculated fatty acid chains of 16 carbons long and 18 carbons long with two double bonds . the cleavage of the phospho - headgroup of pcs gives rise to a fragment at m / z of 184.0752 , which can be used to search for all pc and lpc classes of phospholipid along with sms . to gain further insight into the chemical structure of these lipids , we explored the ms data at high collision energy for identification of other fragments of the precursor ion at the desired retention time . for instance , for the precursor ion at m / z of 758.5685 and retention time of 5.95 min , the low and high energy scans were aligned , and the fragments of the precursor ion were identified . this lead to the identification of fragments at m / z of 478.3211 , which corresponds to the loss of a 16 carbon long fatty acyl chain and a water molecule from the precursor ion . the proposed sn2 reaction that gave rise to the peak at m / z of 478.3211 is depicted in figure 4 ( esi mode ) along with a similar mechanism for the fragment at m / z of 520.3643 . on the basis of these fragments we identified the precursor ion at m / z of 758.5685 as pc(16:0/18:2 ) , as shown in figure 4 . ms data can be carefully mined to gain more structural information on lipids in addition to assigning them to their respective classes . a similar approach was taken to identify fas and lipid species such pes and lpcs within their specified retention time window and mass error window ( < 10 ppm ) . a few examples of lipids identified via this approach are provided in table 3 . ms low and high energy scans of various classes of lipids in sera of mice . the low energy scan highlights the specific retention time window for each class . the high energy scan highlights a specific fragment at m / z of 184.0 that can be used to identify lipids with a phosphate headgroup . base peak of a pc and its chemical structure ( top ) along with its ms / ms spectrum and identified fragments ( bottom ) . the ms / ms spectrum was obtained using described ms method at high collision energy in esi mode . the low and high energy scans were first aligned in the expected retention time window ( a ) . the individual fragments of the precursor ion were determined by mining the high energy scan spectrum ( b ) . fold change was calculated by dividing the relative concentration of a metabolite in post cs exposure serum samples by that in respective control samples . statistical analysis on the identified lipid species revealed that the serum levels of pcs and lpcs were highly affected post - cs - exposure . pcs undergo hydrolysis of their acyl fa chains by the actions of phospholipases ( pl ) a1 and a2 to form lpcs . pc(36:4 ) with m / z of 782.5681 undergoes hydrolysis of its c20:4 acyl chain with m / z of 305.3305 by the actions of pla2 , resulting in the release of arachidonic acid ( c20 ) and lpc(16:0 ) with m / z of 478.3457 , as depicted in figure 5a . arachidonic acid is a known inflammation marker and was determined to have elevated serum levels post - cs - exposure ( figure 5b ) . the calculated ratio of lpc(16:0 ) peak area to the precursor pc(36:4 ) peak area in the serum of cs exposed mice showed an almost four - fold increase when compared with the calculated ratio in the serum of control mice ( figure 5b ) as early as 3 days post - exposure and continued to be elevated throughout the course of the study ( figure 5c ) . this along with elevated levels of arachidonic acid in the serum of cs - exposed mice suggests an increase in the activity of pla2 as a result of inflammatory response to cs exposure . arachidonic is further acted on by lipoxygenases to form leukotrienes or by cyclogenases to form pgs and thromboxanes . we identified leukotriene f4 to be elevated in the sera of cs - exposed mice as expected ( figure 5d ) . ratio of lpc(16:0 ) to pc(36:4 ) and the subsequent formation of arachidonic acid and synthesis of leukotrienes are collectively a measure of phospholipase 2 ( lpa2 ) activity and are used to gauge the level of inflammation . the data suggest an increase in the ratio of lpc / pc , which corresponds to an increase seen in the post - cs - exposure serum levels of arachidonic acid and leukotrienes . an increase in the formation of arachidonic acid via an sn-2 reaction can serve as an inflammation marker . in addition to the above phospholipids , we studied the serum levels of three fatty acids , linoleic , oleic , and palmitic acids , along with acyl - carnitine and acetylcarnitine species . fatty acids are important sources of energy , and they are converted into acyl - coa during -oxidation to be used in the tca cycle . our data suggest a slight decrease in the serum levels of these three fatty acids post - cs - exposure , accompanied by a decrease in the relative concentration of free carnitine ( figure 6 ) . however , the serum levels of acetylcarnitine increase by 35% post cs exposure , which may suggest a decrease in -oxidation . together with our metabolomics data , this indicates a shift in energy production from fatty acids oxidation to glycolysis as a result of cs exposure . it is important to note that the urine metabolomics analysis on these mice revealed opposite changes in the levels of tca cycle metabolites and carnitine species to what we observed in the serum . significant decrease is observed in the serum abundances of fatty acids : palmitic , linoleic , and oleic acid . as a crude measure of fatty acid -oxidation , the ratio of free carnitine to acetylcarnitine was calculated ( peak area ratio shown on y axis ) , which also suggests a decrease in serum - free carnitine levels post - cs - exposure . in this study , we focused on the changes in serum metabolites and lipids from mice exposed to an internal emitter , cs , which follows our previously published work in the urine of these mice . here we took advantage of an untargeted , data - independent , and hybrid technology called ms to rapidly yet comprehensively analyze mouse serum samples after 2 , 3 , 5 , 20 , and 30 days of exposure to cs and gain insight into robust responses to cs exposure . recent metabolomic studies of serum from rats and mice exposed to external gamma irradiation by gas chromatography ( gc)tofms have indicated important changes in the serum levels of amino acids such as serine and lysine , isocitrate , glycerol , stearic acid , steroid hormones such as progesterone , and cholesterol . these studies focused in particular on the volatile small metabolites and steroid hormones via gc tofms providing a specific yet narrow window into metabolic changes in serum of the animals post gamma irradiation . studying the complex network of metabolites and lipids and their exposure - specific responses is a monumental task and can not be covered in any single study ; however , our lc ms approach can provide insight into a wider range of molecules , from small polar metabolites to lipids , with relative quantification in serum . as with previous gamma irradiation studies from external sources , we initially focused on the metabolomic data and the overall metabolomic signature of serum from mice exposed to cs compared with that of serum from control mice . as expected , cs exposure significantly perturbed the levels of many serum metabolites as determined by statistical testing ( metabolyzer , mann whitney u - test p < 0.05 ) as early as 2 days post - exposure at a cumulative dose of 1.95 gy . the magnitude of change in the levels of these metabolites was large enough to affect the differential metabolomic profile of serum in the exposed mice . this is seen from the clear separation of profiles in the pca plot of figure 1a . the heatmap and the volcano plot in figure 1 highlight the specific cs exposure responses of selected metabolites at day 2 compared with their respective controls . the metabolomic profile of serum from cs - exposed mice at all time - points / doses was clearly distinguishable from that of their respective controls ( figure 1d ) . more thorough exploration of the data indicates changes in the metabolites associated with tyrosine metabolism , riboflavin metabolism , glycolysis , and tca cycle . two of the tca cycle metabolites were also found to be significantly perturbed in the urine of these mice , but in the opposite direction . for instance , urinary levels of citrate and malate were found to have decreased , while in serum their levels appeared elevated post - cs - exposure . a key metabolite of glycine and serine metabolism , 2-ketobutyric acid , which feeds into acetyl - coa production , and tca cycle was also found at higher concentration post - cs - exposure throughout the course of the study . this suggests an up - regulation of tca cycle and its feeder pathways in the serum of mice exposed to cs . we additionally found that glucose serum levels in mice post cs exposure were elevated while the average body weight of mice in all of the study groups remained steady ( table 1 ) . thus , the increase in serum levels of glucose and tca cycle associate metabolites is a result of exposure to cs and not food intake . this change is similar to what had been seen in the urine of these mice and those exposed to gamma irradiation . however , taurine was detected at lower concentration in the serum of cs - exposed mice , unlike what had been seen in the urine of these mice and in the urine of mice exposed to gamma irradiation at similar doses . inositol was recently shown by liu et al . to increase in the serum of mice exposed to gamma irradiation in a dose response manner . we also found the levels of this metabolite to be elevated in the serum of cs - exposed mice . inositol serum levels remained elevated throughout the course of the study , with the response being greatest at day 20 and decreasing to levels at earlier time - points by day 30 ( table 2 ) . an increase in serum concentration of inositol is associated with perturbation of lipid metabolism . lipid and fatty acid metabolism were further investigated in the second phase of this study . the statistically significant lipids were assigned to their respective classes using a shotgun approach lc ms method as previously described . we used the elution window and alignment of low and high energy scans as shown in figure 2 to do this . a few examples of lipids in each identified class are shown in table 3 . the pcs were detected at lower concentrations in the serum of cs exposed mice , while lpcs were found at elevated levels . we further investigated the fragmentation pattern of a pc species at m / z of 782.5681 . the phospho- headgroup of all pcs is cleaved at high collision energy scan to give rise to a peak at m / z of 184 . furthermore , the sn1 and sn2 reactions on the two fatty acyl chains of pcs give rise to distinct peaks corresponding to the loss of each acyl chain . as for the high energy scan of pc at 782.5681 , two fragments were aligned with the low energy scan , which corresponded to the loss of an acyl chain with 16 carbons and no double bonds and a 20 carbon long chain with 4 double bonds . thus , the identity assigned to the peak at m / z of 782.5681 was pc(36:4 ) . pcs are converted into lpcs by the action of phospholipase a2 ( pla2 ) and a fatty acid . therefore , we checked for the presence of a lpc(16:0 ) species and a fatty acyl chain of c20:4 , which corresponds to arachidonic acid . both of these molecules were found in the serum of cs - exposed mice at higher levels than pc(36:4 ) . the ratio of concentration of pc(36:4 ) to the formed lpc(16:0 ) thus may be indicative of the activity of pla2 . this ratio is shown in figure 4 along with a proposed pathway for action of this enzyme . furthermore , arachidonic acid is an inflammation marker and its increased levels in the cs - exposed mice may suggest radiation - induced inflammation in these mice and subsequently an increase in the activity of pla2 . we detected leukotriene f4 in the serum at higher concentration in cs - exposed mice ( figure 4 ) . as suggested by our previous pathway analysis , we expected to see perturbations in the levels of linoleic acid . in addition , we studied the serum levels of three fatty acids , linoleic , oleic , and palmitic acids , along with acyl - carnitine and acetylcarnitine species . fatty acids are important sources of energy , and they are converted into acyl - coa during -oxidation in the cytosol and transported into mitochondria via carnitines to ultimately form acetyl - coa to be used in the tca cycle . our data suggest a slight decrease in the serum levels of the three fatty acids post - cs exposure , accompanied by a decrease in the relative concentration of free carnitine . however , the serum levels of acetylcarnitine increased by almost 40% post cs exposure , which may suggest a decrease in -oxidation . together with our metabolomics data , this indicates a shift in energy production from fatty acids oxidation to glycolysis as a result of cs exposure . figure 7 depicts the pathways indicated as significantly perturbed in this study and how they are interconnected in energy production . amino acids such as tyrosine and phenylalanine enter the energy production pathway by forming acetyl - coa . in addition to fatty acids and amino acids , glucose also is converted into acetyl - coa through pyruvate production . all of these pathways help provide a steady flow of acetyl - coa into mitochondria to be used in the tca cycle . it is known that in the presence of environmental stimuli and stress , such as radiation exposures , these pathways become perturbed , which will ultimately affect the function of mitochondria and tca cycle energy production . furthermore , a recent gene profiling study found significant changes in the expression of genes associated with mitochondrial processes such as the electron transport chain , and cellular respiration in white blood cells from the same samples used in our study . a change in mitochondria function can also throw the balance between ros production and scavenging off . our data suggest that exposure to cs may perturb fatty acid oxidation and trigger an increase in tca cycle and its feeder pathways . this may lead to higher levels of ros production and further damage to structural proteins and enzymes . an increase in the serum abundance of dityrosine , a marker of protein oxidation , may further support the proposed mechanism in figure 7 . further exploration of enzymatic activity and chemical assays along with more in - depth and targeted metabolomics and lipidomics is necessary to independently validate these results and paint a more complete biosignature for the effects of cs exposure in serum . pathway illustration of the overall metabolomic and lipidomic changes in mouse serum post cs exposure . at least two metabolites in each noted pathway were found to be significantly perturbed ( p value < 0.05 ) . the overall pathway analysis indicates a shift in energy metabolism from fatty acid oxidation to glycolysis . in this study we used a fast and robust lc ms technique , which allows one to use the elution profile of precursor masses and the fragmentation profiles obtained during high collision energy scans to elucidate structural information on the detected spectral features . by taking advantage of this technique we were able to comprehensively study the effects of exposure to cs , an internal emitter , in the serum of mice . the findings suggest an up - regulation of tca cycle and down - regulation of fatty acid -oxidation as a result of exposure to cs .

in this study ultra performance liquid chromatography ( uplc ) coupled to time - of - flight mass spectrometry in the mse mode was used for rapid and comprehensive analysis of metabolites in the serum of mice exposed to internal exposure by cesium-137 ( 137cs ) . the effects of exposure to 137cs were studied at several time points after injection of 137cscl in mice . over 1800 spectral features were detected in the serum of mice in positive and negative electrospray ionization modes combined . detailed statistical analysis revealed that several metabolites associated with amino acid metabolism , fatty acid metabolism , and the tca cycle were significantly perturbed in the serum of 137cs - exposed mice compared with that of control mice . while metabolites associated with the tca cycle and glycolysis increased in their serum abundances , fatty acids such as linoleic acid and palmitic acid were detected at lower levels in serum after 137cs exposure . furthermore , phosphatidylcholines ( pcs ) were among the most perturbed ions in the serum of 137cs - exposed mice . this is the first study on the effects of exposure by an internal emitter in serum using a uplc mse approach . the results have put forth a panel of metabolites , which may serve as potential serum markers to 137cs exposure .

Introduction Materials and Methods Results Discussion Conclusion

exposure to internal emitters such as cesium-137 is an inevitable consequence of nuclear accidents , such as chernobyl and fukushima daiichi . advances in technology , particularly in the field of mass spectrometry , have enabled our laboratory to make significant progress in determining even the slight and subtle changes in the molecular composition , namely , the metabolome , of biofluids as a result of exposure to external beam and internal emitters using mouse models . this study is complementary to our previous work on the metabolic perturbations in urine of c57/bl6 mice over a 30-day period after exposure to cs , through employing the sensitivity of time - of - flight mass spectrometry ( tofms ) coupled to high resolving power of ultra performance liquid chromatography ( uplc ) . furthermore , we utilized a comprehensive statistical analysis workflow we have specifically developed for metabolomics , called metabolyzer , to determine cs - specific perturbations in metabolites / lipids and their respective pathways . the statistically significant metabolites and lipids were then compared with known external - beam -irradiation markers to determine similarities and differences in terms of responses to these two types of exposure . to date , this is the first time that mass spectrometry has been utilized to study the effects of exposure to an internal emitter in serum of mice . the results of this study and other radiation - induced signaling studies in easily accessible biofluids may help uncover the mechanism behind ir - induced inflammation and injury , which will ultimately lead to the discovery of novel biomarkers of ir exposure . glucose , riboflavin , arachidonic acid , linoleic acid , oleic acid , palmitic acid , hippuric acid , nicotinic acid , lactic acid , uridine , taurine , -ketobutyric acid , uric acid , hydroxyphenylpyruvic acid , acetylcarnitine , and carnitine were purchased from sigma - aldrich ( seelze , germany ) . the concentration range for lysophosphatidylcholine 17:1 ( lpc 17:1 ) and fatty acid 17:1 ( fa 17:1 ) internal standards was at 128 to 1 the internal standards were also spiked into pooled control serum samples and processed via uplc these nonendogenous internal standards were quickly identified based on their unique mass , retention time , and fragmentation profiles . the metabolomic analysis was performed by injecting 2 l of aliquot of each sample into a reverse - phase 50 2.1 mm h - class uplc acquity 1.7-m beh c18 column ( waters corp , milford , ma ) coupled to a time - of - flight mass spectrometry ( tofms ) . the solvents used for the lipidomic analysis were 50% acetonitrile with 0.1% formic acid and 10 mm ammonium formate ( solvent c ) and isopropanol / acetonitrile ( 90:10 v : v ) with 10 mm ammonium formate ( solvent d).the gradient started with 60% solvent c at 0.45 ml / min for the initial 8 min , then switched to 100% solvent d for 1 min , and back to 60% solvent c for the remaining 2 min of the 11 min long gradient . the xevo g2-s qtof mass spectrometer was operated in positive ( esi ) and negative ( esi ) electrospray ionization ( esi ) modes over a mass range of 50 to 1200 da in two channels , ms and ms . metabolyzer allowed for extraction of the ions with nonzero abundance values , which were detected in at least 70% of samples in each study group , called complete - presence ions . the log - transformed data for statistically significant complete - presence ions were then utilized for principal component analysis ( pca ) via singular value decomposition for the purpose of data visualization statistically significant ions were putatively identified via metabolyzer , which utilizes the human metabolome database ( hmdb ) , lipidmaps , and the kyoto encyclopedia of genes and genomes ( kegg ) database . the masses were then compared with the exact mass of small molecules in the databases , from which putative metabolites were identified with a mass error of 20 ppm ( ppm ) or less . in this study we utilized the sensitivity of mass spectrometry ( xevo - g2 , waters ) to detect changes in the sera of mice exposed to cs over the course of 30 days . the comprehensive analysis of serum metabolites and lipids along with their relative abundances were made possible by a feature of the xevo - g2 called ms , where many precursor , neutral loss , and product ion scans are acquired for every injection . these spectral features were used to determine the changes in the serum metabolome of mice after exposure to cs at different time points / doses ( table 1 ) . for example , the overall serum metabolomic profile of mice 2 days after cscl injection at a cumulative dose of 1.95 gy showed significant changes when compared with the serum metabolomic profile of matched control mice , as expected . this is evident from the clear separation in pca of figure 1a , where cs - exposed mice ( red circles ) are tightly clustered and clearly dichotomized from the control mice ( blue triangles ) . the heatmap in figure 1b shows a panel of putative metabolites on the vertical axis , whose serum abundances change significantly ( p < 0.05 ) 2 days post - cs - exposure and contributed the most to the separation seen in the pca of figure 1a . each circle in the volcano plot of panel c represents a putative metabolite , with the boxed red circles on the top half representing those , which were statistically significantly perturbed in the serum of cs - exposed mice 2 days after exposure . to see how the overall metabolomic profiles of all time points / doses compared the resulting proximity - matrix - based mds plot in figure 1d shows that the overall metabolomic profile of serum from control mice is clearly separated from that of cs - exposed mice at all time point / doses based on the top 100 ranked putative metabolite . comparative analysis of serum metabolomic profiles of control mice and those exposed to cs at a cumulative dose of 1.95 gy at 2 days post - exposure . the top half of this heatmap displays metabolites whose levels in serum dropped post - exposure and those of metabolites on the bottom half increased post - exposure after 2 days . panel d is an mds plot generated in random forests showing the spatial separation between the overall metabolomic profiles of serum samples from control mice and those of serum samples from cs - exposed mice at 2 , 3 , 5 , 20 , and 30 days post - exposure . after thorough analysis of the statistically significant metabolites from all of the time - points / doses we were able to group the metabolites into several key pathways , such as riboflavin metabolism and linoleic acid metabolism in esi mode ( supplemental figure 1 in the supporting information ) and glycolysis , tca cycle , tyrosine , and phenylalanine metabolism in esi mode . the results of this analysis suggested that exposure to cs caused an increase in the serum levels of metabolites from these pathways particularly at earlier time points ( day 3 and day 5 post - exposure ) . for instance , two metabolites associated with riboflavin pathway , hydroquinone , and riboflavin ( reduced form ) , showed increased serum levels post - exposure , particularly after 3 and 5 days ( cumulative doses of 2.70 and 4.14 gy ) . changes in the serum abundances of selected metabolites post cs exposure are represented as the ratio of their responses in cs - exposed mice to those in control mice . furthermore , the serum abundance of a few metabolites associated with energy metabolism was determined to be significantly attenuated as a result of exposure to cs . the serum levels of this metabolite increased as a result of exposure to cs in the earlier time points and returned to its pre - exposure levels by the end of the experiment ( day 30 ) . the rise in dityrosine levels during earlier time points matched the increase in serum lactic acid during these time points , which may suggest a link between these two metabolites and internal emitter exposure - induced ros damage . additionally , glucose level was determined to be elevated by almost two - fold in mice exposed to cs starting as early as 2 days post - exposure . together with an increase in serum levels of nicotinic acid , members of the tca cycle such as citrate , malate , and metabolites associated with tyrosine and phenylalanine metabolism ( figure 2 and table 2 ) may suggest an increase in the rate of glycolysis . these metabolites were also found to be significantly perturbed in the urine of cs - exposed mice in this study . we determined that the serum levels of this metabolite increased post cs - exposure ; however , this persistent increase was not dose - specific and may suggest a more systemic perturbation in lipid metabolism as a result of exposure to an internal emitter . for instance , in esi mode , the phosphatidylcholine ( pc ) and lysophosphatidylcholine ( lpc ) standards were used for identification and relative quantification of lipids belonging to these classes , while in esi mode ( phosphatidylethanolamine ( pe ) , lysophosphatidylethanolamine ( lpe ) , phosphatidylglycerol ( pg ) , and ( fatty acid ) fa were used . more than 800 spectral features were detected in both esi modes combined , from which 48 were determined to be pcs , 12 sms , 7 pes , 15 lpcs , 2 lpes , 3 fas , and 1 pg . statistical analysis on the identified lipid species revealed that the serum levels of pcs and lpcs were highly affected post - cs - exposure . the calculated ratio of lpc(16:0 ) peak area to the precursor pc(36:4 ) peak area in the serum of cs exposed mice showed an almost four - fold increase when compared with the calculated ratio in the serum of control mice ( figure 5b ) as early as 3 days post - exposure and continued to be elevated throughout the course of the study ( figure 5c ) . this along with elevated levels of arachidonic acid in the serum of cs - exposed mice suggests an increase in the activity of pla2 as a result of inflammatory response to cs exposure . we identified leukotriene f4 to be elevated in the sera of cs - exposed mice as expected ( figure 5d ) . the data suggest an increase in the ratio of lpc / pc , which corresponds to an increase seen in the post - cs - exposure serum levels of arachidonic acid and leukotrienes . in addition to the above phospholipids , we studied the serum levels of three fatty acids , linoleic , oleic , and palmitic acids , along with acyl - carnitine and acetylcarnitine species . fatty acids are important sources of energy , and they are converted into acyl - coa during -oxidation to be used in the tca cycle . our data suggest a slight decrease in the serum levels of these three fatty acids post - cs - exposure , accompanied by a decrease in the relative concentration of free carnitine ( figure 6 ) . however , the serum levels of acetylcarnitine increase by 35% post cs exposure , which may suggest a decrease in -oxidation . it is important to note that the urine metabolomics analysis on these mice revealed opposite changes in the levels of tca cycle metabolites and carnitine species to what we observed in the serum . significant decrease is observed in the serum abundances of fatty acids : palmitic , linoleic , and oleic acid . as a crude measure of fatty acid -oxidation , the ratio of free carnitine to acetylcarnitine was calculated ( peak area ratio shown on y axis ) , which also suggests a decrease in serum - free carnitine levels post - cs - exposure . in this study , we focused on the changes in serum metabolites and lipids from mice exposed to an internal emitter , cs , which follows our previously published work in the urine of these mice . here we took advantage of an untargeted , data - independent , and hybrid technology called ms to rapidly yet comprehensively analyze mouse serum samples after 2 , 3 , 5 , 20 , and 30 days of exposure to cs and gain insight into robust responses to cs exposure . recent metabolomic studies of serum from rats and mice exposed to external gamma irradiation by gas chromatography ( gc)tofms have indicated important changes in the serum levels of amino acids such as serine and lysine , isocitrate , glycerol , stearic acid , steroid hormones such as progesterone , and cholesterol . these studies focused in particular on the volatile small metabolites and steroid hormones via gc tofms providing a specific yet narrow window into metabolic changes in serum of the animals post gamma irradiation . as with previous gamma irradiation studies from external sources , we initially focused on the metabolomic data and the overall metabolomic signature of serum from mice exposed to cs compared with that of serum from control mice . the metabolomic profile of serum from cs - exposed mice at all time - points / doses was clearly distinguishable from that of their respective controls ( figure 1d ) . more thorough exploration of the data indicates changes in the metabolites associated with tyrosine metabolism , riboflavin metabolism , glycolysis , and tca cycle . two of the tca cycle metabolites were also found to be significantly perturbed in the urine of these mice , but in the opposite direction . a key metabolite of glycine and serine metabolism , 2-ketobutyric acid , which feeds into acetyl - coa production , and tca cycle was also found at higher concentration post - cs - exposure throughout the course of the study . this suggests an up - regulation of tca cycle and its feeder pathways in the serum of mice exposed to cs . we additionally found that glucose serum levels in mice post cs exposure were elevated while the average body weight of mice in all of the study groups remained steady ( table 1 ) . thus , the increase in serum levels of glucose and tca cycle associate metabolites is a result of exposure to cs and not food intake . however , taurine was detected at lower concentration in the serum of cs - exposed mice , unlike what had been seen in the urine of these mice and in the urine of mice exposed to gamma irradiation at similar doses . to increase in the serum of mice exposed to gamma irradiation in a dose response manner . we also found the levels of this metabolite to be elevated in the serum of cs - exposed mice . lipid and fatty acid metabolism were further investigated in the second phase of this study . the pcs were detected at lower concentrations in the serum of cs exposed mice , while lpcs were found at elevated levels . both of these molecules were found in the serum of cs - exposed mice at higher levels than pc(36:4 ) . furthermore , arachidonic acid is an inflammation marker and its increased levels in the cs - exposed mice may suggest radiation - induced inflammation in these mice and subsequently an increase in the activity of pla2 . we detected leukotriene f4 in the serum at higher concentration in cs - exposed mice ( figure 4 ) . in addition , we studied the serum levels of three fatty acids , linoleic , oleic , and palmitic acids , along with acyl - carnitine and acetylcarnitine species . fatty acids are important sources of energy , and they are converted into acyl - coa during -oxidation in the cytosol and transported into mitochondria via carnitines to ultimately form acetyl - coa to be used in the tca cycle . our data suggest a slight decrease in the serum levels of the three fatty acids post - cs exposure , accompanied by a decrease in the relative concentration of free carnitine . however , the serum levels of acetylcarnitine increased by almost 40% post cs exposure , which may suggest a decrease in -oxidation . figure 7 depicts the pathways indicated as significantly perturbed in this study and how they are interconnected in energy production . all of these pathways help provide a steady flow of acetyl - coa into mitochondria to be used in the tca cycle . it is known that in the presence of environmental stimuli and stress , such as radiation exposures , these pathways become perturbed , which will ultimately affect the function of mitochondria and tca cycle energy production . furthermore , a recent gene profiling study found significant changes in the expression of genes associated with mitochondrial processes such as the electron transport chain , and cellular respiration in white blood cells from the same samples used in our study . our data suggest that exposure to cs may perturb fatty acid oxidation and trigger an increase in tca cycle and its feeder pathways . further exploration of enzymatic activity and chemical assays along with more in - depth and targeted metabolomics and lipidomics is necessary to independently validate these results and paint a more complete biosignature for the effects of cs exposure in serum . in this study we used a fast and robust lc ms technique , which allows one to use the elution profile of precursor masses and the fragmentation profiles obtained during high collision energy scans to elucidate structural information on the detected spectral features . by taking advantage of this technique we were able to comprehensively study the effects of exposure to cs , an internal emitter , in the serum of mice . the findings suggest an up - regulation of tca cycle and down - regulation of fatty acid -oxidation as a result of exposure to cs .

human t cell leukemia virus type 1 ( htlv-1 ) is the causative agent of human adult t cell leukemia ( atl ) , a chronic progressive neurological disorder termed htlv-1-associated myelopathy / tropical spastic paraparesis ( ham / tsp ; hinuma et al . , 1981 ; kalyanaraman et al . , 1982 ; osame et al . , 1986 ; jacobson et al . , 1988 ) , and other diseases htlv-1 encodes an unspliced , intron - containing 9 kb rna , which encodes gag and gag pol , a singly spliced 4 kb mrna encoding env , and multiply spliced , intron - free 2 kb mrnas encoding tax and rex ( green and chen , 2001 ) . recently , the htlv-1 leucine zipper factor ( hbz ) gene was identified , which is encoded by a complimentary strand of the htlv-1 genome , driven by a promoter residing in the 3 ltr ( figure 1 ) . tax and hbz are implicated in oncogenesis ( satou et al . , 2006 ; matsuoka and jeang , 2011 ) . historically , rex was the first protein to be identified as a factor that induces the cytoplasmic expression of intron - containing 9 and 4 kb rnas ( inoue et al . , 1986 ; hidaka et al . , rex binds directly to viral rnas containing rex response element ( rxre ) located in the 3 ltr . rxre , which consists of a highly ordered stem - and - loop structure , functions as a rex binding site ( ballaun et al . , 1991 ; bogerd et al . , since all htlv-1 rnas contain rxre , rex also enhances transport of fully spliced 2 kb mrna , although this rna can be transported via the normal route for cellular mrnas that do not require rex ( bai et al . , the sequence elements that retain htlv-1 unspliced rnas in the nucleus have been identified in the pol an important property of rex , which is essential for its role , is its ability to dynamically shuttle between the cytoplasm and nucleus , although rex is predominantly localized in the nucleus and nucleolus ( bogerd et al . , 1996 ; rex is synthesized in the cytoplasm and then imported into the nucleus . in the nucleus , rex directly binds to rxre , after which it escorts the viral rnas into the cytoplasm ( bogerd et al . , 1991 ) . extensive mutational analyses show that rex contains functional domains that are essential for its function ( figure 2 ) . the basic domain is rich in arginine residues and maps between amino acids ( aa ) 119 in the 189 aa rex protein . this domain serves as a nuclear / nucleolar localization signal ( nls ) , and foreign proteins containing these residues are localized to the nucleus / nucleolus ( siomi et al . , 1988 ) . importantly , the same region mediates binding to rxre , thereby determining its specific target rna ( bogerd et al . , 1991 ) . the second essential domain , which maps between aa 84 and 94 of rex , is a leucine - rich activation domain that functions as a nuclear export signal ( nes ; bogerd et al . , 1996 ) . the domain responsible for this was first ascribed to a 6070 aa region of rex ( weichselbraun et al . , 1992 ) ; however , subsequent in vivo two - hybrid assays revealed that a 100120 aa region was also involved in multimerization ( heger et al . , 1998 ) . multimerization of rex on its cognate rna is generally thought to be critical for its ability to export viral rnas , although it is not required for export of the rex protein itself ( heger et al . , 1998 ) . although shuttling proteins have now been identified in a wide range of other viral and cellular proteins , multimerization may distinguish rex from other shuttling proteins that are not involved in rna export since their multimerization is not necessarily required for their functioning ( weichselbraun et al . , 1992 ; bogerd and greene , 1993 ; heger et al . , 1998 ) . a detailed understanding of rex shuttling was obtained from studies that identified the cellular factors binding to the functional domains , and the cellular machinery involved in protein transport into and out of the nucleus ( figure 3 ) . transport of all macromolecules occurs via nuclear pore complexes ( npcs ) , which comprise at least 50 different proteins , termed nucleoporins ( doye and hurt , 1997 ) . the import of proteins containing an nls is mediated by the importin family of import receptors , which have affinity for nucleoporins , and the gtp - bound status of ran ( rangtp ; mattaj and englmeier , 1998 ) . rex directly binds to importin through its nls ( palmeri and malim , 1999 ) . translocation through the pore is thought to be facilitated by sequential direct interactions between importin and various nucleoporins . ran is a small gtpase that can exist in either the gtp or gdp - bound state . rangtp reflects the nuclear localization of chromatin binding protein , rcc1 , which specifically catalyzes the exchange of guanine nucleotides on ran , and rangdp , the predominant form of ran in the cytoplasm , reflects the cytoplasmic location of a gtpase - activating protein , rangap . one role of rangtp is to promote cargo release in the nucleus by dissociating the imported receptor cargo complexes ; rangdp has no affinity for importin ( mattaj and englmeier , 1998 ) . since the nls of rex overlaps with its rna binding domains , binding to importin in the cytoplasm may facilitate the release of viral rnas from rex ( bogerd et al . , 1991 ; siomi et al . , 1988 ; palmeri and malim , 1999 ) . action mechanism of viral transporter rex / rev and involvement of their cellular cofactors are illustrated . crm1 was originally identified as binding the nes of the human immunodeficiency virus ( hiv)-1 rev protein . sequence similarity to importin suggested that hcrm1 was an export receptor ( fornerod et al . subsequently , hcrm1 was shown to form a specific complex with nes ; however , unlike the rex - importin complex , this only occurred in the presence of rangtp ( fornerod et al . , 1997a ) . the association between rex and hcrm1 was confirmed in vivo using a two - hybrid assay ( hakata et al . , 1998 ) . moreover , crm1 directly binds to ran - binding protein 3 ( ranbp3 ) , which binds to rcc1 in a ran - dependent manner and increases the nucleotide exchange activity of rcc1 , resulting in high local concentrations of rangtp . consequently , ranbp3 acts as a scaffold protein through which the components of the export complex are concentrated around the rcc1 site , thereby promoting complex assembly . ranbp3 continues to interact with hcrm1 to stabilize the hcrm1substrate rangtp complex , thereby forming a quaternary complex ( englmeier et al . ranbp3 complex is translocated to the cytoplasm across the npc ; this occurs via possible hydrophobic interactions between hcrm1 and nucleoporins . following this , the rangtp within the complex is converted to rangdp by rangap and ranbp1 ( and probably ranbp2 ) , leading to the dissociation of the complex and release of the substrate into the cytoplasm ( askjaer et al . an important question is whether cellular cofactors , such as hcrm1 , are involved in the multimerization of rex proteins ( hakata et al . therefore , we examined the multimerization of rex proteins using a two - hybrid assay . first , we found that a rex mutant , rexm90 ( which harbors a mutation in the nes ) , lost the ability to bind to hcrm1 and to form multimers . second , we found that the rna transport activity of rexm64 , which harbors a mutation in the multimerization domain , was partially restored by the overexpression of hcrm1 , and this coincided with the partial restoration of multimerization . third , a dominant - negative ( dn ) mutant of rex , termed tagrexm64 , which sequesters rex cofactors ( katahira et al . , 1995 ) , taken together , these results suggest that the multimerization of rex in vivo requires both the intrinsic ability of rex to form multimers , and the hcrm1 protein . moreover , these results also suggest that the multimerization of rex is not a prerequisite for interaction with hcrm1 but , rather , that rex initially interacts with hcrm1 , leading to the multimerization of rex proteins in a step that is favored by its intrinsic capacity to form oligomers on rxre . this , in turn , would favor a structure that facilitates interactions with other hcrm1 molecules on the rna ( hakata et al . , 1998 ) . comparison of human and rat crm1 ( rcrm1 ) mapped the crm1 domain that induces rex multimerization to the central region , particularly a region containing two residues ( aa 411 and 414 ; hakata et al . , 2003 ) ; this domain is different from the region spanning aa 566720 that is involved in binding to the nes ( ossareh - nazari and dargemont , 1999 ) . these data suggest that crm1 not only functions as an export receptor but also participates in the formation of the rna export complex through a high - order interaction with rex . curiously , the crm1 region responsible for the interaction with ranbp3 , which comprises four residues ( aa 411 , 414 , 478 , and 484 ) , and the region responsible for rex multimerization form an overlapping domain ( hakata et al . , 2003 ) . appropriate animal models of htlv-1 infection would allow us to analyze the pathogenesis and oncogenesis of htlv-1-associated diseases , which could lead to the development of therapeutic and preventative measures . htlv-1 is able to infect experimental animals such as monkeys , rabbits , and rats ( akagi et al . , 1985 ; nakamura et al . , 1987 the utility of monkey and rabbit models is limited , however , because of the difficulties in breeding a large number of these animals and the lack of inbred strains . in particular , rat models have been extensively developed to study htlv-1-associated diseases ( ishiguro et al . however , although the current rat models have certainly allowed us to understand htlv-1-associated diseases better , they are still incomplete because of the poor replication of htlv-1 in rats . human t cell leukemia virus type 1 can infect a number of rat cell types , which indicates that rat cells possess the receptors for viral attachment and penetration ( li et al . , 1996 ; sutton and littman , 1996 ) thus , the blockade of viral propagation within the rat cells must occur during subsequent steps . identification of the blocking step and the responsible host factor(s ) could lead to the construction of transgenic rats that express the critical human factor(s ) , which are highly susceptible to htlv-1 infection . the first hint of a blocking step came in a report showing that the viral mrnas encoding gag and env proteins are produced only at low levels in rat cells despite the fact that the mrna for tax / rex protein is abundant ( koya et al . , 1999 ) . indeed , we found that the activity of rex in rat cells is quite low compared with that in human cells . as the functions of rex depend largely on the crm1 protein , we examined whether rcrm1 can act as a cofactor for rex activity as it does in human cells , although crm1 is highly conserved from yeast to mammalians , and hcrm1 can function in yeast cells as an export receptor . actually , only 24 out of 1027 amino acids are different between rat and human crm1 ( hakata et al . , 2003 ) . also , we found that both rcrm1 and hcrm1 could bind to and export the rex protein to the cytoplasm with similar efficiency . however , unlike hcrm1 , rcrm1 could not efficiently support rex function because of its poor ability to induce the rex it was also concluded that the poor ability of rcrm1 to act as a cofactor for rex is responsible for the poor replication of htlv-1 in rats ( hakata et al . , 2001 ) . this notion was further supported by a study showing that a htlv-1-transformed rat cd4 + t cell line , which was a poor producer of htlv-1 , efficiently synthesized functional env and gag proteins , which were then fully processed , and produced infectious htlv-1 progeny viruses at the level comparable to htlv-1-transformed human t cells when it was transduced with a retroviral vector expressing hcrm1 at physiological levels ( zhang et al . , 2006 ) . rex proteins multimerize with help of human crm1 on htlv-1 rna to form transport complex in human cells , and then export it to the cytoplasm . transgenic ( tg ) rats were constructed using an artificial bacterial chromosome clone containing the entire regulatory and coding regions of the crm1 gene ( figure 5 ) , since the expression of the crm1 gene is elaborately regulated by a protein kinase c pathway during lymphocyte activation , initially in a post - transcriptional and subsequently in a transcriptional manner . consequently , tg rats expressed hcrm1 protein in a manner similar to that of intrinsic rcrm1 in various organs . htlv-1-infected t cell lines derived from these tg rats produced 100- to 10,000-fold more htlv-1 than t cells from wild - type rats , and the absolute levels of htlv-1 were similar to those produced by human t cells . we also observed increased dissemination of htlv-1 into the thymuses of tg rats after intraperitoneal inoculation , although the proviral loads were low in both wild - type and tg rats ( takayanagi et al . furthermore , rat regulatory t ( treg ) cells were preferentially transformed by htlv-1 ( takayanagi et al . , 2009 ) , as is the case for human treg cells ( chen et al . , 2006 ) . therefore , the tg rat model forms the basis for an improved animal model for htlv-1 infection . the bac clone harboring entire human crm1 genome was microinjected to eggs of rat f344 strain to construct transgenic rats . t cells prepared from normal and hcrm1 transgenic rats were transformed by mixed culture with mitomycin - treated htlv-1 human t cell mt2 . then the amounts of p19 gag protein in the culture medium were quantified by elisa . human immunodeficiency virus-1 belongs to the subfamily of complex retroviruses and , like htlv-1 , also encodes accessory genes besides gag and env ; however , hiv-1 shows more complex patterns of mrna expression . hiv-1 produces one unspliced 9 kb rna encoding gag and gag pol , five singly spliced 4 kb mrnas encoding vif , vpr , vpu , and env , and 16 multiply spliced 2 kb mrnas encoding tat , rev , and nef ( figure 1 ; freed and martin , 2001 ) . as is the case for htlv-1 rex , the 9- and 4-kb viral rnas of hiv-1 require the viral protein rev for export ( cullen , 1991 ) . however , expression of 2 kb rnas is not augmented by rev , since the rev response element ( rre ) , an approximately 300 nucleotide region with a highly ordered stem - and - loop structure that functions as a rev binding site , is located in the env gene rather than the 3 ltr ( malim et al . , 1990 ; mann et al . , 1994 ) . consequently , during the early phase of infection when rev expression is low , proteins encoded by the 2-kb class of mrnas are expressed . during the late phase , when rev is expressed in sufficient quantities , the remaining viral proteins are produced ( kim et al . , 1989 ) , including the virion constituents and infectivity enhancing and virion - releasing factors , all of which work toward the efficient production and dissemination of progeny virus . rev is a functional homolog of rex , although rev and rex do not show any homology at the amino acid level . however , they do have similar domain structures and function in a similar manner ( figures 2 and 3 ) , as exemplified by the fact that rex can replace rev ( ahmed et al . , 1990 ) . the basic domain rich in arginine residues , which maps between aa 35 and 50 in the 116 aa rev protein , serves as an nls , which recruits importin and binds to the rre ( daly et al . , 1989 ; malim et al . , 1991 ; tiley et al . , 1992 ; madore et al . , 1994 ) . replacing the amino - terminal domain of the rex protein with the rna binding domain of the rev protein yields a chimeric protein that can specifically interact with the rre ( kubota et al . , 1991 ) . a leucine - rich activation domain , which maps between aa 73 and 83 , acts as an nes that associates with crm1 in the presence of rangtp ( fisher et al . , 1995 ; wen et al . , 1995 ) . in vivo randomization selection experiments identified the consensus sequence , which contains relatively evenly spaced bulky hydrophobic residues ( often leucine ) that are crucial for nes function ( bogerd et al . , 1996 ; kim et al . , 1996 ) . in vitro studies of the multimerization of recombinant rev proteins bound to the rre - containing rna defined two essential domains : one between aa 12 and 33 and one between aa 46 and 60 ( malim et al . , 1991 ) . the c - terminal region of rev is highly conserved between hiv-1 strains and supports efficient multimerization and subsequent rev function , probably by stabilizing its conformation . in contrast to rex , rev is able to dimerize without crm1 molecules in vitro , although in vivo two - hybrid assays suggest that crm1 improves dimerization ( chaitanya and belasco , 2001 ; hakata et al . , 2002 ; matthew et al . , 2008 ) early studies suggested that rev binds initially as a monomer to a single high - affinity rev binding site within the rre ( battiste et al . , 1996 ) , but recent studies on the tertiary structure of rev indicate that the dimerization of rev induces conformational changes in the position of the arginine - rich domain to facilitate rna binding ( daugherty et al . , 2010 ) . rna bound to the rre augments further rev multimerization , recruiting up to eight rev molecules on the viral rna , which subsequently recruit several hcrm1 molecules ( mann et al . , 1994 ; d = asp glu ala asp ) box helicase , ddx1 , which is an rna - dependent atpase that binds directly to rev . knockdown of ddx1 greatly reduces rev function , supporting the important role of ddx1 ( fang et al . , 2004 ; robertson - anderson et al . , 2011 ; edgcomb et al . , 2012 ) . after the transport complex is formed , which comprises hiv-1 rna , rev , crm1 , and other cellular components , it moves to the nuclear pores , possibly via the actin filament network ( kimura et al . , 2000 ) . multiple crm1 molecules , which have affinity for nucleoporins , may be required to pass through the nuclear pore because the transport complex could be longer than the depth of the nuclear pore ; thus , the rear portion of the complex is still within the nucleus when the front portion reaches the cytoplasm . crm1 molecules associated with rev within the front portion of the ribonucleoprotein complex ( in the cytoplasm ) will dissociate due to the lack of rangtp ( fornerod et al . thus , additional crm1 molecules may be required to associate with the rear part of the rna complex to enable passage of the whole rna complex out of the nucleus . another dead box protein , ddx3 , which is located outside of the nuclear pore , associates with crm1 directly and facilitates the passage of the complex through the nuclear pore and the release of rna into the cytoplasm by inducing conformational changes in the ribonucleoprotein complex ( yedavalli et al . , 2004 ) . additionally , rip ( also named rab ) , which indirectly binds to rev and has affinity for nuclear pores , is thought to function in releasing the viral rna within the cytoplasm ( sanchez - velar et al . , 2004 ; yu et al . , 2005 ) . both inefficient branch point region and polypyrimidine tract are thought to be responsible for the low splicing efficiency of the hiv-1 tat / rev intron , which comprises most of the hiv-1 env - coding sequences ( staffa and cochrane , 1994 ) . two additional elements , an exon - splicing enhancer ( ese ) and an exon - splicing silencer ( ess ) , both of which modulate the overall efficiency of the 3 tat rev splice site , were identified within the 3 terminal exon ( staffa and cochrane , 1995 ) . it has been proposed that inefficient splicing causes the retention of unspliced , or partially spliced , mrnas since incomplete spliceosome formation at the splice sites inhibits export secondary sequence elements , which retain unspliced hiv-1 rnas within the nucleus , are termed cis - acting repressive sequences ( crss ) or instability sequences ( inss ; cochrane et al . , 1991 ; maldarelli et al . in contrast to fully spliced mrna , which is dispersed throughout both the nucleus and cytoplasm , the location of unspliced gag and env rnas does not coincide with splicing factor sc35-containing nuclear speckles . deletion of the intron sequence containing the crs resulted in a shift from discrete regional localization within the nucleus to a diffuse localization throughout the cell . on the other hand , mutations that affect splicing efficiency do not alter the sequestration of unspliced rnas ( seguin et al . , 1998 ) . based on these results , one may hypothesize that crss ( in conjunction with other elements that cause suboptimal splicing ) cause significant amounts of full - length primary rna , or partially spliced rna , to accumulate . this rna is inaccessible to subsequent splicing due to its localization in discrete regions within the nucleus . this sequestration of intron - containing rnas may be a prerequisite for subsequent export by rev . indeed , addition of the rre to -globin mrna , which contains efficient splice sites , does not make it responsive to rev unless the splice sites are replaced by more inefficient ones , or a crs is introduced into the mrna . the finding that hnrnpa1 and a 50-kda protein associate with the crs suggests a connection between the crs and rna splicing , since hnrnpa1 controls the selection of splice sites ( najera et al . , 1999 ) . current animal models of hiv-1 disease use non - human primates ( veazey et al . , 2005 ; koff et al . , 2006 ) or severe combined immunodeficiency ( scid ) mice transplanted with human hemopoietic stem cells and fetal tissues ( shultz et al . , 2007 these models have made significant contributions to our understanding of lentiviral pathogenesis and have assisted in the development of several therapeutic strategies . however , they also have significant shortcomings , such as limited availability and high cost ( for non - human primates ) , they are permissive only for related retroviruses , and they often show aberrant immune responses . therefore , new animal models are needed . rodent models susceptible to infection by human viruses would be ideal , since the inbred strains are well characterized and can be genetically manipulated . studies on rodent cell - specific defects in the hiv-1 life cycle have facilitated the identification and characterization of host cell gene products that are essential for viral replication , and these may provide a molecular basis for generating fully permissive small animal models . it is thought that this hurdle may be overcome by introducing human cd4 and ccr5 into the animal cells , which would serve as receptors for hiv-1 . however , tg mice and rats expressing human cd4 and ccr5 do not appear to support hiv-1 replication ( browning et al . , 1997 ; keppler et al . , 2002 ; goffinet et al . , 2007 ) , suggesting the presence of additional blocks post - entry . studies of the viral transcription activator , tat , indicate the existence of a profound block at the transcription stage in murine cells ( bieniasz et al . , 1998 ; garber et al . , 1998 ) and in rat t cells but , curiously , not in rat macrophages or fibroblasts ( okada et al . , 2009 ) . expression of the human cyclint1 ( hcyct1 ) , a cellular cofactor for tat , in mouse and rat t cells restored viral transcriptional activity ( okada et al . , a single amino acid difference between human and mouse cyclint1 ( mcyct1 ) , which has a tyrosine at residue 261 in place of the cysteine in hcyct1 , caused almost complete loss of tat cofactor activity ( bieniasz et al . in contrast to the definitive results obtained for rex in rat cells , the existence of a profound block in rev function in rodent cells remains controversial , although a reduced level of the hiv-1 9 kb transcript has been reported ( bieniasz and cullen , 2000 ; keppler et al . , 2001 ) . some studies have reported impaired rev activity ( trono and baltimore , 1990 ; marques et al . , 2003 ) , whereas others have ascribed the reduced transcript levels to over splicing or the reduced stability of unspliced transcripts in murine cells compared with human cells ( malim and cullen , 1991 ) ; a problem corrected by the expression of the human p32 protein ( zheng et al . , 2003 ) . however , malim et al found that the gag protein is still efficiently synthesized in murine cells , indicating that rev is functional , although it is not transported to the plasma membrane , resulting in defective virion formation . when gag mrna is transported via a different route using the cellular transporter , tap , instead of the rev crm1 system , gag protein was synthesized and transported to the surface membrane , leading to efficient virus production ( swanson et al . , 2004 ) . this indicates that the rna export step is a one of the species - specific barriers to hiv-1 propagation , and that the mode of mrna nucleocytoplasmic transport affects subsequent processes occurring in the cytoplasm . even when hiv-1 genes are expressed from transfected plasmids ( which circumvents the block at the entry step ) , virus production in rat cells is still poor . however , it increases markedly in rat macrophages and fibroblasts expressing hcrm1 ( but only marginally in rat t cells ) , which is in sharp contrast to cells transfected with hcyct1 ( okada et al . , 2009 ) . further analyses revealed that hcrm1 enhanced gag mrna export a couple of times at most in rat fibroblasts . the amount of gag protein within the cells increased two to three times , which paralleled the increase in gag mrna in the cytoplasm , although the trafficking of gag to the plasma membrane did not increase . however , the number of viral particles shed into the culture medium increased 20- to 50-fold , accompanied by the increased processing of the p55gag precursor to fully matured gag proteins . even a protease - deficient viral construct that caused accumulation of the p55 gag precursor in the cytoplasm facilitated the release of viral - like particles in the presence of hcrm1 , suggesting increased budding at the plasma membrane . the n - terminal half of hcrm1 was found to be responsible for this as the rcrm1rev interaction was much less efficient . these facts suggest that incomplete formation of transport complexes in the nucleus may affect the mechanisms underlying particle formation ( figure 6 ; nagai - fukataki et al . , 2011 ) . indeed , the interaction between rev and the rre is essential for proper encapsidation of grna into virus particles ( blissenbach et al . moreover , hiv-1 gag proteins gain access to the nucleus because they have nlss ( bukrinsky et al . , 1993 ) and ness that associate with crm1 ( dupont et al . , 1999 ) . thus , it would be interesting to examine the role of intra - nuclear gag proteins in virus morphogenesis . more studies are required to conclude since contradictory results that nls defective mutant gag proteins still support viral - like particle formation has been also reported ( grewe et al . , 2012 ) . schematic presentation of rna transport and particle formation defect of hiv-1 caused by rat crm1 . in rat cells hiv-1 genomic rna and unspliced mrna are inefficiently transported to the cytoplasm . expression of human crm1 corrects both defects , suggesting linking of rna nucleocytoplasmic transport to the subsequent cytoplasmic event . although simple retroviruses , exemplified by the mason - pfizer monkey virus ( mpmv ) , encode only the gag , pol , and env genes located centrally between the two ltrs ( figure 1 ; goff , 2001 ) , they also require the means to circumvent cellular restrictions , since the gag coding region is located within the intron of the pre - mrna encoding the envelope proteins . to express the intron - containing gag mrna and grna , inefficient splicing is necessary ( as in the case of hiv-1 ) , which is demonstrated by the finding that the modification of their splice sites increases their efficiency , resulting in the over splicing of retroviral rnas , which renders the virus replication incompetent ( katz and skalka , 1990 ) . however , inefficient splicing is not sufficient ; a sequence known as the constitutive transport element ( cte ; first identified in the 3-untranslation region of mpmv by bray et al . , 1994 ) since none of the simple retroviruses encode accessory proteins such as rex and rev , cte was thought to recruit cellular proteins . subsequently , a cellular protein called transporter associated with antigen processing ( tap ) was identified , which binds cte directly , and tap sequence similarity to yeast mex67 ( which is involved in mrna transport ) indicates that cte accesses the cellular mrna export pathway ( grter et al . , 1998 ; the fact that the rna export pathway involving cte is independent from those involving rre / rev or rxre / rex was illustrated by its sensitivity to inhibition by a dn mutant , tagrexm64 , which inhibits rev / rex function by sequestering the cellular cofactor , crm1 ( katahira et al . the ribonucleoprotein complex containing simple retroviral rna must also undergo a conformational change during export , which is mediated by rna helicase a ( tang et al . release of viral rna into the cytoplasm is mediated by another rna helicase , dbp5 , which localizes outside the nuclear pores ( leblanc et al . , 2007 ) . an intriguing question is : are grna and unspliced gag mrna exported via different pathways that determine whether they are encapsidated or translated , respectively ? rous sarcoma virus ( rsv ) , a simple avian retrovirus , contains direct repeat ( dr ) elements ( ogert et al . , 1996 ) that mediate the cytoplasmic accumulation of unspliced viral rna in a manner similar to that of mpmv cte , since dn tap and dbp5 mutants abrogate dr - dependent rna expression ( leblanc et al . , 2007 ) . on the other hand , rsv gag is reported to gain access to the nucleus via its nls , which interacts with importin / , transportin sr , and importin 11 ( butterfield - gerson et al . , 2006 ; gudleski et al . , 2010 ) , and is exported via its nes , which is associated with crm1 ( scheifele et al . , 2002 , 2005 ) . gag binds to grna at the packaging element , , and another unidentified site ( dsouza and summers , 2005 ) , and then forms multimers on grna to assemble viral core particles ( scheifele et al . , 2007 ) . thus , it is conceivable that gag proteins drive the transport of viral particles formed in the nucleus . this notion is supported by the following observations : first , transient nuclear trafficking of gag is required for efficient grna encapsidation ( garbitt - hirst et al . , 2009 ) , suggesting that gag may initially bind to grna in the nucleus . second , formation of gag : grna prevents the association between gag and importins , while the binding of gag to grna instead enhances its association with crm1 , consequently promoting the export of the gag grna complex to the cytoplasm ( gudleski et al . , 2010 ; parent , 2011 ) . these facts suggest the intriguing possibility that gag mrna is transported through the tap pathway to be translated , whereas grna ( which appears to be indistinguishable from gag mrna ) , is exported by the gag detailed analyses on mouse mammary tumor virus ( mmtv ) and human endogenous retrovirus k ( herv - k ) that had been initially classified as simple retroviruses revealed doubly spliced mrnas as the case of complex retroviruses . moreover , such mrnas have been found to encode rev like proteins such as rem in mmtv ( indik et al . , 2005 ; mertz et al . , 2005 ) and k - rev in herv - k ( yang et al . , 1999 ) that export their cognate viral rnas through crm1 pathway . the presence of k - rev in herv - k that integrated in primate genome 30 million years ago may evoke the possibility of evolutional relation in the acquisition of rev by exogenous lentiviruses ( yang et al . , 1999 ) . studies on rna transport of retroviruses originally stemmed from molecular biological interest on unusual function of rex and rev that break the cellular blockage against transport of intron - containing rna . a great deal of investigation not only elucidated their own mechanisms but also led discovery of the cellular nucleocytoplasmic transport system . moreover , recent studies showed that the mode of rna export influences subsequent events in the cytoplasm , consequently affecting whole process of viral production including the translation of the cognate mrna , transport of gag proteins to the plasma membrane , and the formation of virus particles . lastly , the discovery on the interaction between the viral and cellular rna transport machinery as a species - specific barrier of hiv-1 and htlv-1 forms the basis to construct convenient animal models of infection , which should facilitate development of more potent therapeutic and preventing means . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

retroviruses have evolved mechanisms for transporting their intron - containing rnas ( including genomic and messenger rnas , which encode virion components ) from the nucleus to the cytoplasm of the infected cell . human retroviruses , such as human immunodeficiency virus ( hiv ) and human t cell leukemia virus type 1 ( htlv-1 ) , encode the regulatory proteins rev and rex , which form a bridge between the viral rna and the export receptor crm1 . recent studies show that these transport systems are not only involved in rna export , but also in the encapsidation of genomic rna ; furthermore , they influence subsequent events in the cytoplasm , including the translation of the cognate mrna , transport of gag proteins to the plasma membrane , and the formation of virus particles . moreover , the mode of interaction between the viral and cellular rna transport machinery underlies the species - specific propagation of hiv-1 and htlv-1 , forming the basis for constructing animal models of infection . this review article discusses recent progress regarding these issues .

Transport of HTLV-1 RNA RNA Export Step as a Major Species-Specific Barrier for HTLV-1 RNA Transport of HIV RNA Export as a Species-Specific Barrier for HIV-1 RNA Export of Simple Retroviruses Conclusion Conflict of Interest Statement

human t cell leukemia virus type 1 ( htlv-1 ) is the causative agent of human adult t cell leukemia ( atl ) , a chronic progressive neurological disorder termed htlv-1-associated myelopathy / tropical spastic paraparesis ( ham / tsp ; hinuma et al . , 1988 ) , and other diseases htlv-1 encodes an unspliced , intron - containing 9 kb rna , which encodes gag and gag pol , a singly spliced 4 kb mrna encoding env , and multiply spliced , intron - free 2 kb mrnas encoding tax and rex ( green and chen , 2001 ) . recently , the htlv-1 leucine zipper factor ( hbz ) gene was identified , which is encoded by a complimentary strand of the htlv-1 genome , driven by a promoter residing in the 3 ltr ( figure 1 ) . historically , rex was the first protein to be identified as a factor that induces the cytoplasmic expression of intron - containing 9 and 4 kb rnas ( inoue et al . , the sequence elements that retain htlv-1 unspliced rnas in the nucleus have been identified in the pol an important property of rex , which is essential for its role , is its ability to dynamically shuttle between the cytoplasm and nucleus , although rex is predominantly localized in the nucleus and nucleolus ( bogerd et al . , 1996 ; rex is synthesized in the cytoplasm and then imported into the nucleus . in the nucleus , rex directly binds to rxre , after which it escorts the viral rnas into the cytoplasm ( bogerd et al . this domain serves as a nuclear / nucleolar localization signal ( nls ) , and foreign proteins containing these residues are localized to the nucleus / nucleolus ( siomi et al . although shuttling proteins have now been identified in a wide range of other viral and cellular proteins , multimerization may distinguish rex from other shuttling proteins that are not involved in rna export since their multimerization is not necessarily required for their functioning ( weichselbraun et al . a detailed understanding of rex shuttling was obtained from studies that identified the cellular factors binding to the functional domains , and the cellular machinery involved in protein transport into and out of the nucleus ( figure 3 ) . transport of all macromolecules occurs via nuclear pore complexes ( npcs ) , which comprise at least 50 different proteins , termed nucleoporins ( doye and hurt , 1997 ) . the import of proteins containing an nls is mediated by the importin family of import receptors , which have affinity for nucleoporins , and the gtp - bound status of ran ( rangtp ; mattaj and englmeier , 1998 ) . rangtp reflects the nuclear localization of chromatin binding protein , rcc1 , which specifically catalyzes the exchange of guanine nucleotides on ran , and rangdp , the predominant form of ran in the cytoplasm , reflects the cytoplasmic location of a gtpase - activating protein , rangap . crm1 was originally identified as binding the nes of the human immunodeficiency virus ( hiv)-1 rev protein . moreover , crm1 directly binds to ran - binding protein 3 ( ranbp3 ) , which binds to rcc1 in a ran - dependent manner and increases the nucleotide exchange activity of rcc1 , resulting in high local concentrations of rangtp . consequently , ranbp3 acts as a scaffold protein through which the components of the export complex are concentrated around the rcc1 site , thereby promoting complex assembly . ranbp3 complex is translocated to the cytoplasm across the npc ; this occurs via possible hydrophobic interactions between hcrm1 and nucleoporins . following this , the rangtp within the complex is converted to rangdp by rangap and ranbp1 ( and probably ranbp2 ) , leading to the dissociation of the complex and release of the substrate into the cytoplasm ( askjaer et al . an important question is whether cellular cofactors , such as hcrm1 , are involved in the multimerization of rex proteins ( hakata et al . second , we found that the rna transport activity of rexm64 , which harbors a mutation in the multimerization domain , was partially restored by the overexpression of hcrm1 , and this coincided with the partial restoration of multimerization . , 1995 ) , taken together , these results suggest that the multimerization of rex in vivo requires both the intrinsic ability of rex to form multimers , and the hcrm1 protein . moreover , these results also suggest that the multimerization of rex is not a prerequisite for interaction with hcrm1 but , rather , that rex initially interacts with hcrm1 , leading to the multimerization of rex proteins in a step that is favored by its intrinsic capacity to form oligomers on rxre . , 2003 ) ; this domain is different from the region spanning aa 566720 that is involved in binding to the nes ( ossareh - nazari and dargemont , 1999 ) . these data suggest that crm1 not only functions as an export receptor but also participates in the formation of the rna export complex through a high - order interaction with rex . curiously , the crm1 region responsible for the interaction with ranbp3 , which comprises four residues ( aa 411 , 414 , 478 , and 484 ) , and the region responsible for rex multimerization form an overlapping domain ( hakata et al . appropriate animal models of htlv-1 infection would allow us to analyze the pathogenesis and oncogenesis of htlv-1-associated diseases , which could lead to the development of therapeutic and preventative measures . human t cell leukemia virus type 1 can infect a number of rat cell types , which indicates that rat cells possess the receptors for viral attachment and penetration ( li et al . identification of the blocking step and the responsible host factor(s ) could lead to the construction of transgenic rats that express the critical human factor(s ) , which are highly susceptible to htlv-1 infection . this notion was further supported by a study showing that a htlv-1-transformed rat cd4 + t cell line , which was a poor producer of htlv-1 , efficiently synthesized functional env and gag proteins , which were then fully processed , and produced infectious htlv-1 progeny viruses at the level comparable to htlv-1-transformed human t cells when it was transduced with a retroviral vector expressing hcrm1 at physiological levels ( zhang et al . rex proteins multimerize with help of human crm1 on htlv-1 rna to form transport complex in human cells , and then export it to the cytoplasm . transgenic ( tg ) rats were constructed using an artificial bacterial chromosome clone containing the entire regulatory and coding regions of the crm1 gene ( figure 5 ) , since the expression of the crm1 gene is elaborately regulated by a protein kinase c pathway during lymphocyte activation , initially in a post - transcriptional and subsequently in a transcriptional manner . htlv-1-infected t cell lines derived from these tg rats produced 100- to 10,000-fold more htlv-1 than t cells from wild - type rats , and the absolute levels of htlv-1 were similar to those produced by human t cells . therefore , the tg rat model forms the basis for an improved animal model for htlv-1 infection . human immunodeficiency virus-1 belongs to the subfamily of complex retroviruses and , like htlv-1 , also encodes accessory genes besides gag and env ; however , hiv-1 shows more complex patterns of mrna expression . as is the case for htlv-1 rex , the 9- and 4-kb viral rnas of hiv-1 require the viral protein rev for export ( cullen , 1991 ) . however , expression of 2 kb rnas is not augmented by rev , since the rev response element ( rre ) , an approximately 300 nucleotide region with a highly ordered stem - and - loop structure that functions as a rev binding site , is located in the env gene rather than the 3 ltr ( malim et al . , 1989 ) , including the virion constituents and infectivity enhancing and virion - releasing factors , all of which work toward the efficient production and dissemination of progeny virus . rev is a functional homolog of rex , although rev and rex do not show any homology at the amino acid level . however , they do have similar domain structures and function in a similar manner ( figures 2 and 3 ) , as exemplified by the fact that rex can replace rev ( ahmed et al . the basic domain rich in arginine residues , which maps between aa 35 and 50 in the 116 aa rev protein , serves as an nls , which recruits importin and binds to the rre ( daly et al . in vitro studies of the multimerization of recombinant rev proteins bound to the rre - containing rna defined two essential domains : one between aa 12 and 33 and one between aa 46 and 60 ( malim et al . , 1996 ) , but recent studies on the tertiary structure of rev indicate that the dimerization of rev induces conformational changes in the position of the arginine - rich domain to facilitate rna binding ( daugherty et al . rna bound to the rre augments further rev multimerization , recruiting up to eight rev molecules on the viral rna , which subsequently recruit several hcrm1 molecules ( mann et al . after the transport complex is formed , which comprises hiv-1 rna , rev , crm1 , and other cellular components , it moves to the nuclear pores , possibly via the actin filament network ( kimura et al . multiple crm1 molecules , which have affinity for nucleoporins , may be required to pass through the nuclear pore because the transport complex could be longer than the depth of the nuclear pore ; thus , the rear portion of the complex is still within the nucleus when the front portion reaches the cytoplasm . crm1 molecules associated with rev within the front portion of the ribonucleoprotein complex ( in the cytoplasm ) will dissociate due to the lack of rangtp ( fornerod et al . thus , additional crm1 molecules may be required to associate with the rear part of the rna complex to enable passage of the whole rna complex out of the nucleus . another dead box protein , ddx3 , which is located outside of the nuclear pore , associates with crm1 directly and facilitates the passage of the complex through the nuclear pore and the release of rna into the cytoplasm by inducing conformational changes in the ribonucleoprotein complex ( yedavalli et al . additionally , rip ( also named rab ) , which indirectly binds to rev and has affinity for nuclear pores , is thought to function in releasing the viral rna within the cytoplasm ( sanchez - velar et al . both inefficient branch point region and polypyrimidine tract are thought to be responsible for the low splicing efficiency of the hiv-1 tat / rev intron , which comprises most of the hiv-1 env - coding sequences ( staffa and cochrane , 1994 ) . two additional elements , an exon - splicing enhancer ( ese ) and an exon - splicing silencer ( ess ) , both of which modulate the overall efficiency of the 3 tat rev splice site , were identified within the 3 terminal exon ( staffa and cochrane , 1995 ) . in contrast to fully spliced mrna , which is dispersed throughout both the nucleus and cytoplasm , the location of unspliced gag and env rnas does not coincide with splicing factor sc35-containing nuclear speckles . deletion of the intron sequence containing the crs resulted in a shift from discrete regional localization within the nucleus to a diffuse localization throughout the cell . this sequestration of intron - containing rnas may be a prerequisite for subsequent export by rev . indeed , addition of the rre to -globin mrna , which contains efficient splice sites , does not make it responsive to rev unless the splice sites are replaced by more inefficient ones , or a crs is introduced into the mrna . current animal models of hiv-1 disease use non - human primates ( veazey et al . however , they also have significant shortcomings , such as limited availability and high cost ( for non - human primates ) , they are permissive only for related retroviruses , and they often show aberrant immune responses . studies on rodent cell - specific defects in the hiv-1 life cycle have facilitated the identification and characterization of host cell gene products that are essential for viral replication , and these may provide a molecular basis for generating fully permissive small animal models . , a single amino acid difference between human and mouse cyclint1 ( mcyct1 ) , which has a tyrosine at residue 261 in place of the cysteine in hcyct1 , caused almost complete loss of tat cofactor activity ( bieniasz et al . in contrast to the definitive results obtained for rex in rat cells , the existence of a profound block in rev function in rodent cells remains controversial , although a reduced level of the hiv-1 9 kb transcript has been reported ( bieniasz and cullen , 2000 ; keppler et al . however , malim et al found that the gag protein is still efficiently synthesized in murine cells , indicating that rev is functional , although it is not transported to the plasma membrane , resulting in defective virion formation . when gag mrna is transported via a different route using the cellular transporter , tap , instead of the rev crm1 system , gag protein was synthesized and transported to the surface membrane , leading to efficient virus production ( swanson et al . this indicates that the rna export step is a one of the species - specific barriers to hiv-1 propagation , and that the mode of mrna nucleocytoplasmic transport affects subsequent processes occurring in the cytoplasm . however , it increases markedly in rat macrophages and fibroblasts expressing hcrm1 ( but only marginally in rat t cells ) , which is in sharp contrast to cells transfected with hcyct1 ( okada et al . the amount of gag protein within the cells increased two to three times , which paralleled the increase in gag mrna in the cytoplasm , although the trafficking of gag to the plasma membrane did not increase . however , the number of viral particles shed into the culture medium increased 20- to 50-fold , accompanied by the increased processing of the p55gag precursor to fully matured gag proteins . even a protease - deficient viral construct that caused accumulation of the p55 gag precursor in the cytoplasm facilitated the release of viral - like particles in the presence of hcrm1 , suggesting increased budding at the plasma membrane . these facts suggest that incomplete formation of transport complexes in the nucleus may affect the mechanisms underlying particle formation ( figure 6 ; nagai - fukataki et al . indeed , the interaction between rev and the rre is essential for proper encapsidation of grna into virus particles ( blissenbach et al . moreover , hiv-1 gag proteins gain access to the nucleus because they have nlss ( bukrinsky et al . in rat cells hiv-1 genomic rna and unspliced mrna are inefficiently transported to the cytoplasm . although simple retroviruses , exemplified by the mason - pfizer monkey virus ( mpmv ) , encode only the gag , pol , and env genes located centrally between the two ltrs ( figure 1 ; goff , 2001 ) , they also require the means to circumvent cellular restrictions , since the gag coding region is located within the intron of the pre - mrna encoding the envelope proteins . to express the intron - containing gag mrna and grna , inefficient splicing is necessary ( as in the case of hiv-1 ) , which is demonstrated by the finding that the modification of their splice sites increases their efficiency , resulting in the over splicing of retroviral rnas , which renders the virus replication incompetent ( katz and skalka , 1990 ) . subsequently , a cellular protein called transporter associated with antigen processing ( tap ) was identified , which binds cte directly , and tap sequence similarity to yeast mex67 ( which is involved in mrna transport ) indicates that cte accesses the cellular mrna export pathway ( grter et al . release of viral rna into the cytoplasm is mediated by another rna helicase , dbp5 , which localizes outside the nuclear pores ( leblanc et al . rous sarcoma virus ( rsv ) , a simple avian retrovirus , contains direct repeat ( dr ) elements ( ogert et al . on the other hand , rsv gag is reported to gain access to the nucleus via its nls , which interacts with importin / , transportin sr , and importin 11 ( butterfield - gerson et al . , 2010 ) , and is exported via its nes , which is associated with crm1 ( scheifele et al . thus , it is conceivable that gag proteins drive the transport of viral particles formed in the nucleus . , 2009 ) , suggesting that gag may initially bind to grna in the nucleus . second , formation of gag : grna prevents the association between gag and importins , while the binding of gag to grna instead enhances its association with crm1 , consequently promoting the export of the gag grna complex to the cytoplasm ( gudleski et al . these facts suggest the intriguing possibility that gag mrna is transported through the tap pathway to be translated , whereas grna ( which appears to be indistinguishable from gag mrna ) , is exported by the gag detailed analyses on mouse mammary tumor virus ( mmtv ) and human endogenous retrovirus k ( herv - k ) that had been initially classified as simple retroviruses revealed doubly spliced mrnas as the case of complex retroviruses . moreover , such mrnas have been found to encode rev like proteins such as rem in mmtv ( indik et al . studies on rna transport of retroviruses originally stemmed from molecular biological interest on unusual function of rex and rev that break the cellular blockage against transport of intron - containing rna . a great deal of investigation not only elucidated their own mechanisms but also led discovery of the cellular nucleocytoplasmic transport system . moreover , recent studies showed that the mode of rna export influences subsequent events in the cytoplasm , consequently affecting whole process of viral production including the translation of the cognate mrna , transport of gag proteins to the plasma membrane , and the formation of virus particles . lastly , the discovery on the interaction between the viral and cellular rna transport machinery as a species - specific barrier of hiv-1 and htlv-1 forms the basis to construct convenient animal models of infection , which should facilitate development of more potent therapeutic and preventing means .

despite the laborious efforts of scientists and the pharmaceutical industry to develop new and more effective antimicrobial therapies , systemic infections remain a serious health issue even in well - developed and wealthy countries of the western hemisphere . this situation is at least partially a result of mechanisms that enable microorganisms to quickly modify their genomes and acquire resistance to newly developed antibiotics . therefore , the race between drug discovery and the lethal effects of virulent pathogens on millions of patients worldwide seems to be endless . the problems posed by infectious diseases have become even more serious as a result of the increasing number of patients who are receiving immunosuppressive treatment because of disseminated cancer or organ transplantation . in addition , the significant rise in average life expectancy in recent decades has contributed to an increase in the number of patients suffering from chronic diseases , which increase the vulnerability of these individuals to serious complications of infectious disease . under these circumstances , an ongoing discussion among scientists and physicians concerning infections and sepsis is both timely and necessary . in this review , we discuss the mechanisms that contribute to sepsis - associated activation of the complement system . complement constitutes a crucial line of defence against microbial invasion , but it is also often identified as an inducer of excessive inflammatory host responses , which are thought to increase mortality from sepsis [ 5 , 6 ] . the currently accepted definition of sepsis describes it as a systemic inflammatory response syndrome ( sirs ) induced by presumed or confirmed infection . sirs is defined on the basis of clinical criteria , which include body temperature , heart and respiratory rate , blood pc02 and white blood cell count . under unfavourable circumstances septic shockis described as a combination of sepsis - induced hypotension that is unresponsive to adequate fluid resuscitation and the presence of perfusion abnormalities . according to studies published in 2003 , the prevalence of sepsis in the united states was estimated to be approximately 750,000 cases per year . considering the steady rise in the number of septic patients diagnosed each year , it has been predicted that this number will reach over 1 million cases by 2020 . despite the decrease in sepsis mortality rates over the past 20 years , the increasing number of sepsis cases has resulted in a tripling of the actual number of sepsis - associated deaths , to 215,000 deaths per year ; remarkably , sepsis is responsible for 9.3% of all deaths in the united states . these frightening numbers clearly indicate that the threat of infectious disease is one of the main problems facing twenty - first century medicine . contrary to the popular understanding of sepsis biology , which links this syndrome to infection with bacteria , the aetiology of sepsis is actually variable and includes fungi , parasites and viruses as well as bacteria . sepsis development is often preceded by localized respiratory or abdominal infections , although other organ systems , including the urogenital tract , can also be a source . currently , gram - positive organisms outnumber gram - negative bacteria as causative microflora , and an increasing number of sepsis cases are associated with fungal infections . in a small but appreciable number of patients with a clinical presentation of sepsis , causative organisms can not be identified . however even in the absence of a clear aetiology , these individuals should be treated as septic patients . the key events in sepsis pathogenesis involve complex multidimensional pathogen - host interactions , which are not only responsible for the clinical manifestations of sepsis but also strongly contribute to the clinical course , prognosis and complications , and potentially to the associated mortality . therefore , the overall picture of a septic patient is influenced by various factors that include aetiology , preexisting clinical conditions ( comor - bidities ) , the extent of the inflammatory and immune responses to pathogen invasion , and the disturbances of homeostasis that are induced by pathogen- and host - derived factors . the reciprocal interactions among all these variables contribute to the complexity of sepsis pathophysiology and further complicate our efforts to gain insight into the mechanisms regulating the host response during sepsis . the challenges that have limited our understanding of these complex processes are well illustrated by the recent evolution of thought concerning the role of inflammatory reactions in adverse outcomes in septic patients . it has long been accepted that death from sepsis - associated multi - organ failure is the result of an excessive inflammatory response to pathogens . therefore , anti - inflammatory therapies have been seen as beneficial in decreasing the rate of sepsis - associated complications [ 1214 ] . however , virtually all clinical trials of anti - inflammatory therapies have failed to yield improved outcomes for septic patients . these disappointing results seem to be a consequence of inappropriately applying data obtained from animal studies or inadequate design of clinical trials . in some experiments , animals were infused with large doses of bacteria or bacterial products , which , as expected , induced a brisk inflammatory response . the factors contributing to this inflammation were found to be directly responsible for the death of experimental animals . however , the direct translation of results obtained from animal studies to the clinic led to the premature conclusion that anti - inflammatory treatment would be of benefit to all septic patients , regardless of the severity of their symptoms . this prediction did not take into account the fact that the character of sepsis - associated inflammatory reactions evolves with time and that the intense , dysregulated inflammation observed in severe sepsis is absent from patients with less pronounced symptoms . furthermore , inhibiting inflammatory reactions in the early phases of sepsis can , in fact , reduce the host 's capability to cope with the invading pathogens . obviously , the simple hypothesis that an excessive inflammatory reaction is the main factor responsible for an adverse outcome in sepsis must be reconsidered . the complement system is an important contributor to the inflammatory reaction that occurs during sepsis . in mammals , the complement system has three well - characterized initial pathways of activation : the classical , lectin and alternative pathways . although traditionally complement activation is attributed to the presence of invading pathogens , this process can also occur as a response to tissue damage . therefore , complement can be currently viewed as an alarm system , which is capable of recognizing structures ( danger - associated molecular patterns [ damps ] ) associated with a risk of the disturbance of homeostasis of either infectious or non - infectious origin . the classical pathway is activated by antibody - antigen complexes consisting of natural or immune response - elicited immunoglobulins bound to multivalent antigens on the surfaces of pathogens or altered host cells . the complement protein complex c1 ( consisting of c1q with two molecules of c1 r and two molecules of c1s ) binds to antibodies within these immune complexes , and this binding stimulates the c1s - mediated cleavage of the c4 complement component to c4a and c4b . in turn the cleavage of c4 by c1s also exposes the binding site for c2 , which , once bound , is also cleaved by c1s . these initial steps lead to the formation of the c3 convertase , c4b2a , on pathogen or host cell surfaces . c1q is a pattern recognition receptor that is capable of distinguishing between self and non - self antigens through association with pathogen - associated molecular patterns ( pamps ) . in addition , c1q serves as sensor of danger of non - infectious origin by binding to fragments of cellular or subcellular membranes ( e.g. mito - chondrial membranes ) and other modified host proteins and phospholipids [ 1821 ] . furthermore , c1q can activate the classical pathway through its interaction with pentraxin pattern recognition receptors , such as c - reactive protein ( crp ) and serum amyloid protein ( sap ) . c3 convertase composed of c4b and c2a fragments is also generated as a result of complement activation through the lectin pathway . this pathway is triggered when pattern recognition receptors such as mannose - binding lectin ( mbl ) or ficolins bind to pamps or apoptotic host cells . this binding activates mbl - associated serine proteases ( masps ) , which are known to exist in three forms ( masp1 , masp2 and masp3 ) . like c1s , masp2 cleaves c4 , leading to the formation of c3 convertase ( masp1 has also recently been shown to promote c4 cleavage through activation of masp2 ) . the convertases formed by the classical or lectin pathways cleave c3 , the central component of the complement system , into the anaphylatoxin c3a and the opsonin c3b . the resulting c3b binds to the c4b2a complex , creating the c5 convertase ( c4b2a3b ) , which then cleaves c5 into c5a ( a potent anaphylatoxin ) and c5b . in evolutionary terms , the alternative pathway is considered to be the oldest pathway of complement activation . activation of complement through this pathway leads to the formation of a c3 convertase that is significantly different from those formed via the classical and lectin pathways . the internal thioester of c3 is spontaneously hydrolysed ( referred to as the tickover of c3 ) at a slow rate , leading to the formation of c3(h20 ) , a conformationally altered form of c3 . the binding of c3(h20 ) to the complement protein factor b changes the conformation of factor b , making it susceptible to cleavage by the constitutively active serum protease factor d into ba and bb fragments . these reactions result in the formation of the alternative pathway c3 convertase ( c3(h20)bb ) . like the c4b2a convertase , this complex cleaves c3 into c3a and c3b , with small amounts of c3b binding to the hydroxyl or amino groups on susceptible surfaces ; several studies show spontaneous c3b deposition on microorganisms and tumour cells ( reviewed in ref . ) . surface - bound c3b can bind directly to factor b , and the factor b - c3b complexes can then be cleaved by factor d to form the alternative pathway c3 convertase ( c3bbb ) . second c3 convertase represents an amplification loop that results in further cleavage of c3 , which may augment the process of complement activation induced by the classical or lectin pathways . properdin ( p ) stabilizes the c3bbb convertase , and additional molecules of c3b resulting from c3 cleavage bind to this enzymatic complex , forming the alternative pathway c5 convertase , c3b3bbbr like the c5 convertase produced by the classical or lectin pathways , the alternative pathway c5 convertase can cleave c5 to yield c5a and c5b fragments . cleavage of c5 by the various convertases and the binding of c5b to c6 begin the downstream terminal pathway of complement . c5b6 binds to c7 , creating an amphiphilic complex that can be incorporated into the lipid bilayer of cell membranes . one c8 molecule is bound by one c5b-7 complex , which then binds one or more c9 molecules . the resulting c5b-9 complex , or membrane attack complex ( mac ) , creates a physical pore in the membrane that results in leakage and cell activation ( at sublethal doses ) or cell lysis . interestingly , these downstream events do not occur ( i.e. no mac is formed ) when complement is activated by crp . it is assumed that ligands bound by crp , such as cell wall components of streptococcus pneumonia , are opsonized by c3 fragments generated by crp - induced complement activation and are targeted for phagocytosis . many recently published studies have demonstrated that complement activation can also occur through mechanisms that differ in several aspects from the traditionally recognized pathways of complement activation . for example , a new immunoglobulin - independent mechanism for activation of the classical pathway during s. pneumoniae infection has been described . complement activation through this mechanism is a result of c1q binding to the c - type lectin sign - r1 . this is the first known example of a cell - surface lectin directly initiating the classical pathway . sign - r1 is expressed at high levels by macrophages of the spleen marginal zone and lymph nodes [ 31 , 32 ] and is the principal receptor for the s. pneumoniae capsular pneumococcal polysaccharide ( cps ) [ 33 , 34 ] . mice lacking sign - r1 are more susceptible to pneumococcal septicemia , have deficits in c3 catabolism when challenged with s. pneumoniae or cps , and lack proper localization of cps to follicular dendritic cells ( fdcs ) in the follicles of the white pulp of the spleen [ 30 , 33 , 34 ] . it has also been shown that cps does not bind to fdcs in c3-deficient mice , suggesting that both sign - r1 and c3 are necessary for cps binding to fdcs . thus , it appears that sign - r1 contributes to c3 fixation and , therefore , to the ability of the spleen to defend the host against certain encapsulated pathogens . it has also recently been shown that properdin is able to directly activate the alternative pathway of complement . individuals with properdin deficiency are more susceptible to infection by neisseria and suffer mortality rates of 4365% from resulting meningococcal disease . properdin was shown to bind directly to c3b in vitro , with formation of the c3bbbp convertase after treatment with factor b and factor d . indeed , properdin - treated n. gonhorrheae bound c3b and formed the alternative pathway convertase in the presence of factor b and factor d . this binding was likely through interaction with neisseria lipo - oligosaccharide ( los ) , a component of lipopolysaccharide ( lps ) found on other bacteria such as e. coli . however , the los component is normally masked in lps ; thus , properdin does not bind to lps - expressing bacteria . when e. coli strains that differed in their ability to bind properdin , due to their various mutant forms of lps , were tested , it was seen that those with stronger properdin binding had faster complement activation ( including c3b deposition and conversion to ic3b ) through the alternative pathway . similarly , lps from different types of bacteria had various abilities to bind to human properdin . in another study , it was observed that alternative pathway complement activation in sera from properdin - deficient mice was not induced by lps from salmonella typhosa , salmonella minnesota ( s ) or e. coli . interestingly , both los and lps were unable to induce complement activation in properdin - deficient sera . however , injection of los caused systemic complement activation in wild - type but not properdin - deficient mice , while lps - induced systemic complement activation was still seen ( though at reduced levels ) in properdin - deficient animals . thus , los appears to activate the alternative pathway in vivo , likely through interaction with properdin , whereas lps activates complement through both alternative pathway - dependent and -independent mechanisms . finally , recent work has demonstrated that neutrophil - derived properdin binds to early apoptotic t cells and initiates c3b deposition through alternative pathway activation . properdin - tagged t cells can also be taken up by phagocytes without prior complement activation , through the direct interaction of properdin with the phagocytic cells . all of these studies point to an important role for properdin not only in supporting alternative pathway activation of complement by stabilizing the convertase , but also in directly activating this pathway in response to some pathogens . a growing body of research has also suggested that the complement cascade can be initiated by factors that contribute to hemostasis such as platelets and coagulation or fibrynolytic factors [ 3941 ] . thrombin has been shown to indirectly induce complement activation in rabbits via the classical pathway by activating platelets [ 4244 ] . this mechanism may be important for the pathogenesis of sepsis , because platelets are activated during sepsis ; furthermore , it is known that once platelets are activated , they release a serine / threonine mn / ca / mg - dependent protein kinase ( likely a casein kinase type i ) that phosphorylates residues of the c3d region of c3 . thus , both c3 and several of its degradation fragments ( c3b , ic3b and c3dg ) are phosphorylated . the phosphorylation of c3b prevents its cleavage into ic3b by factor i , resulting in prolonged complement activation . therefore , parallel activation of complement and platelets during sepsis can lead to phosphorylation of activated complement components , a modification that may be important for regulating their activity . activation of complement can occur as a result of a direct cleavage of c3 or c5 , without the involvement of convertases , through what has recently been termed the extrinsic protease pathway [ 5 , 46 ] . various in vitro studies have indicated that factors related to the kinin and coagulation cascades , as well as to fibrinolysis , are able to cleave complement proteins . kallikrein isolated from rabbit plasma can cleave c5 , resulting in the generation of active c5a , which induces neutrophil chemotaxis . in addition , the hageman factor fragment is capable of inducing complement activation through direct interaction with c1 , which activates the c1r and c1s subunits . incubation of thrombin with c5 leads to c5a generation , and thrombin is also known to stimulate the cleavage of c5 into c5a in vivo . during acute lung inflammation , c5a is produced in c3-deficient mice , which lack c3 cleavage products ( i.e. c3b ) . it has also been shown that in the absence of c3 , thrombin becomes the dominant enzyme during the inflammatory process that generates biologically active c5a . thus , in addition to playing an indirect role in regulating the complement pathway through platelet activation , thrombin can directly cleave c5 to initiate the downstream terminal pathway of complement ( a ) c1 can bind to various factors to initiate the classical pathway ( cp ) . these include , most commonly antibody - antigen complexes , but also danger - associated molecular patterns ( damps ) such as membrane fragments and proteins associated with tissue damage . c1 also binds to c - reactive protein ( crp ) and serum amyloid protein ( sap ) , which recognize pathogen - associated molecular patterns ( pamps ) present on the surfaces of many pathogens . binding by the c1 q subunit of c1 activates c1 r and c1 s , which results in the cleavage of c2 and c4 by c1s ( i ) . binding of mannose - binding lectin ( mbl ) or ficolins to pamps or apoptotic host cells activates mbl - associated serine proteases ( masps ) , which cleave c4 ( ii ) . the result of activation through either pathway is that c4a is released and c2a and c4b form the cp c3 convertase on the surface of the pathogen or apoptotic cell ( iii ) , resulting in cleavage of c3 into c3a and c3b fragments . the alternative pathway ( ap ) can be initiated by spontaneous hydrolysis of c3 ( tickover ) to form c3(h20 ) ( iv ) . c3(h20 ) binds to factor b ( fb ) , which is cleaved by factor d ( fd ) into ba and bb fragments , resulting in formation of the initial ap c3 convertase ( v ) . like the cp convertase the anaphylatoxin c3a induces chemotaxis and inflammation , while some c3b binds to the cell surface ( opsonization ) ( vi ) , which promotes phagocytosis by cr3-bearing cells . surface - bound c3b binds to factor b , and the resulting complex is cleaved by factor d to form the ap c3 convertase ( vii ) . this convertase is stabilized by the binding of properdin ( p ) . through an amplification loop , the ap c3 convertase cleaves more c3 to augment complement activation induced by the classical or lectin pathways ( viii ) . ( b ) after c3 is cleaved by the cp c3 convertase , c3b binds to the cell surface but also can bind to the c4b2a complex to form the cp c5 convertase ( i ) . similarly , c3b resulting from activity of the ap c3 convertase can bind to c3bbbp to form the ap c5 convertase ( ii ) . these convertases cleave c5 , leading to the generation of c5a , which acts similarly to c3a to promote inflammation and chemotaxis , and c5b . c5b is bound by c6 and c7 , which can insert into the cell membrane and bind c8 . one or multiple c9 molecules then bind , resulting in formation of the membrane attack complex ( mac ) ( iii ) . ( c ) c1 can bind to sign - r1 on marginal zone macrophages to result in formation of the cp c3 convertase , and subsequent c3 cleavage ( i ) . p can bind directly to c3b through interaction with neisseria lipo - oligosaccharide ( los ) and , in the presence of fb and fd , can form the ap c3 convertase ( ii ) . p from neutrophils can bind to early apoptotic t cells to activate the ap and initiate c3b deposition , facilitating phagocytosis ( iii ) . phagocytosis can also be promoted through a direct interaction of p with phagocytes ( without complement activation ) when it binds to t cells ( iv ) . ( d ) c1 can interact with the hageman factor fragment , which results in complement activation through the cp ( i ) . thrombin induces c3 cleavage through the cp through the activation of platelets ( ii ) . activated platelets release a serine / threonine ( ser / thr ) kinase that can phosphorylate ( p ) c3b to block its cleavage into ic3b by factor i ( fl ) ( iii ) . kallikrein and thrombin can directly cleave both c3 ( v ) and c5 ( vi ) to generate cleavage products . at first glance , the role of complement in sepsis pathogenesis might appear ambiguous or paradoxical : on the one hand , c3 deficiency , which eliminates most complement effector functions , clearly increases sepsis - associated mortality in animals [ 5153 ] ; these studies have emphasized the importance of complement as a defence mechanism against invading microbes . conversely , other data have indicated that inhibition of c5a signalling improves the survival of experimental animals . this apparent inconsistency between various studies may actually be an indication of the diversity of complement functions during the development and progression of sepsis . during the early stages of widespread bacterial infections , complement 's pro - inflammatory and antimicrobial properties are critical for protecting the host from the detrimental effects of an uncontrolled spread of microbes , whereas in the later stages of sepsis development , c5a , in concert with cytokines , contributes to the development of multi - organ failure and circulatory insufficiency . the complement system , which was originally viewed as an arm of humoural immunity , is now perceived as a central constituent of innate immunity , defending the host against infections , orchestrating inflammatory responses and connecting the innate and adaptive immune responses . this broad spectrum of complement activities , together with the abundance of complement proteins in the plasma , enables this system to cope with local and systemic infections . in addition to plasma proteins that interact with various cells and mediators of the immune system , several membrane - bound regulators and receptors constitute an efficient regulatory module of the complement system that is designed to limit the activation of complement to pathogen surfaces or altered host cells . the anti - microbial properties of complement can be divided into three distinct categories : ( i ) opsonization and subsequent killing of microorganism by phagocytes , ( ii ) lysis of pathogens ( neisseria species ) and ( iii ' ) coordination of inflammatory events associated with the response to infection . opsonization involves the coating of bacterial surfaces with complement proteins such as c3b and ic3b . these cleavage products of c3 are covalently bound to the pathogen surfaces and act as ligands for receptors expressed by phagocytes . engagement of these receptors with their ligands significantly facilitates the phagocytosis and killing of bacteria by neutrophils and macrophages . various defects in opsonization are known to contribute to an increased susceptibility to infections caused by pyogenic bacteria such as haemophilus influenzae and s. pneumoniae . lysis of pathogens occurs as a result of sequential activation of complement proteins on pathogen surfaces , forming the mac , which then creates pores in the bacterial cell wall and ultimately leads to bacterial lysis . although direct lysis of pathogens by mac is a rare form of defence against invading pathogens , it has been shown that inherited deficiencies in components of the mac are associated with an increased susceptibility to neisserial diseases , particularly neisseria meningitides . complement - mediated lysis is a major mechanism for neutralizing neisseria species , which are capable of intracellular survival . the complement system plays an invaluable role in promoting and coordinating the inflammatory process that is triggered in response to pathogens . the anaphylatoxins c3a and c5a are actively involved in the regulation of various critical events during an inflammatory response , such as changes in vascular flow and blood vessel calibre , increased vascular permeability , and leukocyte extravasation and chemotaxis . these processes are essential for recruiting and activating the cells that are involved in the innate immune response , including neutrophils , monocytes and macrophages . anaphylatoxins not only directly coordinate cellular inflammatory responses by binding to the reciprocal receptors expressed by peripheral blood leukocytes or macrophages but also provide coordination in an indirect manner by controlling cytokine production and secretion . . the essential goal of inflammatory reactions is to eliminate hazardous factors , which in the case of infection are microbes . the contribution of the complement system is necessary to achieving this goal . when invading pathogens are successfully eliminated through innate and/or adaptive immune responses , acute inflammation subsides . regulatory complement proteins can immediately shut down the activation process in the absence of threatening infection . however , under unfavourable circumstances , pathogens can escape surveillance by the immune system . in these situations , frustrated inflammatory response continues its efforts to neutralize the danger , leading to the destruction of host tissue and further exacerbation of inflammation . this large - scale inflammatory response , currently referred to as sirs , is a hallmark of sepsis . when this response occurs , a steady activation of complement and the release of other inflammatory mediators , combined with an inability to destroy pathogens , creates a vicious circle leading ultimately to multiple organ dysfunction and immunosuppression . disturbances in various organ and circulatory system functions accompany severe sepsis . at this stage , the decline in the overall condition of the patient and a decrease in the patient 's ability to cope with the existing infection together facilitate the spread of microbes , contributing to a worsening of the individual 's clinical status . with severe sepsis , a crucial requirement for successful sepsis therapy is the capacity to break this vicious cycle of progressive infection and exacerbating inflammation . the most effective way to do so is through elimination of the infectious agent . in many cases , therapy based on the empiric selection of antibiotics is sufficient to prevent severe sepsis complications . however , early surgical intervention is also critical in controlling and eliminating the focus of infection if sepsis is related to perforation or obstruction of the gastrointestinal , biliary or urinary tract , or if the abscess , which is the source of pathogens , requires drainage . extensive clinical experience has confirmed that early intervention is essential for successful therapy . in order to have the best chance of curing septic patients , currently , therapeutic interventions targeting the complex network of inflammatory and immune responses appear to be a risky approach , given that inhibition of these responses may significantly impair the ability of the host to naturally cope with infections . however , the combination of appropriate antibiotic therapy introduced early during sepsis combined with inhibition of inflammation in the late stages of the disease process appears to be a promising mode of treatment of septic patients in the near future . although various factors influence the activation of complement during sepsis , the specific aetiology of the syndrome in a particular individual has a decisive impact on the initiation of the complement cascade . in the last three decades , gram - positive microorganisms have become the leading etiological factors in sepsis , with staphylococcus aureus and s. pneumoniae being the most commonly isolated pathogens . in the group of gram - negative bacteria , e. coli , recently published data from human studies have indicated that complement activation is initiated differently by gram - positive and gram - negative bacteria that cause bacterial septicemia . in patients with confirmed gram - positive bacteria - induced sepsis , there is a significant consumption of c1q , but not mbl , in the acute phase of the disease ; the opposite pattern is seen in sepsis caused by gram - negative bacteria . these data suggest that the classical pathway of complement activation plays an essential role in the sepsis induced by gram - positive pathogens . c1q can bind directly to bacterial surfaces or to immune complexes formed by bacterial antigens and antibodies . in the case of gram - negative pathogens , the activation of complement is induced in sepsis through the binding of mbl ( the lectin pathway ) predominantly to lps , which is a component of the outer cell wall of gram - negative bacteria . this relatively clear picture becomes obscured when we consider other reports relating to the mechanisms of complement activation by particular species of bacteria . gram - positive s. aureus , which has developed numerous mechanisms to evade complement attack , has been reported to activate the complement cascade through the lectin pathway , with no involvement of alternative pathway amplification . in line with these findings , it has also been reported that l - ficolin , which initiates the lectin pathway , binds to lipoteichoic acid ( lta ) , a cell wall component found in a majority of gram - positive bacteria . other studies , however , have suggested that the lectin pathway preferentially facilitates complement - mediated opsonophagocytosis of the fungus candida albicans , but not bacteria . both gram - positive bacterial strains ( e.g. s. aureus and s. pneumoniae ) and gram - negative e. coli have been shown to activate the c1q - dependent classical pathway . in accordance with these findings , the classical pathway of complement activation has been shown to be essential for innate immune responses to s. pneumoniae . interestingly , studies using various complement - deficient mouse strains and mice lacking secretory igm have demonstrated that the proportion of a population of s. pneumoniae bound by c3 depends on the classical pathway , whereas the intensity of this binding is alternative pathway - dependent . the classical pathway of complement activation is initiated by the binding of natural igm to s. pneumoniae surfaces . the lack of igm in deficient mice results in rapidly progressing sepsis and alterations in macrophage function . however , it appears that an antibody - independent mechanism of classical pathway activation by s. pneumoniae is equally important . as described earlier , it has recently been reported that the direct binding of c1q to sign - r1 , a c - type lectin that is an uptake receptor expressed at high levels by spleen marginal zone and lymph node macrophages , contributes significantly to complement activation and opsonization of s. pneumoniae by c3 cleavage products . a detailed discussion of the large number of other studies investigating the mechanisms of complement activation by various pathogens goes far beyond the scope of this review . however , the overall picture that emerges from these investigations indicates the enormous diversity in the mechanisms of complement activation that contribute to the response to infection . it appears that the simple assignment of a single pathway to particular pathogen is an inappropriate approach that can lead to an oversimplification of this process . in addition , activation of complement can also be enhanced in a pathogen - independent manner by acute phase proteins , whose levels increase during sepsis . for example , as mentioned earlier , the acute phase protein crp can bind to c1q , and this interaction triggers the classical pathway . sepsis - associated coagulopathy can occur in various forms , including disseminated intravascular coagulation ( dic ) , a particularly severe complication that significantly and adversely affects the prognosis in sepsis . dic is a condition in which an increased tendency toward coagulation leads to the formation of multiple thrombi in the microcirculation . this massive formation of thrombi is responsible for the consumption of clotting factors , which in turn leads to haemorrhagic diathesis . clinical studies have suggested that dic contributes to the development of multi - organ dysfunction , ultimately increasing mortality in patients with sepsis . in addition , treatment regimens that attenuate coagulation have been postulated to improve overall survival . increased thrombogenicity in sepsis is a result of the upregulation of tissue factor ( tf ) expression on circulating leukocytes and endothelial cells in the blood . under normal physiological conditions , contact between tf and blood clotting factors is largely prevented by anatomical barriers , and leukocytes and endothelial cells do not express tf . however , when these cells are stimulated by inflammatory mediators , including complement effectors , they become key contributors to the increased tendency toward clotting in various clinical conditions that are associated with inflammation . several studies have demonstrated that complement activation can be triggered by the activation of the coagulation or contact systems , as described in the previous section of this review . thus , cross - talk between the coagulation and complement systems represents another way of amplifying complement activation in sepsis . the existence of rare human complement deficiencies and the results of various animal studies have established an essential role for the complement system in the defence against infections , including those that may eventually lead to sepsis . complement creates several barriers that prevent the spread of microorganisms and contribute to their clearance . at virtually every step in the development of the infectious process , including the activation of adaptive immunity , complement proteins are engaged in the battle against pathogens . these multiple tasks require prompt and efficient activation of the complement cascade in coordination with other immune mechanisms , resulting in the generation of a number of effector molecules that participate in the response to infection . this review has highlighted how such activation is achieved by multiple pathways acting in concert to ensure both the development of a response that is proportional to the scale of danger and a timely delivery of complement effectors to the sites of infection . it also appears that in addition to the previously well - studied pathways of complement activation , various newly discovered mechanisms can either initiate or amplify the activation of the complement cascade in sepsis .

the complement system is one of the key players in the defence against infections . its activation during the innate immune response leads to the generation of several proteins that contribute to the lysis and opsonization of microorganisms , regulate inflammatory reactions and bridge innate immunity with the subsequent adaptive immune response . complement is also activated in overwhelming bacterial infections that lead to sepsis , and its protective functions play a role in this frequently lethal disorder . however , despite its role in protection , complement can also contribute to the development of severe complications that significantly worsen the prognosis of septic patients . therefore , an understanding of the mechanisms involved in the activation of complement during sepsis is essential to our efforts to introduce rational therapies targeting complement to the treatment of patients suffering from this condition . this review presents a current view of the mechanisms involved in the activation of complement in sepsis , in the context of the multiple interactions between complement and other biological systems that are involved in the pathogenesis of this disorder .

Introduction Sepsis terminology and basic facts The pathways of complement activation The role of the complement system in the pathogenesis of sepsis Aetiology-dependent mechanisms of complement activation in sepsis Sepsis-associated coagulopathy and complement activation Concluding remarks

therefore , the race between drug discovery and the lethal effects of virulent pathogens on millions of patients worldwide seems to be endless . the problems posed by infectious diseases have become even more serious as a result of the increasing number of patients who are receiving immunosuppressive treatment because of disseminated cancer or organ transplantation . in addition , the significant rise in average life expectancy in recent decades has contributed to an increase in the number of patients suffering from chronic diseases , which increase the vulnerability of these individuals to serious complications of infectious disease . under these circumstances , an ongoing discussion among scientists and physicians concerning infections and sepsis is both timely and necessary . in this review , we discuss the mechanisms that contribute to sepsis - associated activation of the complement system . complement constitutes a crucial line of defence against microbial invasion , but it is also often identified as an inducer of excessive inflammatory host responses , which are thought to increase mortality from sepsis [ 5 , 6 ] . considering the steady rise in the number of septic patients diagnosed each year , it has been predicted that this number will reach over 1 million cases by 2020 . despite the decrease in sepsis mortality rates over the past 20 years , the increasing number of sepsis cases has resulted in a tripling of the actual number of sepsis - associated deaths , to 215,000 deaths per year ; remarkably , sepsis is responsible for 9.3% of all deaths in the united states . these frightening numbers clearly indicate that the threat of infectious disease is one of the main problems facing twenty - first century medicine . contrary to the popular understanding of sepsis biology , which links this syndrome to infection with bacteria , the aetiology of sepsis is actually variable and includes fungi , parasites and viruses as well as bacteria . sepsis development is often preceded by localized respiratory or abdominal infections , although other organ systems , including the urogenital tract , can also be a source . in a small but appreciable number of patients with a clinical presentation of sepsis , causative organisms can not be identified . however even in the absence of a clear aetiology , these individuals should be treated as septic patients . the key events in sepsis pathogenesis involve complex multidimensional pathogen - host interactions , which are not only responsible for the clinical manifestations of sepsis but also strongly contribute to the clinical course , prognosis and complications , and potentially to the associated mortality . therefore , the overall picture of a septic patient is influenced by various factors that include aetiology , preexisting clinical conditions ( comor - bidities ) , the extent of the inflammatory and immune responses to pathogen invasion , and the disturbances of homeostasis that are induced by pathogen- and host - derived factors . the reciprocal interactions among all these variables contribute to the complexity of sepsis pathophysiology and further complicate our efforts to gain insight into the mechanisms regulating the host response during sepsis . the challenges that have limited our understanding of these complex processes are well illustrated by the recent evolution of thought concerning the role of inflammatory reactions in adverse outcomes in septic patients . however , virtually all clinical trials of anti - inflammatory therapies have failed to yield improved outcomes for septic patients . however , the direct translation of results obtained from animal studies to the clinic led to the premature conclusion that anti - inflammatory treatment would be of benefit to all septic patients , regardless of the severity of their symptoms . this prediction did not take into account the fact that the character of sepsis - associated inflammatory reactions evolves with time and that the intense , dysregulated inflammation observed in severe sepsis is absent from patients with less pronounced symptoms . furthermore , inhibiting inflammatory reactions in the early phases of sepsis can , in fact , reduce the host 's capability to cope with the invading pathogens . obviously , the simple hypothesis that an excessive inflammatory reaction is the main factor responsible for an adverse outcome in sepsis must be reconsidered . the complement system is an important contributor to the inflammatory reaction that occurs during sepsis . in mammals , the complement system has three well - characterized initial pathways of activation : the classical , lectin and alternative pathways . although traditionally complement activation is attributed to the presence of invading pathogens , this process can also occur as a response to tissue damage . therefore , complement can be currently viewed as an alarm system , which is capable of recognizing structures ( danger - associated molecular patterns [ damps ] ) associated with a risk of the disturbance of homeostasis of either infectious or non - infectious origin . the complement protein complex c1 ( consisting of c1q with two molecules of c1 r and two molecules of c1s ) binds to antibodies within these immune complexes , and this binding stimulates the c1s - mediated cleavage of the c4 complement component to c4a and c4b . in turn the cleavage of c4 by c1s also exposes the binding site for c2 , which , once bound , is also cleaved by c1s . these initial steps lead to the formation of the c3 convertase , c4b2a , on pathogen or host cell surfaces . c3 convertase composed of c4b and c2a fragments is also generated as a result of complement activation through the lectin pathway . like c1s , masp2 cleaves c4 , leading to the formation of c3 convertase ( masp1 has also recently been shown to promote c4 cleavage through activation of masp2 ) . the convertases formed by the classical or lectin pathways cleave c3 , the central component of the complement system , into the anaphylatoxin c3a and the opsonin c3b . activation of complement through this pathway leads to the formation of a c3 convertase that is significantly different from those formed via the classical and lectin pathways . the binding of c3(h20 ) to the complement protein factor b changes the conformation of factor b , making it susceptible to cleavage by the constitutively active serum protease factor d into ba and bb fragments . these reactions result in the formation of the alternative pathway c3 convertase ( c3(h20)bb ) . surface - bound c3b can bind directly to factor b , and the factor b - c3b complexes can then be cleaved by factor d to form the alternative pathway c3 convertase ( c3bbb ) . second c3 convertase represents an amplification loop that results in further cleavage of c3 , which may augment the process of complement activation induced by the classical or lectin pathways . many recently published studies have demonstrated that complement activation can also occur through mechanisms that differ in several aspects from the traditionally recognized pathways of complement activation . for example , a new immunoglobulin - independent mechanism for activation of the classical pathway during s. pneumoniae infection has been described . mice lacking sign - r1 are more susceptible to pneumococcal septicemia , have deficits in c3 catabolism when challenged with s. pneumoniae or cps , and lack proper localization of cps to follicular dendritic cells ( fdcs ) in the follicles of the white pulp of the spleen [ 30 , 33 , 34 ] . thus , it appears that sign - r1 contributes to c3 fixation and , therefore , to the ability of the spleen to defend the host against certain encapsulated pathogens . properdin - tagged t cells can also be taken up by phagocytes without prior complement activation , through the direct interaction of properdin with the phagocytic cells . all of these studies point to an important role for properdin not only in supporting alternative pathway activation of complement by stabilizing the convertase , but also in directly activating this pathway in response to some pathogens . a growing body of research has also suggested that the complement cascade can be initiated by factors that contribute to hemostasis such as platelets and coagulation or fibrynolytic factors [ 3941 ] . this mechanism may be important for the pathogenesis of sepsis , because platelets are activated during sepsis ; furthermore , it is known that once platelets are activated , they release a serine / threonine mn / ca / mg - dependent protein kinase ( likely a casein kinase type i ) that phosphorylates residues of the c3d region of c3 . therefore , parallel activation of complement and platelets during sepsis can lead to phosphorylation of activated complement components , a modification that may be important for regulating their activity . activation of complement can occur as a result of a direct cleavage of c3 or c5 , without the involvement of convertases , through what has recently been termed the extrinsic protease pathway [ 5 , 46 ] . kallikrein isolated from rabbit plasma can cleave c5 , resulting in the generation of active c5a , which induces neutrophil chemotaxis . incubation of thrombin with c5 leads to c5a generation , and thrombin is also known to stimulate the cleavage of c5 into c5a in vivo . it has also been shown that in the absence of c3 , thrombin becomes the dominant enzyme during the inflammatory process that generates biologically active c5a . thus , in addition to playing an indirect role in regulating the complement pathway through platelet activation , thrombin can directly cleave c5 to initiate the downstream terminal pathway of complement ( a ) c1 can bind to various factors to initiate the classical pathway ( cp ) . like the cp convertase the anaphylatoxin c3a induces chemotaxis and inflammation , while some c3b binds to the cell surface ( opsonization ) ( vi ) , which promotes phagocytosis by cr3-bearing cells . similarly , c3b resulting from activity of the ap c3 convertase can bind to c3bbbp to form the ap c5 convertase ( ii ) . these convertases cleave c5 , leading to the generation of c5a , which acts similarly to c3a to promote inflammation and chemotaxis , and c5b . one or multiple c9 molecules then bind , resulting in formation of the membrane attack complex ( mac ) ( iii ) . ( c ) c1 can bind to sign - r1 on marginal zone macrophages to result in formation of the cp c3 convertase , and subsequent c3 cleavage ( i ) . p can bind directly to c3b through interaction with neisseria lipo - oligosaccharide ( los ) and , in the presence of fb and fd , can form the ap c3 convertase ( ii ) . ( d ) c1 can interact with the hageman factor fragment , which results in complement activation through the cp ( i ) . thrombin induces c3 cleavage through the cp through the activation of platelets ( ii ) . at first glance , the role of complement in sepsis pathogenesis might appear ambiguous or paradoxical : on the one hand , c3 deficiency , which eliminates most complement effector functions , clearly increases sepsis - associated mortality in animals [ 5153 ] ; these studies have emphasized the importance of complement as a defence mechanism against invading microbes . this apparent inconsistency between various studies may actually be an indication of the diversity of complement functions during the development and progression of sepsis . during the early stages of widespread bacterial infections , complement 's pro - inflammatory and antimicrobial properties are critical for protecting the host from the detrimental effects of an uncontrolled spread of microbes , whereas in the later stages of sepsis development , c5a , in concert with cytokines , contributes to the development of multi - organ failure and circulatory insufficiency . the complement system , which was originally viewed as an arm of humoural immunity , is now perceived as a central constituent of innate immunity , defending the host against infections , orchestrating inflammatory responses and connecting the innate and adaptive immune responses . this broad spectrum of complement activities , together with the abundance of complement proteins in the plasma , enables this system to cope with local and systemic infections . in addition to plasma proteins that interact with various cells and mediators of the immune system , several membrane - bound regulators and receptors constitute an efficient regulatory module of the complement system that is designed to limit the activation of complement to pathogen surfaces or altered host cells . the anti - microbial properties of complement can be divided into three distinct categories : ( i ) opsonization and subsequent killing of microorganism by phagocytes , ( ii ) lysis of pathogens ( neisseria species ) and ( iii ' ) coordination of inflammatory events associated with the response to infection . lysis of pathogens occurs as a result of sequential activation of complement proteins on pathogen surfaces , forming the mac , which then creates pores in the bacterial cell wall and ultimately leads to bacterial lysis . although direct lysis of pathogens by mac is a rare form of defence against invading pathogens , it has been shown that inherited deficiencies in components of the mac are associated with an increased susceptibility to neisserial diseases , particularly neisseria meningitides . the complement system plays an invaluable role in promoting and coordinating the inflammatory process that is triggered in response to pathogens . the anaphylatoxins c3a and c5a are actively involved in the regulation of various critical events during an inflammatory response , such as changes in vascular flow and blood vessel calibre , increased vascular permeability , and leukocyte extravasation and chemotaxis . these processes are essential for recruiting and activating the cells that are involved in the innate immune response , including neutrophils , monocytes and macrophages . the essential goal of inflammatory reactions is to eliminate hazardous factors , which in the case of infection are microbes . the contribution of the complement system is necessary to achieving this goal . regulatory complement proteins can immediately shut down the activation process in the absence of threatening infection . in these situations , frustrated inflammatory response continues its efforts to neutralize the danger , leading to the destruction of host tissue and further exacerbation of inflammation . when this response occurs , a steady activation of complement and the release of other inflammatory mediators , combined with an inability to destroy pathogens , creates a vicious circle leading ultimately to multiple organ dysfunction and immunosuppression . at this stage , the decline in the overall condition of the patient and a decrease in the patient 's ability to cope with the existing infection together facilitate the spread of microbes , contributing to a worsening of the individual 's clinical status . however , early surgical intervention is also critical in controlling and eliminating the focus of infection if sepsis is related to perforation or obstruction of the gastrointestinal , biliary or urinary tract , or if the abscess , which is the source of pathogens , requires drainage . in order to have the best chance of curing septic patients , currently , therapeutic interventions targeting the complex network of inflammatory and immune responses appear to be a risky approach , given that inhibition of these responses may significantly impair the ability of the host to naturally cope with infections . however , the combination of appropriate antibiotic therapy introduced early during sepsis combined with inhibition of inflammation in the late stages of the disease process appears to be a promising mode of treatment of septic patients in the near future . although various factors influence the activation of complement during sepsis , the specific aetiology of the syndrome in a particular individual has a decisive impact on the initiation of the complement cascade . in the last three decades , gram - positive microorganisms have become the leading etiological factors in sepsis , with staphylococcus aureus and s. pneumoniae being the most commonly isolated pathogens . in patients with confirmed gram - positive bacteria - induced sepsis , there is a significant consumption of c1q , but not mbl , in the acute phase of the disease ; the opposite pattern is seen in sepsis caused by gram - negative bacteria . these data suggest that the classical pathway of complement activation plays an essential role in the sepsis induced by gram - positive pathogens . in the case of gram - negative pathogens , the activation of complement is induced in sepsis through the binding of mbl ( the lectin pathway ) predominantly to lps , which is a component of the outer cell wall of gram - negative bacteria . this relatively clear picture becomes obscured when we consider other reports relating to the mechanisms of complement activation by particular species of bacteria . other studies , however , have suggested that the lectin pathway preferentially facilitates complement - mediated opsonophagocytosis of the fungus candida albicans , but not bacteria . in accordance with these findings , the classical pathway of complement activation has been shown to be essential for innate immune responses to s. pneumoniae . as described earlier , it has recently been reported that the direct binding of c1q to sign - r1 , a c - type lectin that is an uptake receptor expressed at high levels by spleen marginal zone and lymph node macrophages , contributes significantly to complement activation and opsonization of s. pneumoniae by c3 cleavage products . a detailed discussion of the large number of other studies investigating the mechanisms of complement activation by various pathogens goes far beyond the scope of this review . however , the overall picture that emerges from these investigations indicates the enormous diversity in the mechanisms of complement activation that contribute to the response to infection . it appears that the simple assignment of a single pathway to particular pathogen is an inappropriate approach that can lead to an oversimplification of this process . in addition , activation of complement can also be enhanced in a pathogen - independent manner by acute phase proteins , whose levels increase during sepsis . sepsis - associated coagulopathy can occur in various forms , including disseminated intravascular coagulation ( dic ) , a particularly severe complication that significantly and adversely affects the prognosis in sepsis . dic is a condition in which an increased tendency toward coagulation leads to the formation of multiple thrombi in the microcirculation . this massive formation of thrombi is responsible for the consumption of clotting factors , which in turn leads to haemorrhagic diathesis . clinical studies have suggested that dic contributes to the development of multi - organ dysfunction , ultimately increasing mortality in patients with sepsis . increased thrombogenicity in sepsis is a result of the upregulation of tissue factor ( tf ) expression on circulating leukocytes and endothelial cells in the blood . however , when these cells are stimulated by inflammatory mediators , including complement effectors , they become key contributors to the increased tendency toward clotting in various clinical conditions that are associated with inflammation . several studies have demonstrated that complement activation can be triggered by the activation of the coagulation or contact systems , as described in the previous section of this review . the existence of rare human complement deficiencies and the results of various animal studies have established an essential role for the complement system in the defence against infections , including those that may eventually lead to sepsis . complement creates several barriers that prevent the spread of microorganisms and contribute to their clearance . at virtually every step in the development of the infectious process , including the activation of adaptive immunity , complement proteins are engaged in the battle against pathogens . these multiple tasks require prompt and efficient activation of the complement cascade in coordination with other immune mechanisms , resulting in the generation of a number of effector molecules that participate in the response to infection . this review has highlighted how such activation is achieved by multiple pathways acting in concert to ensure both the development of a response that is proportional to the scale of danger and a timely delivery of complement effectors to the sites of infection . it also appears that in addition to the previously well - studied pathways of complement activation , various newly discovered mechanisms can either initiate or amplify the activation of the complement cascade in sepsis .

recognition of antigen as mhc peptide ( mhcp ) complexes by the t cell receptor ( tcr ) is the first stage of t cell activation , and therefore critical to all adaptive immune responses to pathogens and cancers , as well as to autoimmunity . the binding of tcr to mhcp initiates a protein kinase signaling cascade that branches out as it proceeds , to encompass various signaling pathways leading to changes in cell shape , adhesion , and motility , as well as to changes in gene transcription . there are several models for how tcr interaction with mhcp can lead to initial triggering of the signaling cascade . these have been comprehensively reviewed ( germain , 2001 ; krogsgaard and davis , 2005 ; van der merwe and dushek , 2011 ) , so will only be briefly mentioned here . they fall into several groups , aggregation , conformation / architectural changes , and separation models . evidence from studies of t cells activated by apc or by soluble mhcp indicate that some kind of crosslinking or aggregation of two or more tcrs is required ( boniface et al . , 1998 ; cochran et al . tcrs are grouped into microclusters during antigen recognition ( yokosuka et al . , 2005 ; varma et al . , 2006 ) , and there is also some evidence for pre - formed aggregations of tcrs existing on the cell surface ( schamel et al . clustering of the tcrs by antigen recognition aided by weak interactions with endogenous mhcp and the recruitment of co - receptor - bound lck to the site of recognition would then start the signaling cascade ( krogsgaard and davis , 2005 ; gascoigne , 2008 ) . conformational changes have been proposed as a mechanism for signal transduction , but apart from movement in the cdrs during binding to mhcp , conformation change upon antigen binding has only been found as a movement of part of the c domain , in a region predicted to interact with cd3 ( kjer - nielsen et al . , 2003 ; beddoe et al . , conformation changes in intracellular domains of cd3 seem to be involved in allowing signaling adaptor proteins to interact with cd3 ( gil et al . , 2002 ) or for accessibility for immunoreceptor tyrosine - based activation motif ( itam ) phosphorylation ( xu et al . , 2008 ) . a conformational / architectural model has received strong recent support whereby mechanical pulling by the tcr mhcp interaction puts torque on the cd3 subunits , leading to signal transduction ( kim et al . , 2009 ; , the interaction between short molecules like tcr and mhc at the immunological synapse requires that larger molecules such as cell adhesion molecules and the phosphatase cd45 are pushed out to the periphery . removal of negative regulators of signaling could thus aid phosphorylation steps at the center of the synapse ( davis and van der merwe , 2006 ) . these different classes of model are not necessarily mutually exclusive , and indeed elements of each class seem to operate in t cell activation ( van der merwe and dushek , 2011 ) . the very first stage of the tcr signaling cascade after tcr mhcp binding is the phosphorylation of the cd3 subunits of the tcr . complex consists of the tcr and chains that recognize mhcp , and the cd3 , , and dimers that form the signal transduction modules of the tcr . all of these subunits are cell surface transmembrane proteins and all have at least one itam in their cytoplasmic domain . cd3 , , and each have one itam , while cd3 has three . at the start of the signaling cascade , the cd3 itams are phosphorylated , and are then bound by another kinase called zap70 . each itam has dual tyrosines so that when they are both phosphorylated , they form a high affinity binding site for the dual sh2 domains of zap70 . after zap70 binds to cd3 , the co - receptors cd4 or cd8 , which are associated with the src - family kinase lck , become associated with the tcr cd3 complex and bind to mhc . mhcp interaction , and lck continues the phosphorylation of cd3 elements , zap70 and the many other downstream targets . a recent biophysical study on tcr and cd8 binding to mhcp demonstrated that the initial binding of tcr to mhcp induces , in a src - family kinase - dependent manner , the binding of cd8 to the mhcp ( jiang et al . , 2011 ) . this illustrates two related and important problems in understanding t cell activation that have never been fully resolved . firstly , how is the first cd3 phosphorylation step accomplished , and by which src - family kinase(s ) ? secondly , if the co - receptor is brought to the activated tcr before the co - receptor binds to mhc , but after tcr signaling has started , how is this initial recruitment to the tcr accomplished ? rather little is known about these earliest steps in t cell activation . lck is a major target for drug discovery , as small molecule inhibitors could be used as immunosuppressives , for example in autoimmunity , rheumatoid arthritis , or asthma . current inhibitors are not very selective , also inhibiting other src - family kinases or other kinases ( martin and machacek , 2010 ) . it is therefore important to understand exactly how src - family kinases work in t cell activation . it was shown in the early 1990s that stimulation of the tcr enhances the binding of cd8 to mhc class i ( mhci ) , and that this increase in binding requires tyrosine kinase activity ( orourke et al . , 1990 ; orourke and mescher , 1992 ) . recently , a cell - based adhesion frequency assay was developed that enables investigation of molecular interactions at the cell surface with very fast time resolution ( huang et al . this method is based on touching a t cell to a red blood cell coated with appropriate mhcp ligands , then retracting it , and measuring whether or not the red blood cell membrane is distorted . this process is repeated multiple times with the interaction allowed to continue for certain fixed periods . for each of these interaction times , an adhesion frequency is calculated . this method showed clearly that tcr and cd8 interactions with mhcp are biphasic ( jiang et al . , 2011 ) . the first phase the initial tcr interaction with ligand starts within the first 0.25 s after the cell surfaces make contact ( figure 1 ) . after about 1 s of contact , a second , stronger , binding phase occurs . initiation of the second phase requires a src - family tyrosine kinase , as it does not occur when the inhibitor pp2 is present . the first phase is co - receptor - independent but the second phase is co - receptor dependent , as the second phase is blocked by anti - cd8 antibodies and does not occur when the cd8 binding site of mhci is non - functional . this is followed by a second phase of tcr - induced cd8 binding ( jiang et al . , 2011 ) . mhcp interaction , but like that , it has single - stage kinetics and reaches a plateau at about 1 s ( huang et al . , 2007 ) . this cd8mhci interaction is sufficient to concentrate cd8 at the interface between a t cell and an antigen - presenting cell ( yachi et al . , 2005 , 2006 ; gascoigne et al . , 2010 ) . a model for initiation of tcr cd3 phosphorylation , and the recruitment of the co - receptor . lck and/or fyn are associated with the plasma membrane , and lck also exists associated with cd8 . upon tcr binding to mhcp ( b ) the fyn and/or non - cd8 cd3 complex , and phosphorylates cd3 itams and then zap70 which binds to the phosphorylated cd3 itams . ( c ) the sh2 domain of cd8 bound lck then interacts with the phospho - zap70 , pulling cd8 into proximity with the mhcp - interacting tcr . ( d ) the extracellular immunoglobulin domains then bind to the mhcp , stabilizing the tcr this two - step activation ( jiang et al . , 2011 ) does not fit with the classical model of tcr triggering , where the co - receptor binding to mhcp is required for lck to phosphorylate cd3 itams . it is compatible with aggregation models such as the pseudodimer or co - receptor pre - concentration models , where the co - receptor s extracellular domains bind to non - cognate mhcp but the co - receptor - bound lck interacts with the antigen binding tcr / cd3 within the cell ( krogsgaard et al . it is also consistent with those models requiring a conformation / architectural change due to antigen binding , providing that the initial phosphorylation of cd3 recruits the co - receptor through the interaction of lck with phospho - itams . several lines of evidence indicate that the initial phosphorylation of cd3 itams and zap70 is caused by a src - family kinase ( lck and/or fyn ) that is not bound to cd4 or cd8 , yet the mechanism of this process that initiates the tcr signaling cascade is poorly defined ( acuto et al . , 2008 ) . it is clear that co - receptors are not absolutely required for tcr signaling as some t cells are independent of co - receptor , and the requirement for cd8 has been shown to be inversely related to the affinity of the tcr . tcrs with affinity above a certain threshold do not need co - receptor ( cho et al . another piece of evidence is that thymocyte development requires the presence of lck , but co - receptors are not absolutely required for thymocyte development ( schilham et al . , 1993 ; van laethem et al . , 2007 in addition , the fact that cd8 does not bind mhc to stabilize tcr binding until after signaling has started argues this point strongly ( jiang et al . , 2011 ) . lck can be co - precipitated with tcr / cd3 in mild detergents ( schraven et al . 2003 ) , but although lck can be co - capped with co - receptor ( after crosslinking by suitable antibody ) , it does not co - cap with tcr ( ehrlich et al . , 2002 ) . fyn can also be co - precipitated with the tcr in mild detergents ( samelson et al . thus there is evidence that both lck and fyn can associate in some way with tcr , but at very low stoichiometry . also , because these experiments used detergents that do not disrupt lipid rafts , it is possible that the interactions were artifacts due to the detergent lysis method causing interactions between normally separate lipid domains . fyn or lck individually can both perform the constitutive phosphorylation of tcr / cd3 required for maintenance of cell viability in vivo , but lck - deficient cells have stronger defects in most aspects of signaling than fyn - deficient cells . the signaling differences between t cells from fyn and lck knockout mice are summarized in table 1 . because of the block in positive selection in lckmice , data on lck - deficiency in mature t cells have come from a very elegant inducible transgenic system bred onto the lck ko background . this transgene is turned on to allow normal t cell development , then off so that lck expression is gradually lost . transgenic lck expression in mature t cells is , however , only about 1020% of the normal amount ( seddon et al . , 2000 ; for the most part , the defects in fyn - deficient t cells were rather modest , but recent studies have shown that fyn may in fact be a negative regulator of tcr signaling . stimulation with antigen ( or with crosslinking anti - cd3 plus anti - cd8 ) gave stronger and faster responses in the absence of fyn ( filby et al . this is in contrast to earlier studies that showed that cells lacking fyn gave lower responses than normal to anti - cd3 stimulation ( as opposed to antigen or anti - tcr plus anti - co - receptor stimulation ; reviewed in palacios and weiss , 2004 ; salmond et al . data on mature t cells deficient in lck expression is from mouse with inducible lck expression , which can be turned off after development ( legname et al . most interestingly , in lck knockout mature t cells , fyn directs relatively strong erk activation , but reduced lat phosphorylation , while plc1 phosphorylation and ca signaling do not occur ( lovatt et al . , 2006 ; a direct interaction of grb2sos with cd3 has been suggested ( chau and madrenas , 1999 ; methi et al . , 2007 ) , which could perhaps provide such a pathway . however , t cell activation is very strongly dependent on the rasgrp pathway to activate ras and then erk , and it is unclear if sos has any crucial role ( dower et al . , 2000 ; priatel et al . , 2002 ) . it may also be important to consider that these studies in lck or fyn - deficient cell lines or knockout mice have not distinguished free lck from that bound to the co - receptor . resting t cells and thymocytes contain substantial quantities of both activated lck and fyn , and stimulation through tcr or co - receptor does not increase the proportion of the activated kinases ( nika et al . , 2010 ) . both lck and fyn are targeted to the inner leaflet of the plasma membrane in lipid rafts through palmitoylation and myristoylation of their amino termini . this fatty acylation is required for fyn to interact with and phosphorylate cd3 itams ( vant hof and resh , 1999 ) . lipid - raft targeting by fatty acylation is also the case for lyn , a src - family kinase that is important in initiation of bcr signaling . a recent experiment showed that lyn interacts with the bcr after bcr crosslinking by binding to antigen , when ag - induced bcr microclusters interact with lipid rafts ( sohn et al . , 2008 ) . a similar mechanism is likely to apply for the initiation of tcr signaling by lck and/or fyn . yet another complexity comes from single - molecule studies of the movement of cell membrane - associated molecules . stimulation by very small clusters of anti - cd3 antibodies on a solid surface showed that a raft - targeted construct ( the amino terminal of lck with the fatty acylation sites ) or full - length lck both concentrated quickly at the stimulation site and were retained there ( ike et al . , 2003 ) . diffusion was slower closer to the site of stimulation , and faster away from these sites . directed movement of lck was not seen , indicating that lck was not brought to the signaling region by actin , but concentration at the stimulation site was blocked by drugs that either poisoned the cytoskeleton or dispersed lipid rafts . clustering of the actin filaments at the site of activation ( wulfing and davis , 1998 ) would slow diffusion away from the activation site , either because the actin mesh impedes free diffusion of the membrane - bound protein , or because of a picket fence of transmembrane proteins associated with the cytoskeleton . these phenomena would lead to corralling of lck ( and rafts ) at the activation site ( ike et al . , others have described a similar phenomenon of microdomains caused by proteins rather than lipids , where it was noted that cd45 was excluded from microdomains that contained lck ( douglass and vale , 2005 ) . this study also demonstrated that various t cell signaling proteins were able to move quickly from one microdomain to another . a protein such as annexin a6 , which binds negatively charged phospholipids , lipid rafts , and f - actin , could organize and bridge different kinds of microdomains and/or rafts around signaling receptors ( cornely et al . , 2011 ) . the data on biphasic tcr and cd8 interactions with mhcp show that in a very quick sequence , tcr binds mhcp , causing src - family kinase activity , then cd8 binds to the mhc and stabilizes the interaction between the three molecules ( jiang et al . , 2011 ) . if cd8 does not bind to mhcp until the second stage , then how is the co - receptor initially recruited to the tcr ? it has generally been assumed that this is accomplished by cd8 binding to mhc , but this does not fit with the new data . it has been suggested that cd8 is brought into the signaling complex passively , through an interaction of the lck molecule attached to cd8 with one of the phospho - tyrosines produced by the initial signal on cd3 , or to phospho - zap70 ( jiang et al . , 2011 ) . a study using a chimera between cd4 and lck demonstrated that the sh2 domain of lck was involved in cd4 co - receptor activity : a mutation stopping the lck sh2 from binding phosphotyrosine partially reduced co - receptor activity ( xu and littman , 1993 ) . this suggested that a protein protein interaction mediated by lck s sh2 domain is important for co - receptor function , and indeed it has since been shown that after tcr stimulation , the lck sh2 domain binds to phospho - zap70 bound to phospho - cd3 , and that this interaction causes the co - receptor to come into proximity with tcr ( thome et al . , 1995 , 1996 ) . as mentioned above , much of the lck in resting t cells is activated kinase , and this activity is not affected by tcr stimulation ( nika et al . , 2010 ) , but kinase activity is not required for the co - receptor function of cd4 ( collins and burakoff , 1993 ; xu and littman , 1993 ) . the cd4 co - receptor is bound to both active and inactive lck ( nika et al . , 2010 ) , indicating that there is no simple explanation . the protein cd8 exists as both an homodimer and as the heterodimer that is the main co - receptor for t cell activation . both forms are equivalent in their ability to bind to classical mhci , but cd8 binds much more strongly than cd8 to the non - classical mhci molecule h2-tl ( cheroutre and lambolez , 2008 ) . cd8 is a much stronger co - receptor than cd8 , probably because of preferential localization of cd8 to lipid rafts where it is more likely to interact with lck . we tested the ability of the two forms expressed on the same cell to be recruited to the site of antigen recognition , and found that there was very little difference between them ( rybakin et al . , 2011 ) . if h2-tl was present on the antigen - presenting cell , then the cd8 form was preferentially recruited , leading us to the conclusion that the ability of the cd8 isoforms to be recruited to the immunological synapse was not directly related to their co - receptor ability , but simply to the strength of their interaction with mhci . in previous studies from this lab , we used foerster resonance energy transfer ( fret ) microscopy in live cells to investigate the interaction of both cd4 and cd8 with tcr , using cd3cfp as fret donor and cd4yfp or cd8yfp as fret acceptors ( zal et al . , 2002 ; we showed that the co - receptors interact closely with the tcr only when tcr recognizes antigen , even though both cd4 and cd8 can be recruited to the immunological synapse without any requirement for antigen recognition . this was most striking for cd8 , which binds to non - antigenic mhci molecules and can be concentrated in the immunological synapse in proportion to the number of mhci proteins available ( yachi et al . , 2005 ; gascoigne et al . , 2010 ) . cd3 complex ( cd3cfp cd8yfp fret ) corresponded closely to strength of t cell activation by particular peptides , and that differences in fret kinetics during recognition of different mhcp ligands correlated closely to the ability of a particular ligand to induce positive or negative selection in thymocytes ( yachi et al . , 2006 ; mallaun et al . , 2008 ) . however , the kinetics of the close apposition of tcr and cd8 were slower than expected from other measures of t cell activation , as they developed over several minutes , peaking at about 10 min ( yachi et al . , 2005 , 2006 ) . early signaling events such as lat phosphorylation or calcium flux occur within a few seconds , although other events like cytoskeletal rearrangement for example , are on a longer timescale of a few minutes ( huse et al . , 2007 ) . in more recent higher - resolution experiments using a supported lipid bilayer system , we have been able to image tcr cd8 fret in small regions within the immunological synapse ( j. casas and nicholas r. j. gascoigne , unpublished ) . these were too localized to show a signal above the background when fret signals were averaged over the whole synapse , explaining why they were not noticed before . this leads us to believe that the strong fret signals measured previously in synapses with antigen - presenting cells that were on the timescale of minutes ( yachi et al . , 2005 , 2006 ) , represent the result of stabilization of tcr mhcp binding by the co - receptor the second phase of interaction identified in the cell cell adhesion experiments ( jiang et al . , 2011 ) , rather than the first . the tcr -chain connecting peptide motif ( -cpm ) is a highly conserved membrane - proximal extracellular sequence motif in the tcr -chain . it is important for the ability of cd8 to associate with tcr to enhance t cell responses ( naeher et al . , 2002 ; 2008 ) , and for t cell activation and positive selection ( werlen et al . , 2000 ) . mutation of the -cpm reduced the stable fret response between tcr and cd8 ( mallaun et al . the -cpm is believed to work by interacting with membrane - proximal regions of cd8 in a co - receptor zipper mechanism , enabling lck to completely phosphorylate the cd3 itams ( palmer and naeher , 2009 ) . 2003 ) . the molecular basis of the interaction between the -cpm and cd8 is not clear , as it is unlikely that cd8 could approach closely enough to the -cpm to interact with it directly , due to the presence of the cd3 subunits . however , the transmembrane region of cd3 interacts with that of tcr ( call et al . , 2002 ) , and structural models place cd3 and tcr apposed to each other ( sun et al . , it has been suggested that movement within the tcr s -cpm could apply torque to cd3 in the architectural / conformational change model of tcr triggering described earlier ( kim et al . , 2010 ) . it will be interesting to find out how the -cpm mutation affects the two - stage tcr binding ( jiang et al . , 2011 ) . cd8 interaction after the initial tcr mhcp binding , so that mutation of the -cpm will inhibit the second phase of tcr mhcp binding without affecting the first phase . the molecular details of the apparent adaptor function of lck need to be more clearly worked out , and indeed , a similar function may exist in other src - family kinases . lck and fyn molecules have overlapping but distinct roles at the beginning of tcr signal transduction , but the mechanisms for their initial interaction with tcr to start the signaling cascade are still somewhat obscure . improvements in our ability to image tcr cd8 fret will allow us to investigate separately the early and later phases of the tcr cd8 interactions . the early phase likely corresponds to the passive recruitment of cd8 to tcr by the lck interacting with phospho - cd3 and/or phospho - zap70 , whereas the later and stronger phase of fret is likely the result of stabilization of tcr mhcp binding by the co - receptor . in this regard , it is worth noting that the two - stage binding model that shows co - receptor stabilization of the tcr indeed , modeling studies suggest that the major role of co - receptors , particularly cd4 , is simply to ferry lck toward the site of tcr activation ( artyomov et al . , 2010 ) . we are currently analyzing the different functions of cd8 and lck using mutational analyses and fret microscopy . the future application of super - resolution microscopy techniques such as palm and storm ( stochastic optical reconstruction microscopy ) will enable a nanometer - scale understanding of the molecular interactions in the early phases of t cell activation . 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 .

recent data with cd8 + t cells show that the initial phase of t cell receptor ( tcr ) binding to mhc peptide ( mhcp ) is quickly followed by a second , stronger , binding phase representing the binding of cd8 to the mhcp . this second phase requires signaling by a src - family kinase such as lck . these data point out two aspects of the initial stage of tcr signaling that have not yet been clearly resolved . firstly , how and by which src - family kinase , is the initial phosphorylation of cd3 accomplished , given that the lck associated with the co - receptors ( cd4 or cd8 ) is not yet available . secondly , what is the mechanism by which the co - receptor is brought close to the bound tcr before the co - receptor binds to mhcp ?

Introduction TCR Binding to MHCp Augments CD8 Binding to MHC Initial Phosphorylation of TCRCD3 in T Cell Activation How is CD8 Recruited to the Activated TCR Before CD8 Interacts with MHC? Different Phases in the TCR Interaction with Co-Receptor Future Directions Conflict of Interest Statement

recognition of antigen as mhc peptide ( mhcp ) complexes by the t cell receptor ( tcr ) is the first stage of t cell activation , and therefore critical to all adaptive immune responses to pathogens and cancers , as well as to autoimmunity . the binding of tcr to mhcp initiates a protein kinase signaling cascade that branches out as it proceeds , to encompass various signaling pathways leading to changes in cell shape , adhesion , and motility , as well as to changes in gene transcription . there are several models for how tcr interaction with mhcp can lead to initial triggering of the signaling cascade . evidence from studies of t cells activated by apc or by soluble mhcp indicate that some kind of crosslinking or aggregation of two or more tcrs is required ( boniface et al . clustering of the tcrs by antigen recognition aided by weak interactions with endogenous mhcp and the recruitment of co - receptor - bound lck to the site of recognition would then start the signaling cascade ( krogsgaard and davis , 2005 ; gascoigne , 2008 ) . conformational changes have been proposed as a mechanism for signal transduction , but apart from movement in the cdrs during binding to mhcp , conformation change upon antigen binding has only been found as a movement of part of the c domain , in a region predicted to interact with cd3 ( kjer - nielsen et al . , 2009 ; , the interaction between short molecules like tcr and mhc at the immunological synapse requires that larger molecules such as cell adhesion molecules and the phosphatase cd45 are pushed out to the periphery . removal of negative regulators of signaling could thus aid phosphorylation steps at the center of the synapse ( davis and van der merwe , 2006 ) . these different classes of model are not necessarily mutually exclusive , and indeed elements of each class seem to operate in t cell activation ( van der merwe and dushek , 2011 ) . the very first stage of the tcr signaling cascade after tcr mhcp binding is the phosphorylation of the cd3 subunits of the tcr . at the start of the signaling cascade , the cd3 itams are phosphorylated , and are then bound by another kinase called zap70 . after zap70 binds to cd3 , the co - receptors cd4 or cd8 , which are associated with the src - family kinase lck , become associated with the tcr cd3 complex and bind to mhc . mhcp interaction , and lck continues the phosphorylation of cd3 elements , zap70 and the many other downstream targets . a recent biophysical study on tcr and cd8 binding to mhcp demonstrated that the initial binding of tcr to mhcp induces , in a src - family kinase - dependent manner , the binding of cd8 to the mhcp ( jiang et al . this illustrates two related and important problems in understanding t cell activation that have never been fully resolved . firstly , how is the first cd3 phosphorylation step accomplished , and by which src - family kinase(s ) ? secondly , if the co - receptor is brought to the activated tcr before the co - receptor binds to mhc , but after tcr signaling has started , how is this initial recruitment to the tcr accomplished ? rather little is known about these earliest steps in t cell activation . current inhibitors are not very selective , also inhibiting other src - family kinases or other kinases ( martin and machacek , 2010 ) . it is therefore important to understand exactly how src - family kinases work in t cell activation . it was shown in the early 1990s that stimulation of the tcr enhances the binding of cd8 to mhc class i ( mhci ) , and that this increase in binding requires tyrosine kinase activity ( orourke et al . this method is based on touching a t cell to a red blood cell coated with appropriate mhcp ligands , then retracting it , and measuring whether or not the red blood cell membrane is distorted . this process is repeated multiple times with the interaction allowed to continue for certain fixed periods . after about 1 s of contact , a second , stronger , binding phase occurs . initiation of the second phase requires a src - family tyrosine kinase , as it does not occur when the inhibitor pp2 is present . the first phase is co - receptor - independent but the second phase is co - receptor dependent , as the second phase is blocked by anti - cd8 antibodies and does not occur when the cd8 binding site of mhci is non - functional . this is followed by a second phase of tcr - induced cd8 binding ( jiang et al . this cd8mhci interaction is sufficient to concentrate cd8 at the interface between a t cell and an antigen - presenting cell ( yachi et al . a model for initiation of tcr cd3 phosphorylation , and the recruitment of the co - receptor . lck and/or fyn are associated with the plasma membrane , and lck also exists associated with cd8 . upon tcr binding to mhcp ( b ) the fyn and/or non - cd8 cd3 complex , and phosphorylates cd3 itams and then zap70 which binds to the phosphorylated cd3 itams . ( c ) the sh2 domain of cd8 bound lck then interacts with the phospho - zap70 , pulling cd8 into proximity with the mhcp - interacting tcr . ( d ) the extracellular immunoglobulin domains then bind to the mhcp , stabilizing the tcr this two - step activation ( jiang et al . , 2011 ) does not fit with the classical model of tcr triggering , where the co - receptor binding to mhcp is required for lck to phosphorylate cd3 itams . it is compatible with aggregation models such as the pseudodimer or co - receptor pre - concentration models , where the co - receptor s extracellular domains bind to non - cognate mhcp but the co - receptor - bound lck interacts with the antigen binding tcr / cd3 within the cell ( krogsgaard et al . it is also consistent with those models requiring a conformation / architectural change due to antigen binding , providing that the initial phosphorylation of cd3 recruits the co - receptor through the interaction of lck with phospho - itams . several lines of evidence indicate that the initial phosphorylation of cd3 itams and zap70 is caused by a src - family kinase ( lck and/or fyn ) that is not bound to cd4 or cd8 , yet the mechanism of this process that initiates the tcr signaling cascade is poorly defined ( acuto et al . it is clear that co - receptors are not absolutely required for tcr signaling as some t cells are independent of co - receptor , and the requirement for cd8 has been shown to be inversely related to the affinity of the tcr . tcrs with affinity above a certain threshold do not need co - receptor ( cho et al . another piece of evidence is that thymocyte development requires the presence of lck , but co - receptors are not absolutely required for thymocyte development ( schilham et al . lck can be co - precipitated with tcr / cd3 in mild detergents ( schraven et al . 2003 ) , but although lck can be co - capped with co - receptor ( after crosslinking by suitable antibody ) , it does not co - cap with tcr ( ehrlich et al . fyn can also be co - precipitated with the tcr in mild detergents ( samelson et al . also , because these experiments used detergents that do not disrupt lipid rafts , it is possible that the interactions were artifacts due to the detergent lysis method causing interactions between normally separate lipid domains . fyn or lck individually can both perform the constitutive phosphorylation of tcr / cd3 required for maintenance of cell viability in vivo , but lck - deficient cells have stronger defects in most aspects of signaling than fyn - deficient cells . the signaling differences between t cells from fyn and lck knockout mice are summarized in table 1 . because of the block in positive selection in lckmice , data on lck - deficiency in mature t cells have come from a very elegant inducible transgenic system bred onto the lck ko background . transgenic lck expression in mature t cells is , however , only about 1020% of the normal amount ( seddon et al . , 2000 ; for the most part , the defects in fyn - deficient t cells were rather modest , but recent studies have shown that fyn may in fact be a negative regulator of tcr signaling . stimulation with antigen ( or with crosslinking anti - cd3 plus anti - cd8 ) gave stronger and faster responses in the absence of fyn ( filby et al . this is in contrast to earlier studies that showed that cells lacking fyn gave lower responses than normal to anti - cd3 stimulation ( as opposed to antigen or anti - tcr plus anti - co - receptor stimulation ; reviewed in palacios and weiss , 2004 ; salmond et al . data on mature t cells deficient in lck expression is from mouse with inducible lck expression , which can be turned off after development ( legname et al . most interestingly , in lck knockout mature t cells , fyn directs relatively strong erk activation , but reduced lat phosphorylation , while plc1 phosphorylation and ca signaling do not occur ( lovatt et al . however , t cell activation is very strongly dependent on the rasgrp pathway to activate ras and then erk , and it is unclear if sos has any crucial role ( dower et al . it may also be important to consider that these studies in lck or fyn - deficient cell lines or knockout mice have not distinguished free lck from that bound to the co - receptor . resting t cells and thymocytes contain substantial quantities of both activated lck and fyn , and stimulation through tcr or co - receptor does not increase the proportion of the activated kinases ( nika et al . both lck and fyn are targeted to the inner leaflet of the plasma membrane in lipid rafts through palmitoylation and myristoylation of their amino termini . lipid - raft targeting by fatty acylation is also the case for lyn , a src - family kinase that is important in initiation of bcr signaling . a recent experiment showed that lyn interacts with the bcr after bcr crosslinking by binding to antigen , when ag - induced bcr microclusters interact with lipid rafts ( sohn et al . a similar mechanism is likely to apply for the initiation of tcr signaling by lck and/or fyn . yet another complexity comes from single - molecule studies of the movement of cell membrane - associated molecules . diffusion was slower closer to the site of stimulation , and faster away from these sites . directed movement of lck was not seen , indicating that lck was not brought to the signaling region by actin , but concentration at the stimulation site was blocked by drugs that either poisoned the cytoskeleton or dispersed lipid rafts . clustering of the actin filaments at the site of activation ( wulfing and davis , 1998 ) would slow diffusion away from the activation site , either because the actin mesh impedes free diffusion of the membrane - bound protein , or because of a picket fence of transmembrane proteins associated with the cytoskeleton . a protein such as annexin a6 , which binds negatively charged phospholipids , lipid rafts , and f - actin , could organize and bridge different kinds of microdomains and/or rafts around signaling receptors ( cornely et al . the data on biphasic tcr and cd8 interactions with mhcp show that in a very quick sequence , tcr binds mhcp , causing src - family kinase activity , then cd8 binds to the mhc and stabilizes the interaction between the three molecules ( jiang et al . if cd8 does not bind to mhcp until the second stage , then how is the co - receptor initially recruited to the tcr ? it has generally been assumed that this is accomplished by cd8 binding to mhc , but this does not fit with the new data . it has been suggested that cd8 is brought into the signaling complex passively , through an interaction of the lck molecule attached to cd8 with one of the phospho - tyrosines produced by the initial signal on cd3 , or to phospho - zap70 ( jiang et al . a study using a chimera between cd4 and lck demonstrated that the sh2 domain of lck was involved in cd4 co - receptor activity : a mutation stopping the lck sh2 from binding phosphotyrosine partially reduced co - receptor activity ( xu and littman , 1993 ) . this suggested that a protein protein interaction mediated by lck s sh2 domain is important for co - receptor function , and indeed it has since been shown that after tcr stimulation , the lck sh2 domain binds to phospho - zap70 bound to phospho - cd3 , and that this interaction causes the co - receptor to come into proximity with tcr ( thome et al . as mentioned above , much of the lck in resting t cells is activated kinase , and this activity is not affected by tcr stimulation ( nika et al . , 2010 ) , but kinase activity is not required for the co - receptor function of cd4 ( collins and burakoff , 1993 ; xu and littman , 1993 ) . the cd4 co - receptor is bound to both active and inactive lck ( nika et al . the protein cd8 exists as both an homodimer and as the heterodimer that is the main co - receptor for t cell activation . both forms are equivalent in their ability to bind to classical mhci , but cd8 binds much more strongly than cd8 to the non - classical mhci molecule h2-tl ( cheroutre and lambolez , 2008 ) . cd8 is a much stronger co - receptor than cd8 , probably because of preferential localization of cd8 to lipid rafts where it is more likely to interact with lck . we tested the ability of the two forms expressed on the same cell to be recruited to the site of antigen recognition , and found that there was very little difference between them ( rybakin et al . if h2-tl was present on the antigen - presenting cell , then the cd8 form was preferentially recruited , leading us to the conclusion that the ability of the cd8 isoforms to be recruited to the immunological synapse was not directly related to their co - receptor ability , but simply to the strength of their interaction with mhci . , 2002 ; we showed that the co - receptors interact closely with the tcr only when tcr recognizes antigen , even though both cd4 and cd8 can be recruited to the immunological synapse without any requirement for antigen recognition . this was most striking for cd8 , which binds to non - antigenic mhci molecules and can be concentrated in the immunological synapse in proportion to the number of mhci proteins available ( yachi et al . cd3 complex ( cd3cfp cd8yfp fret ) corresponded closely to strength of t cell activation by particular peptides , and that differences in fret kinetics during recognition of different mhcp ligands correlated closely to the ability of a particular ligand to induce positive or negative selection in thymocytes ( yachi et al . however , the kinetics of the close apposition of tcr and cd8 were slower than expected from other measures of t cell activation , as they developed over several minutes , peaking at about 10 min ( yachi et al . early signaling events such as lat phosphorylation or calcium flux occur within a few seconds , although other events like cytoskeletal rearrangement for example , are on a longer timescale of a few minutes ( huse et al . this leads us to believe that the strong fret signals measured previously in synapses with antigen - presenting cells that were on the timescale of minutes ( yachi et al . , 2005 , 2006 ) , represent the result of stabilization of tcr mhcp binding by the co - receptor the second phase of interaction identified in the cell cell adhesion experiments ( jiang et al . the tcr -chain connecting peptide motif ( -cpm ) is a highly conserved membrane - proximal extracellular sequence motif in the tcr -chain . it is important for the ability of cd8 to associate with tcr to enhance t cell responses ( naeher et al . mutation of the -cpm reduced the stable fret response between tcr and cd8 ( mallaun et al . the -cpm is believed to work by interacting with membrane - proximal regions of cd8 in a co - receptor zipper mechanism , enabling lck to completely phosphorylate the cd3 itams ( palmer and naeher , 2009 ) . the molecular basis of the interaction between the -cpm and cd8 is not clear , as it is unlikely that cd8 could approach closely enough to the -cpm to interact with it directly , due to the presence of the cd3 subunits . however , the transmembrane region of cd3 interacts with that of tcr ( call et al . , it has been suggested that movement within the tcr s -cpm could apply torque to cd3 in the architectural / conformational change model of tcr triggering described earlier ( kim et al . cd8 interaction after the initial tcr mhcp binding , so that mutation of the -cpm will inhibit the second phase of tcr mhcp binding without affecting the first phase . the molecular details of the apparent adaptor function of lck need to be more clearly worked out , and indeed , a similar function may exist in other src - family kinases . lck and fyn molecules have overlapping but distinct roles at the beginning of tcr signal transduction , but the mechanisms for their initial interaction with tcr to start the signaling cascade are still somewhat obscure . improvements in our ability to image tcr cd8 fret will allow us to investigate separately the early and later phases of the tcr cd8 interactions . the early phase likely corresponds to the passive recruitment of cd8 to tcr by the lck interacting with phospho - cd3 and/or phospho - zap70 , whereas the later and stronger phase of fret is likely the result of stabilization of tcr mhcp binding by the co - receptor . in this regard , it is worth noting that the two - stage binding model that shows co - receptor stabilization of the tcr indeed , modeling studies suggest that the major role of co - receptors , particularly cd4 , is simply to ferry lck toward the site of tcr activation ( artyomov et al . the future application of super - resolution microscopy techniques such as palm and storm ( stochastic optical reconstruction microscopy ) will enable a nanometer - scale understanding of the molecular interactions in the early phases of t cell activation . 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 .

centrioles are evolutionarily conserved organelles that are pivotal for the formation of cilia , flagella , and centrosomes , and are thus critical for fundamental cellular processes such as signaling , polarity , motility , and division ( reviewed in azimzadeh and marshall , 2010 , bornens , 2012 , jana et al . , 2014 , gnczy , 2012 ) . owing to their central role in such diverse cellular processes , centriole aberrations contribute to a range of severe human diseases , including ciliopathies , primary microcephaly , and cancer ( reviewed in nigg and raff , 2009 , gnczy , 2015 ) . microtubules are the major constituent of centrioles and are arranged in a radial nine - fold symmetrical array , typically of triplet microtubules , which forms a barrel - shaped centriolar wall ( reviewed in azimzadeh and marshall , 2010 , bornens , 2012 , jana et al . centriolar microtubules are unique in exhibiting exceptionally slow growth rates of a few tens of nanometers per hour on average , and in being very stable after their formation ( kuriyama and borisy , 1981 , chretien et al . , 1997 ) . such properties likely contribute to setting centriole length , which is well conserved across evolution . the behavior of centriolar microtubules is in stark contrast to that of their cytoplasmic counterparts , which assemble up to four orders of magnitude faster and are highly dynamic ( kinoshita et al . , 2001 ) . the molecular mechanisms that impart the exceptional slow growth rate and stability of centriolar microtubules are not known . several centriolar proteins that can directly interact with tubulin and/or microtubules have been identified , including cep120 , cep135 , centrobin , and cpap ( gudi et al . , 2011 , lin et al . cpap ( sas-4 in worms and flies ) is of particular interest , since it is the only component among the ones listed above that is present and essential for centriole formation from worm to man ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , 2009 , the importance of cpap is further substantiated by the fact that homozygous mutations in the corresponding gene lead to autosomal recessive primary microcephaly , a devastating human disease with drastically reduced neuron numbers and , thus , brain size ( bond et al . , 2005 ) . interestingly , cpap overexpression induces overly long centrioles in human cells ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , 2009 ) , thus interfering with cell division ( kohlmaier et al . , 2009 ) . together , these observations suggest that cpap somehow regulates centriolar microtubule growth to produce proper centrioles . cpap comprises a tubulin - binding domain ( pn2 - 3 ) , a positively charged microtubule - binding domain ( mbd ) , a coiled - coil dimerization domain , and a c - terminal g box ( figure 1a ) . the pn2 - 3 domain is of prime interest as this region is highly conserved across evolution and found exclusively in cpap / sas-4 proteins . pn2 - 3 sequesters tubulin dimers and has been shown to destabilize microtubules both in vitro and in cells ( hsu et al . , 2008 , how this observation can be reconciled with the presence of overly long centrioles upon cpap overexpression , which is suggestive of the protein - enhancing centriolar microtubule elongation , has remained puzzling . here , to elucidate the fundamental mechanism of action of cpap / sas-4 proteins , we set out to decipher how cpap affects microtubules using structural , biophysical , and cell biological approaches . we first report a high - resolution structure of -tubulin in complex with the pn2 - 3 domain of cpap . this structural information guided the design of experiments aimed at understanding the key role of cpap in regulating centriolar microtubule behavior . using reconstitution experiments , we demonstrate that cpap autonomously recognizes and tracks growing microtubule plus ends . there , cpap suppresses microtubule polymerization and increases microtubule stability by inhibiting catastrophes and promoting rescues . we further establish that the pn2 - 3 domain of cpap is critical in the cellular context to restrict the extent of centriolar microtubule elongation by acting as a molecular cap . to gain insight into the molecular mechanism of tubulin binding by cpap / sas-4 proteins , we sought to crystallize the pn2 - 3 domain of human cpap in complex with tubulin ( see table s1 for all constructs generated in the course of this study ) . extensive crystallization trials with complexes of tubulin and pn2 - 3 variants were not met with success . however , adding the -tubulin - binding darpin ( d1 ; pecqueur et al . , 2012 ) allowed us to solve the ternary complex formed between pn2 - 3 , -tubulin , and d1 ( denoted the d1-tubulin - pn2 - 3 complex ) to 2.2 resolution by x - ray crystallography ( figures 1a1c and table s2 ) . as reported previously for other structures , the d1 molecule was bound at the tip of -tubulin in the d1-tubulin - pn2 - 3 complex structure , a location involved in longitudinal tubulin - tubulin contacts within microtubules ( pecqueur et al . , 2012 ) . in addition , we found electron densities for residues 373385 of pn2 - 3 , which correspond to the evolutionarily conserved sac domain of cpap ( for similar in sas-4 and cpap ; leidel and gnczy , 2003 , hsu et al . 2011 ) ( figure 1b ) . in the pn2 - 3-tubulin - d1 complex structure , we found that sac residues are engaged with both - and -tubulin subunits without affecting the overall conformation of the dimer ( root - mean - square deviation of tubulin in the tubulin - d1 [ pdb : 4drx ] and pn2 - 3-tubulin - d1 complex structures : 0.49 over 760 c atoms ) . the sac - binding site on tubulin is shaped by hydrophobic and polar residues of helices h11 , h3 , h5 , and h12 , and loops h8-s7 and h11-h12 ( figure 1c ) . the sac - tubulin interaction is characterized by an extensive water and non - water - mediated hydrogen - bonding network , as well as by hydrophobic contacts established between both main - chain and side - chain atoms of sac and tubulin residues . prominent sac side - chain contacts involve phe375 ( asp414 , met416 , glu417 , glu420 ) , leu376 ( his192 , qln193 , glu196 ) , lys377 ( glu420 ) , arg378 ( glu420 , asn426 ) , and phe385 ( h406 , trp407 , pro263 ) . pull - down experiments had implicated lys377 and arg378 in tubulin binding ( hsu et al . , 2008 ) , and our findings reveal the structural basis for their importance in this interaction . previous studies suggested that pn2 - 3 inhibits exchange of the nucleotide on -tubulin and that residues situated n - terminal to sac also interact with tubulin ( cormier et al . the d1-pn2 - 3-tubulin structure revealed that the n terminus of sac is located close to the d1-binding site at the tip of -tubulin ( figure 1d ) . we therefore reasoned that the presence of d1 might hinder access of this region of cpap to its binding site . accordingly , isothermal titration calorimetry ( itc ) experiments demonstrated that whereas pn2 - 3 bound tubulin with an equilibrium dissociation constant ( kd ) of 47 6 nm , the affinity of pn2 - 3 for tubulin - d1 dropped one order of magnitude ( figure 2a ) . likewise , eribulin and maytansine , two drugs that bind near the d1- and nucleotide - binding site , and which also inhibit longitudinal tubulin - tubulin contacts in microtubules ( pecqueur et al . , 2012 , , 2014 , alday and correia , 2009 ) , significantly impaired the affinity of pn2 - 3 for tubulin ( figure 2a ) . we conclude that the n - terminal part of pn2 - 3 binds to a site on the tip of -tubulin engaged in longitudinal tubulin - tubulin interactions ( cormier et al . , 2009 ) ; we therefore named this putative domain lid . to test whether the hydrolysis state of the exchangeable nucleotide bound to -tubulin is important for tubulin - pn2 - 3 complex formation , as previously suggested for pn2 - 3 of drosophila dmsas-4 ( gopalakrishnan et al . , 2012 ) , we performed additional itc experiments . as shown in figure s1 , we found similar kd values for the interaction between human cpap pn2 - 3 and guanosine diphosphate ( gdp)- , guanosine triphosphate ( gtp)- , or gmpcpp - tubulin ( maximal difference of 1.3-fold ) . we conclude that the hydrolysis state of the nucleotide bound to -tubulin has at most a minor effect on tubulin - pn2 - 3 complex formation . to assess whether sac and lid can bind tubulin independently , we generated two corresponding peptides , sacp and lidp , and analyzed their tubulin - binding properties by itc . kd values in the low micromolar range were obtained for the interactions between tubulin and either sacp or lidp ( figure 2b ) . to investigate the importance of selected sac and lid residues for tubulin binding , we conducted further itc experiments with mutant variants of the pn2 - 3 domain . mutation of the tubulin - interacting sac residues lys377 and arg378 to glutamic acid ( kr / ee ) , or of phe375 and phe385 to alanine ( ff / aa ) , reduced the affinity of pn2 - 3 for tubulin by two orders of magnitude ( figure 2c ; compare with wild - type pn2 - 3 in figure 2a ) . we also tested a pn2 - 3 mutant in which three residues in a conserved region of lid ( phe338 , glu339 , tyr341 ; figure 1a ) were simultaneously mutated to alanine ( fey / aaa ) , and also in this case obtained a kd in the low micromolar range ( figure 2c ) . these results suggest that both sac and lid can bind independently to tubulin with low micromolar affinities , and that they cooperate to give rise to a 100-fold tighter interaction with tubulin when present together . to test whether sac and lid could bind in the context of microtubules , we used an atomic model of a microtubule based on a cryoelectron microscopy reconstruction at 3.5- resolution ( zhang et al . , 2015 ) . interestingly , this analysis showed that both sac and lid binding interfaces are located on the outer surface , at the distal tip of the microtubule , which has exposed -tubulin subunits ( figure 2d ) . this result indicates that cpap could specifically target microtubule plus ends via its pn2 - 3 domain . to test the idea that cpap targets microtubule plus ends , we performed in vitro reconstitution experiments whereby dynamic microtubules were grown from gmpcpp - stabilized seeds and imaged using a total internal reflection fluorescence ( tirf ) microscopy - based assay ( bieling et al . since purified full - length cpap was insoluble in our hands , we engineered a soluble chimeric protein in which the pn2 - 3-mbd moiety was fused to the leucine zipper domain of the yeast transcriptional activator gcn4 ( o'shea et al . , 1991 ) to mimic the dimerization imparted by the endogenous coiled - coil domain of cpap ( zhao et al . , 2010 ) , which is required for cpap function in centriole duplication ( kitagawa et al . , 2011 ) , as well as to gfp ( the resulting protein has been dubbed cpapmini ; figures 3a and s2a ) . we found that cpapmini bound weakly to the microtubule lattice ; importantly , in addition , we found that cpapmini localized to , and tracked , one end of dynamic microtubules specifically ( figures 3b3e ) . we determined that cpapmini bound the plus end of microtubules , distinguished as such because it is negative for the minus - end targeting protein camsap3 ( figure 3d ) ( jiang et al . , 2014 ) . cpapmini co - localized at growing microtubule tips with the plus - end tracking protein eb3 ( figure 3f ) . however , in contrast to eb3 , the plus - end accumulation of which critically depends on microtubule growth ( bieling et al . , 2007 , montenegro gouveia et al . , 2010 ) , cpapmini was enriched at one end of gmpcpp - stabilized microtubules even when soluble tubulin was present at a concentration insufficient for microtubule elongation ( 5 instead of 15 m tubulin ; figure s2b ) . these data reveal that microtubule growth is not required for plus - end recognition by cpapmini . in the absence of soluble tubulin , cpapmini localized along the entire length of gmpcpp - stabilized microtubules ( data not shown ) , indicating that the interaction of cpapmini with soluble tubulin suppresses its binding to the microtubule lattice . overall , we conclude that cpapmini is an autonomous microtubule plus - end tracking protein . strikingly , we found that cpapmini strongly reduced the rate of microtubule growth ( figures 3e and 3h ) . this effect could not be explained by tubulin sequestration , because the concentration of cpapmini in these experiments ( 75100 nm ) was much lower than that of tubulin ( 15 m ) . furthermore , cpapmini dramatically reduced the frequency of catastrophes and promoted rescues , together leading to highly processive microtubule polymerization ( figures 3e , 3i , and 3j ) . this effect was also observed in the presence of eb3 , despite the fact that eb3 itself causes a 1.5-fold increase in microtubule growth rate and a 3-fold increase in catastrophe frequency ( figures 3f and 3k3 m ) . to test whether the artificial dimerization domain of gcn4 in cpapmini has an influence on microtubule dynamics , we purified a cpap construct that contains the endogenous coiled coil ( cpaplong ; figures s2a and s2c ) . although cpaplong had a higher propensity for degradation and aggregation , it also bound to microtubule plus ends , very similarly to cpapmini , and had a similar effect on microtubule dynamics : it reduced both the growth rate and the catastrophe frequency , and increased the rescue frequency ( figures s2d these data demonstrate that gcn4 links cpapmini polypeptide chains in a fashion similar to that of the endogenous coiled - coil domain . next , we tested the effect of mutations that disrupt either the tubulin - sac ( kr / ee , ff / aa ) or the tubulin - lid interaction ( lid , fey / aaa ) on the ability of cpapmini to regulate microtubule plus - end dynamics ( figures 1a and s2a ) . we found that all four mutants abrogated tip enrichment , as well as the effects on microtubule growth and catastrophes ( figures 3g3l and s2h ) . in contrast , all mutants , as well as the dimeric version of mbd alone ( pn2 - 3 ) , were still able to bind to the microtubule lattice and induce rescues to an extent similar to that of wild - type cpapmini ( figures 3g3 m ) . this result suggests that the mbd has microtubule - stabilizing properties . to further assess the mechanism underlying the activity of cpapmini , we investigated how its concentration affects microtubule dynamics . as mentioned above , at 75 nm of cpapmini , though not at lower concentrations , both the microtubule growth rate and the catastrophe frequency were strongly reduced ( figures 3h3l , 4a , and 4b ) . a 4-fold increase in concentration of cpapmini had little additional effect on the microtubule growth rate and catastrophe frequency , indicating that the effect of cpapmini on microtubule elongation in vitro saturates at 75 nm ( figures 4a and 4b ) . we next set out to determine the number of cpapmini molecules at microtubule plus ends . single - molecule fluorescence intensity analysis of cpapmini in comparison with gfp confirmed that cpapmini is a dimer ( figure 4c ) . to determine the number of cpapmini molecules at one microtubule tip , we immobilized single cpapmini molecules on the surface of one flow chamber and performed the in vitro reconstitution assay in the adjacent chamber on the same coverslip . images of unbleached cpapmini single molecules were acquired first , after which time - lapse imaging of the in vitro assay with cpapmini was performed using the same illumination and imaging conditions . we found that approximately two dimers of cpapmini were present at microtubule tips in the presence of 100 or 300 nm protein , respectively ( figures 4d and 4e ) . our tubulin - pn2 - 3 structural model suggests that cpapmini , which contains two pn2 - 3 moieties , interacts with microtubule plus ends by binding to terminal -tubulin subunits ( figure 2d ) . our reconstitution data thus indicate that cpapmini can reach its full activity with respect to microtubule growth inhibition and catastrophe suppression by binding to approximately four protofilaments . to acquire mechanistic insights into cpapmini association with the microtubule tip and lattice , we mixed 5 nm cpapmini - gfp ( figure 4f ) with 100 nm cpapmini - mcherry . this approach allowed us to observe the behavior of single cpapmini - gfp molecules in conditions whereby the protein inhibits microtubule growth . rapid imaging of such samples showed that in most cases cpapmini directly associated with the microtubule plus end , where it remained stationary and then detached . occasionally , we observed cpapmini molecules diffusing toward or away from the microtubule tip ( figure 4f ) . the analysis of binding events of cpapmini at growing microtubule plus ends yielded an exponential dwell - time distribution with a mean value of 1.7 s ( corrected for photobleaching [ helenius et al . the dwell time for cpapmini was longer than those observed previously for eb1 , eb3 , and clip-170 in similar conditions ( values ranged between 0.05 and 0.3 s [ bieling et al . we also observed binding and unbinding of cpapmini to the microtubule lattice , with an average dwell time of 0.5 s ( figures 4 g , s3a , and s3b ) . on the lattice we noted that a single cpapmini molecule could switch between the two types of behavior ( figure 4f ) . automated single - particle tracking combined with mean squared displacement ( msd ) analysis indicated that the mobile cpapmini molecule population undergoes one - dimensional diffusion , as the increase of the msd value over time was linear ( figures s3c s3e ) ( qian et al . , 1991 ) . the diffusion coefficient of cpapmini bound to microtubule lattices was 0.03 0.0004 m s ( table s3 ) . this value is three times lower than that for eb3 and similar to the one for the kinesin-13 mcak , obtained under similar conditions ( montenegro gouveia et al . , 2010 ) . taken together , these results demonstrate that cpapmini localizes to microtubule plus ends mostly through direct binding and remains largely immobile at the microtubule tip until detachment . this behavior , as well as the longer dwell times at the microtubule plus end compared with the lattice , can be explained by the presence of the lid domain , which can attach cpapmini to the tip of a protofilament . our data further show that cpapmini can either diffuse along the lattice or bind to it in a stationary manner . we think that these two types of cpapmini behavior might be due to the presence of the mbd and sac domains , which can independently interact with microtubule lattices . finally , our results demonstrate that cpapmini suppresses microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues . in combination with the structural and biophysical data , our reconstitution data suggest that cpapmini ensures slow processive microtubule growth by capping and stabilizing the plus ends of protofilaments ( figure 4h ) . our in vitro observations raised the possibility that cpap could contribute to centriole formation in two ways : the mbd could promote centriolar microtubule assembly by stabilizing microtubules , while lid , together with sac , might limit centriolar microtubule elongation by slowing growth of their plus ends . to test these predictions in the cellular context , we generated cell lines conditionally expressing yfp - tagged cpap transgenes resistant to rnai targeting endogenous cpap ( kitagawa et al . , 2011 ) . we analyzed mitotic cells using the centriolar marker centrin 2 as a readout of successful centriole assembly . whereas > 90% of control cells contained 4 centrioles , this was the case for fewer than 10% of cells depleted of endogenous cpap ( figures 5a , 5b , 5i , and s4a ) ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . yfp - cpap rescued this phenotype to a large extent , with > 80% of cells having successfully duplicated their centrioles ( figures 5c , 5i , and s4a ) ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , 2009 ) . strikingly , we found that the removal of mbd ( mbd ) completely abolished centriole assembly , to levels that are even lower than depletion of cpap alone , likely due to dominant - negative effects following heterodimerization of yfp - cpap-mbd with any residual endogenous cpap ( figures 5d , 5i , and s4a ) . we conclude that the microtubule - stabilizing effect of mbd observed in vitro is essential for centriole biogenesis . we found also that cpap constructs lacking the lid domain or with key residues mutated , yfp - cpap-lid and fey / aaa , respectively , were able to sustain centriole assembly in 65% of cells ( figures 5g5i ) , indicating that lid contributes to , but is not essential for , this process . in contrast , cpap function was severely compromised upon mutation of sac ( kr / ee or ff / aa ; figures 5e , 5f , and 5i ) , in line with previous findings ( hsu et al . , 2008 , impaired function of all cpap mutants likely reflects an impact on protein activity , because localization and turnover at the centrosome were unaffected ( figures 5c5h and s4b ) . overall , these results establish that mbd and sac are critical for centriole assembly , whereas lid appears to play an accessory role in this process . we further investigated the function of sac and lid by analyzing cells depleted of endogenous cpap and overexpressing yfp - cpap variants , which provided us with an assay system to address the contribution of distinct domains of cpap to centriole biogenesis . in these conditions , wild - type yfp - cpap leads to the formation of overly long centrioles that contain not only cpap but also other centriolar markers such as centrin 2 along their entire length ( figures 6a and 6d ) , as reported for cells overexpressing wild - type cpap in addition to having the endogenous protein ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , in contrast , we found no overly long centrioles upon yfp - cpap-mbd overexpression ( figures s4c and s4d ) , as anticipated from the fact that mbd is essential for centriole assembly . mutation of sac ( kr / ee or ff / aa ) led to a reduction in the proportion of cells with overly long centrioles while deletion of lid had no such effect , again reflecting the respective impact of these variants on the ability of cpap to sustain regular centriole assembly ( figures 6a , 6d , s4c , and s4d ) . intriguingly , close examination of cells depleted of endogenous cpap and overexpressing either yfp - cpap - ff / aa or yfp - cpap-lid revealed the presence of extended yfp - positive fibers that stemmed from centrosomes , but which did not contain centriolar markers along their length ( figures 6a and 6c ) . to determine whether such centrosomal fibers originated from centrioles or from the pericentriolar matrix , we conducted immunofluorescence experiments with a marker surrounding the proximal end of parental centrioles ( cep63 ) and one marking the distal end of all centrioles ( centrin 2 ) . this analysis revealed that 80% of centrosomal fibers appeared to be continuations of the distal end of regular centrioles or of overly long centrioles , and were thus termed centriole fibers ( figures 6e and 6f ) . these fibers , lacking centrin , were often considerably longer than overly long centrioles bearing centrin ( figure 6h ) and were positive for -tubulin ( figures 6i and 6j ) , raising the possibility that they correspond to abnormal extensions of centriolar microtubules . to test this hypothesis , we carried out correlative light and electron microscopy ( clem ; figures 7 and s5 ) . in line with previous observations ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , 2009 ) , we found that cells overexpressing yfp - cpap and depleted of endogenous cpap harbored overly long centrioles , in which microtubules extended from the distal end of the centriole ( figures 7b and 7f ; compare with 7a and 7e ) . strikingly , cells expressing yfp - cpap-lid also exhibited microtubule extensions from the distal end of the centriole , which attained several microns in some cases ( figures 7c and 7 g ) . to ensure that such figures represented centriole fibers ( i.e. , devoid of centrin ) and not overly long centrioles ( i.e. , bearing centrin ) , we carried out further clem experiments using tagrfp - centrin 1 as an additional marker . in agreement with our immunofluorescence analysis , we found that yfp - cpap-lid centriole fibers were indeed microtubules extending from the distal end of centrioles ( figures 7d and 7h ) . we conclude that the activities of lid and sac can prevent aberrant overextension of centriolar microtubules . our study provides fundamental insights into the mechanisms by which the evolutionarily conserved family of cpap / sas-4 proteins regulate the growth of centriolar microtubules , thus contributing to setting organelle size . whereas the crystal structure of the g box of cpap , either alone or in complex with a peptide derived from the centriolar protein stil , has been solved ( zheng et al . , 2014 , , 2013 , hatzopoulos et al . , 2013 ) , high - resolution structural information on the evolutionarily conserved domains interacting with tubulin and microtubules was lacking , precluding full understanding of how cpap regulates centriole biogenesis . the structural and biophysical data presented here reveal a dual binding mode between tubulin and the lid plus sac domains of pn2 - 3 , explaining its tubulin - sequestering and microtubule - destabilizing activities ( hsu et al . the finding that lid binds at the tip of -tubulin , in combination with the structural model suggesting that pn2 - 3 can bind the terminal -tubulin subunits on the outside of a microtubule , strongly suggested that pn2 - 3 represents an autonomous microtubule plus - end targeting domain in cpap . to test whether this is the case , we performed dynamic microtubule reconstitution experiments and indeed found that the sequential arrangement of lid , sac , and mbd in a dimeric configuration localizes cpapmini to growing microtubule plus ends . the plus - end localization of cpapmini is unlikely to depend on co - polymerization with tubulin , because tip localization is observed at cpapmini concentrations that are several orders of magnitude lower than that of tubulin . analysis of the behavior of single cpapmini molecules in our in vitro reconstitution system showed that the majority of cpapmini molecules bound to microtubule plus ends directly . cpapmini also displayed one - dimensional diffusion along the microtubule lattice , as described previously for other microtubule - end - interacting proteins ( brouhard et al . 2010 ) ; however , diffusion did not seem to be a major contributing factor responsible for the recruitment of cpapmini to microtubule plus ends . notably , in the context of a centriole , cpap is expected to be organized in a precise manner along the inside of the centriole wall where it binds stil , and is thus very likely not diffusive ( hatzopoulos et al . , 2013 ) . our reconstitution data revealed that depending on the conditions , microtubule - tip - localized cpapmini slows down microtubule growth 5- to 8-fold and that only a few cpapmini molecules suffice to induce this effect . based on the structural and biophysical results , we propose that the lid and sac domains of cpapmini bind terminal -tubulin subunits exposed at the microtubule plus ends and thereby occlude the binding sites for the incoming tubulin dimers . the ability of pn2 - 3 to block longitudinal tubulin - tubulin interactions is reminiscent of the tubulin - sequestering protein stathmin and the microtubule plus - end capping ligands darpin d1 , eribulin , and maytansine ( gigant et al . , 2000 , pecqueur et al . , 2012 , smith et al . , 2010 , prota et al . , 2014 ) however , in striking contrast to these microtubule - destabilizing and polymerization - blocking agents , cpapmini stabilizes microtubules by potently suppressing catastrophes and promoting rescues . the latter property can be attributed to the mbd , which binds strongly to the microtubule lattice and can autonomously induce rescues . the mbd is positively charged , and its binding to negatively charged microtubules likely involves an electrostatic mechanism , similar to that of microtubule lattice - binding regions of cytoplasmic microtubule regulators such as xmap215/ch - tog ( brouhard et al . , 2008 , widlund et al . , 2011 ) . we assume that the mbd of cpapmini binds along , as well as between , protofilaments to stabilize microtubules . based on these considerations , we conclude that this particular mode of binding to microtubule plus ends , due to the combination of lid , sac , and mbd , can cap and stabilize terminal tubulin dimers ( figure 4h ) . we postulate that in this state , a capped protofilament can only be elongated by an incoming tubulin dimer if the lid domain is released and frees the interaction site needed for establishing longitudinal tubulin - tubulin contacts . the release of lid in turn weakens the cpapmini microtubule plus - end interaction and either promotes cpapmini detachment or enables the liberated pn2 - 3 domain to cap the newly elongated protofilament or a neighboring one . notably , slowing microtubule growth normally promotes catastrophes by reducing the size of the gtp cap ( walker et al . , 1988 , janson et al . , 2003 however , exceptions to this rule are known , including the kinesin-4 family members xklp1/kif4 and kif21a which , similarly to cpapmini , can suppress both microtubule growth and catastrophes ( bringmann et al . the molecular basis of kinesin-4-mediated microtubule growth inhibition is not understood , but is very likely distinct from that of cpapmini , because both xklp1/kif4 and kif21a are molecular motors that can move along microtubules and accumulate at microtubule plus ends due to their processive motility ( bieling et al . , 2010 , in contrast , cpap presents a unique arrangement of tubulin - binding and microtubule - lattice - binding domains , which together have the potential to recognize , cap , and stabilize microtubule plus ends , a mechanism that to our knowledge has not been previously described for any microtubule regulator . in cells , centriolar microtubules grow very slowly and processively as judged from the analysis of specimens prepared from different stages of the cell cycle ( kuriyama and borisy , 1981 , chretien et al . our study reveals that cpap plays a critical dual role in ensuring that this is the case . first , our data indicate that a pivotal function of cpap is to stabilize centriolar microtubules via its mbd , which likely explains why this domain is essential for centriole assembly . second , although the capacity of lid to cap microtubule plus ends is not essential for this stabilization function , and thus for centriole formation , it exerts a negative role by limiting microtubule extension from the distal end of centrioles . the sac domain , which interacts with the lateral side of tubulin dimers , can contribute to both microtubule stabilization and capping functions , potentially explaining why mutations in sac affect centriole formation more strongly than those in lid . these considerations can at least partially explain why centrioles overelongate when cpap dosage is increased ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , 2009 ) ; however , additional mechanisms likely control the activity of cpap domains in cells . our in vitro data revealed that approximately two cpapmini dimers are sufficient to limit the growth rate of a single microtubule , suggesting that cpap works at substoichiometric levels . in the context of the centriolar microtubule wall , we expect approximately two cpap dimers to be able to access the microtubule tips of each triplet from the luminal side of the centriole ( figure s6 ) ( hatzopoulos et al . , 2013 , sonnen et al . , 2012 , overall , our results reveal that cpap is a highly specialized microtubule plus - end regulator that acts as a molecular cap to ensure slow and processive growth of centriolar microtubules . given that endogenous cpap is present primarily in the proximal region of mature centrioles ( kohlmaier et al . , 2009 , schmidt et al . , 2009 , tang et al . , 2009 ) , we propose that cpap activity is most critical during the early stages of centriole elongation . upon overexpression of the wild - type protein , excess cpap molecules localize along the entire length of centrioles and could cause overelongation of centriolar microtubules due to the microtubule stabilization activity of ectopic cpap . furthermore , we speculate that upon overexpression of a mutant protein lacking lid - domain function , overelongation of centriolar microtubules is exacerbated due to the lack of capping function ( figure s6 ) . we note that microtubule growth rates obtained in vitro upon cpapmini addition are still some three orders of magnitude faster than those estimated for centriolar microtubules in cells ( kuriyama and borisy , 1981 , chretien et al . , 1997 ) . regions of cpap outside of the microtubule- and tubulin - binding domains , including the g box , that may organize cpap molecules along the entire length of the microtubule wall in a precise manner ( hatzopoulos et al . , 2013 ) , as well as post - translational modifications , could be important to further bolster cpap activity ; these features are not recapitulated by the cpapmini and cpaplong constructs used in our study . in addition , other centriolar proteins have been implicated in controlling centriole length , including the cpap - interacting proteins cep120 , cep135 , and centrobin ( gudi et al . , 2011 , lin et al . , 2013a , lin et al . it will be of prime interest to assess on a mechanistic level how these proteins cooperate to impact on cpap function or otherwise contribute to regulate organelle size . proteins were buffer - exchanged to brb80 ( 80 mm pipes - koh [ ph 6.8 ] supplemented with 1 mm mgcl2 and 1 mm egta ) supplemented with 0.5 mm tris(2-carboxyethyl)phosphine . 0.10.4 mm pn2 - 3 variants in the syringe were injected stepwise into 1020 m tubulin solutions in the cell . the resulting heats were integrated and fitted in origin ( originlab ) using the standard structure solution by x - ray crystallography is described in full in the supplemental experimental procedures . in brief , equimolar amounts of d1 , pn2 - 3 , and subtilisin - treated tubulin were mixed and the pn2 - 3-tubulin - d1 complex was concentrated to 20 mg / ml . pn2 - 3-tubulin - d1 samples were complemented with 0.2 mm gdp , 1 mm colchicine , and 5 mm dtt before setting up sitting - drop vapor diffusion crystallization trials . crystals were obtained in a condition containing 20% polyethylene glycol ( peg ) 550 monomethyl ether and 0.1 m 2-(n - morpholino)ethanesulfonic acid ( ph 6.5 ) . x - ray diffraction data were collected at 100 k at beamline x06da at the swiss light source . the pn2 - 3-tubulin - d1 structure was solved by molecular replacement using the -tubulin - d1 complex structure as a search model ( pdb : 4drx ) . protein purification for in vitro reconstitution assays is described in full in supplemental experimental procedures . reconstitution of microtubule growth dynamics in vitro was performed as described previously ( montenegro gouveia et al . , 2010 ) . in brief , flow chambers were functionalized by sequential incubation with 0.2 mg / ml pll - peg - biotin ( susos ) and 1 mg / ml neutravidin ( invitrogen ) in mrb80 buffer ( 80 mm piperazine - n , n-bis(2-ethanesulfonic acid ) [ ph 6.8 ] supplemented with 4 mm mgcl2 and 1 mm egta ) . gmpcpp - microtubule seeds were attached to coverslips through biotin - neutravidin interactions . the reaction mix with or without cpapmini proteins ( mrb80 buffer supplemented with 15 m tubulin , 0.5 m rhodamine - tubulin , 50 mm kcl , 1 mm guanosine triphosphate , 0.2 mg / ml -casein , 0.1% methylcellulose , and oxygen scavenger mix [ 50 mm glucose , 400 g / ml glucose oxidase , 200 g / ml catalase , and 4 mm dtt ] ) was added to the flow chamber . flow chambers were sealed and dynamic microtubules were imaged immediately at 30c using tirf microscopy . intensity analysis for cpapmini along microtubules , single - molecule fluorescence intensity analysis of cpapmini , cpapmini molecule counting at microtubule tips , analysis of microtubule plus - end dynamics , and statistical analysis procedures are all described in full in supplemental experimental procedures . cells were grown on glass coverslips and fixed in methanol for 7 min at 20c . cold treatment was carried out by incubating cells in ice - cold pbs , on ice , for 1 hr before fixation . triton x-100 , washed in pbs and 0.01% triton x-100 , and blocked for 1 hr in pbs , 1% bsa , and 2% fetal calf serum . all antibodies were diluted in the blocking solution and incubated for either 1 hr at room temperature or 12 hr at 4c . primary antibodies were mouse anti--tubulin ( dm1a , sigma ) , mouse 20h5 anti - centrin-2 ( a gift from j. salisbury ) , rabbit anti - hpoc5 ( azimzadeh et al . , 2009 ; a gift from m. bornens ) , rabbit anti - cep63 ( millipore 06 - 1292 ) , goat anti - gfp ( abcam ab6673 ) , and rabbit anti - gfp ( a gift from v. simanis ) . secondary antibodies were goat anti - rabbit alexa fluor 488 , goat anti - mouse alexa fluor 568 , donkey anti - goat alexa fluor 488 , donkey anti - mouse alexa fluor 568 , and donkey anti - rabbit alexa fluor 647 ( invitrogen ) . all primary antibodies were diluted 1,000-fold , except centrin 2 ( 2000 ) and anti--tubulin ( 5000 ) . samples were washed three times between , and after , antibody incubations and incubated with 1 g / ml hoechst in pbs prior to mounting in pbs , 90% glycerol , and 4% n - propyl gallate . confocal imaging was carried out using a zeiss lsm 700 microscope with a plan - apochromat 63 oil - immersion objective , na 1.40 . all images shown are maximum - intensity projections and were processed using fiji ( schindelin et al . , 2012 ) , maintaining relative intensities within a series . endogenous cpap was depleted by rnai for 72 hr , simultaneous with induction of the transgene . for dual marker experiments , cells were transfected with a tagrfp - centrin 1 expression vector 16 hr prior to fixation . cells were fixed , then washed thoroughly with 0.1 m cacodylate buffer ( ph 7.4 ) , and imaged by light microscopy . immediately afterward , samples were post - fixed for 40 min in 1.0% osmium tetroxide , then 30 min in 1.0% uranyl acetate in water , before being dehydrated through increasing concentrations of alcohol and then embedded in durcupan acm resin ( fluka ) . the coverslips were then placed face down on a glass slide coated with mold - releasing agent ( glorex ) , with approximately 1 mm of resin separating the two . these regions were mounted on blank resin blocks with acrylic glue and trimmed with glass knives to form a block ready for serial sectioning . series of between 150 and 300 thin sections ( 50 nm thickness ) were cut with a diamond knife mounted on an ultramicrotome ( leica uc7 ) . these sections were contrasted with lead citrate and uranyl acetate and images taken using an fei spirit transmission electron microscope equipped with an eagle ccd camera .

summarycentrioles are fundamental and evolutionarily conserved microtubule - based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules . several centriolar proteins can interact with tubulin or microtubules , but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious . here , we bring together crystallographic , biophysical , and reconstitution assays to demonstrate that the human centriolar protein cpap ( sas-4 in worms and flies ) binds and caps microtubule plus ends by associating with a site of -tubulin engaged in longitudinal tubulin - tubulin interactions . strikingly , we uncover that cpap activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues . we further establish that the capping function of cpap is important to limit growth of centriolar microtubules in cells . our results suggest that cpap acts as a molecular lid that ensures slow assembly of centriolar microtubules and , thereby , contributes to organelle length control .

Introduction Results Discussion Experimental Procedures Author Contributions

centrioles are evolutionarily conserved organelles that are pivotal for the formation of cilia , flagella , and centrosomes , and are thus critical for fundamental cellular processes such as signaling , polarity , motility , and division ( reviewed in azimzadeh and marshall , 2010 , bornens , 2012 , jana et al . centriolar microtubules are unique in exhibiting exceptionally slow growth rates of a few tens of nanometers per hour on average , and in being very stable after their formation ( kuriyama and borisy , 1981 , chretien et al . the behavior of centriolar microtubules is in stark contrast to that of their cytoplasmic counterparts , which assemble up to four orders of magnitude faster and are highly dynamic ( kinoshita et al . the molecular mechanisms that impart the exceptional slow growth rate and stability of centriolar microtubules are not known . several centriolar proteins that can directly interact with tubulin and/or microtubules have been identified , including cep120 , cep135 , centrobin , and cpap ( gudi et al . cpap ( sas-4 in worms and flies ) is of particular interest , since it is the only component among the ones listed above that is present and essential for centriole formation from worm to man ( kohlmaier et al . , 2009 , the importance of cpap is further substantiated by the fact that homozygous mutations in the corresponding gene lead to autosomal recessive primary microcephaly , a devastating human disease with drastically reduced neuron numbers and , thus , brain size ( bond et al . together , these observations suggest that cpap somehow regulates centriolar microtubule growth to produce proper centrioles . cpap comprises a tubulin - binding domain ( pn2 - 3 ) , a positively charged microtubule - binding domain ( mbd ) , a coiled - coil dimerization domain , and a c - terminal g box ( figure 1a ) . here , to elucidate the fundamental mechanism of action of cpap / sas-4 proteins , we set out to decipher how cpap affects microtubules using structural , biophysical , and cell biological approaches . using reconstitution experiments , we demonstrate that cpap autonomously recognizes and tracks growing microtubule plus ends . there , cpap suppresses microtubule polymerization and increases microtubule stability by inhibiting catastrophes and promoting rescues . we further establish that the pn2 - 3 domain of cpap is critical in the cellular context to restrict the extent of centriolar microtubule elongation by acting as a molecular cap . to gain insight into the molecular mechanism of tubulin binding by cpap / sas-4 proteins , we sought to crystallize the pn2 - 3 domain of human cpap in complex with tubulin ( see table s1 for all constructs generated in the course of this study ) . as reported previously for other structures , the d1 molecule was bound at the tip of -tubulin in the d1-tubulin - pn2 - 3 complex structure , a location involved in longitudinal tubulin - tubulin contacts within microtubules ( pecqueur et al . in addition , we found electron densities for residues 373385 of pn2 - 3 , which correspond to the evolutionarily conserved sac domain of cpap ( for similar in sas-4 and cpap ; leidel and gnczy , 2003 , hsu et al . in the pn2 - 3-tubulin - d1 complex structure , we found that sac residues are engaged with both - and -tubulin subunits without affecting the overall conformation of the dimer ( root - mean - square deviation of tubulin in the tubulin - d1 [ pdb : 4drx ] and pn2 - 3-tubulin - d1 complex structures : 0.49 over 760 c atoms ) . the sac - tubulin interaction is characterized by an extensive water and non - water - mediated hydrogen - bonding network , as well as by hydrophobic contacts established between both main - chain and side - chain atoms of sac and tubulin residues . previous studies suggested that pn2 - 3 inhibits exchange of the nucleotide on -tubulin and that residues situated n - terminal to sac also interact with tubulin ( cormier et al . the d1-pn2 - 3-tubulin structure revealed that the n terminus of sac is located close to the d1-binding site at the tip of -tubulin ( figure 1d ) . accordingly , isothermal titration calorimetry ( itc ) experiments demonstrated that whereas pn2 - 3 bound tubulin with an equilibrium dissociation constant ( kd ) of 47 6 nm , the affinity of pn2 - 3 for tubulin - d1 dropped one order of magnitude ( figure 2a ) . likewise , eribulin and maytansine , two drugs that bind near the d1- and nucleotide - binding site , and which also inhibit longitudinal tubulin - tubulin contacts in microtubules ( pecqueur et al . we conclude that the n - terminal part of pn2 - 3 binds to a site on the tip of -tubulin engaged in longitudinal tubulin - tubulin interactions ( cormier et al . to test whether the hydrolysis state of the exchangeable nucleotide bound to -tubulin is important for tubulin - pn2 - 3 complex formation , as previously suggested for pn2 - 3 of drosophila dmsas-4 ( gopalakrishnan et al . we conclude that the hydrolysis state of the nucleotide bound to -tubulin has at most a minor effect on tubulin - pn2 - 3 complex formation . to assess whether sac and lid can bind tubulin independently , we generated two corresponding peptides , sacp and lidp , and analyzed their tubulin - binding properties by itc . mutation of the tubulin - interacting sac residues lys377 and arg378 to glutamic acid ( kr / ee ) , or of phe375 and phe385 to alanine ( ff / aa ) , reduced the affinity of pn2 - 3 for tubulin by two orders of magnitude ( figure 2c ; compare with wild - type pn2 - 3 in figure 2a ) . these results suggest that both sac and lid can bind independently to tubulin with low micromolar affinities , and that they cooperate to give rise to a 100-fold tighter interaction with tubulin when present together . this result indicates that cpap could specifically target microtubule plus ends via its pn2 - 3 domain . to test the idea that cpap targets microtubule plus ends , we performed in vitro reconstitution experiments whereby dynamic microtubules were grown from gmpcpp - stabilized seeds and imaged using a total internal reflection fluorescence ( tirf ) microscopy - based assay ( bieling et al . , 1991 ) to mimic the dimerization imparted by the endogenous coiled - coil domain of cpap ( zhao et al . we found that cpapmini bound weakly to the microtubule lattice ; importantly , in addition , we found that cpapmini localized to , and tracked , one end of dynamic microtubules specifically ( figures 3b3e ) . overall , we conclude that cpapmini is an autonomous microtubule plus - end tracking protein . strikingly , we found that cpapmini strongly reduced the rate of microtubule growth ( figures 3e and 3h ) . furthermore , cpapmini dramatically reduced the frequency of catastrophes and promoted rescues , together leading to highly processive microtubule polymerization ( figures 3e , 3i , and 3j ) . although cpaplong had a higher propensity for degradation and aggregation , it also bound to microtubule plus ends , very similarly to cpapmini , and had a similar effect on microtubule dynamics : it reduced both the growth rate and the catastrophe frequency , and increased the rescue frequency ( figures s2d these data demonstrate that gcn4 links cpapmini polypeptide chains in a fashion similar to that of the endogenous coiled - coil domain . next , we tested the effect of mutations that disrupt either the tubulin - sac ( kr / ee , ff / aa ) or the tubulin - lid interaction ( lid , fey / aaa ) on the ability of cpapmini to regulate microtubule plus - end dynamics ( figures 1a and s2a ) . this result suggests that the mbd has microtubule - stabilizing properties . as mentioned above , at 75 nm of cpapmini , though not at lower concentrations , both the microtubule growth rate and the catastrophe frequency were strongly reduced ( figures 3h3l , 4a , and 4b ) . we next set out to determine the number of cpapmini molecules at microtubule plus ends . our tubulin - pn2 - 3 structural model suggests that cpapmini , which contains two pn2 - 3 moieties , interacts with microtubule plus ends by binding to terminal -tubulin subunits ( figure 2d ) . the analysis of binding events of cpapmini at growing microtubule plus ends yielded an exponential dwell - time distribution with a mean value of 1.7 s ( corrected for photobleaching [ helenius et al . the dwell time for cpapmini was longer than those observed previously for eb1 , eb3 , and clip-170 in similar conditions ( values ranged between 0.05 and 0.3 s [ bieling et al . taken together , these results demonstrate that cpapmini localizes to microtubule plus ends mostly through direct binding and remains largely immobile at the microtubule tip until detachment . finally , our results demonstrate that cpapmini suppresses microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues . in combination with the structural and biophysical data , our reconstitution data suggest that cpapmini ensures slow processive microtubule growth by capping and stabilizing the plus ends of protofilaments ( figure 4h ) . our in vitro observations raised the possibility that cpap could contribute to centriole formation in two ways : the mbd could promote centriolar microtubule assembly by stabilizing microtubules , while lid , together with sac , might limit centriolar microtubule elongation by slowing growth of their plus ends . whereas > 90% of control cells contained 4 centrioles , this was the case for fewer than 10% of cells depleted of endogenous cpap ( figures 5a , 5b , 5i , and s4a ) ( kohlmaier et al . strikingly , we found that the removal of mbd ( mbd ) completely abolished centriole assembly , to levels that are even lower than depletion of cpap alone , likely due to dominant - negative effects following heterodimerization of yfp - cpap-mbd with any residual endogenous cpap ( figures 5d , 5i , and s4a ) . we conclude that the microtubule - stabilizing effect of mbd observed in vitro is essential for centriole biogenesis . we found also that cpap constructs lacking the lid domain or with key residues mutated , yfp - cpap-lid and fey / aaa , respectively , were able to sustain centriole assembly in 65% of cells ( figures 5g5i ) , indicating that lid contributes to , but is not essential for , this process . we further investigated the function of sac and lid by analyzing cells depleted of endogenous cpap and overexpressing yfp - cpap variants , which provided us with an assay system to address the contribution of distinct domains of cpap to centriole biogenesis . mutation of sac ( kr / ee or ff / aa ) led to a reduction in the proportion of cells with overly long centrioles while deletion of lid had no such effect , again reflecting the respective impact of these variants on the ability of cpap to sustain regular centriole assembly ( figures 6a , 6d , s4c , and s4d ) . to determine whether such centrosomal fibers originated from centrioles or from the pericentriolar matrix , we conducted immunofluorescence experiments with a marker surrounding the proximal end of parental centrioles ( cep63 ) and one marking the distal end of all centrioles ( centrin 2 ) . these fibers , lacking centrin , were often considerably longer than overly long centrioles bearing centrin ( figure 6h ) and were positive for -tubulin ( figures 6i and 6j ) , raising the possibility that they correspond to abnormal extensions of centriolar microtubules . we conclude that the activities of lid and sac can prevent aberrant overextension of centriolar microtubules . our study provides fundamental insights into the mechanisms by which the evolutionarily conserved family of cpap / sas-4 proteins regulate the growth of centriolar microtubules , thus contributing to setting organelle size . whereas the crystal structure of the g box of cpap , either alone or in complex with a peptide derived from the centriolar protein stil , has been solved ( zheng et al . , 2013 ) , high - resolution structural information on the evolutionarily conserved domains interacting with tubulin and microtubules was lacking , precluding full understanding of how cpap regulates centriole biogenesis . the structural and biophysical data presented here reveal a dual binding mode between tubulin and the lid plus sac domains of pn2 - 3 , explaining its tubulin - sequestering and microtubule - destabilizing activities ( hsu et al . the finding that lid binds at the tip of -tubulin , in combination with the structural model suggesting that pn2 - 3 can bind the terminal -tubulin subunits on the outside of a microtubule , strongly suggested that pn2 - 3 represents an autonomous microtubule plus - end targeting domain in cpap . to test whether this is the case , we performed dynamic microtubule reconstitution experiments and indeed found that the sequential arrangement of lid , sac , and mbd in a dimeric configuration localizes cpapmini to growing microtubule plus ends . the plus - end localization of cpapmini is unlikely to depend on co - polymerization with tubulin , because tip localization is observed at cpapmini concentrations that are several orders of magnitude lower than that of tubulin . analysis of the behavior of single cpapmini molecules in our in vitro reconstitution system showed that the majority of cpapmini molecules bound to microtubule plus ends directly . based on the structural and biophysical results , we propose that the lid and sac domains of cpapmini bind terminal -tubulin subunits exposed at the microtubule plus ends and thereby occlude the binding sites for the incoming tubulin dimers . the ability of pn2 - 3 to block longitudinal tubulin - tubulin interactions is reminiscent of the tubulin - sequestering protein stathmin and the microtubule plus - end capping ligands darpin d1 , eribulin , and maytansine ( gigant et al . , 2014 ) however , in striking contrast to these microtubule - destabilizing and polymerization - blocking agents , cpapmini stabilizes microtubules by potently suppressing catastrophes and promoting rescues . based on these considerations , we conclude that this particular mode of binding to microtubule plus ends , due to the combination of lid , sac , and mbd , can cap and stabilize terminal tubulin dimers ( figure 4h ) . we postulate that in this state , a capped protofilament can only be elongated by an incoming tubulin dimer if the lid domain is released and frees the interaction site needed for establishing longitudinal tubulin - tubulin contacts . , 2003 however , exceptions to this rule are known , including the kinesin-4 family members xklp1/kif4 and kif21a which , similarly to cpapmini , can suppress both microtubule growth and catastrophes ( bringmann et al . the molecular basis of kinesin-4-mediated microtubule growth inhibition is not understood , but is very likely distinct from that of cpapmini , because both xklp1/kif4 and kif21a are molecular motors that can move along microtubules and accumulate at microtubule plus ends due to their processive motility ( bieling et al . , 2010 , in contrast , cpap presents a unique arrangement of tubulin - binding and microtubule - lattice - binding domains , which together have the potential to recognize , cap , and stabilize microtubule plus ends , a mechanism that to our knowledge has not been previously described for any microtubule regulator . in cells , centriolar microtubules grow very slowly and processively as judged from the analysis of specimens prepared from different stages of the cell cycle ( kuriyama and borisy , 1981 , chretien et al . first , our data indicate that a pivotal function of cpap is to stabilize centriolar microtubules via its mbd , which likely explains why this domain is essential for centriole assembly . second , although the capacity of lid to cap microtubule plus ends is not essential for this stabilization function , and thus for centriole formation , it exerts a negative role by limiting microtubule extension from the distal end of centrioles . , 2009 ) ; however , additional mechanisms likely control the activity of cpap domains in cells . , 2012 , overall , our results reveal that cpap is a highly specialized microtubule plus - end regulator that acts as a molecular cap to ensure slow and processive growth of centriolar microtubules . , 2009 ) , we propose that cpap activity is most critical during the early stages of centriole elongation . furthermore , we speculate that upon overexpression of a mutant protein lacking lid - domain function , overelongation of centriolar microtubules is exacerbated due to the lack of capping function ( figure s6 ) . we note that microtubule growth rates obtained in vitro upon cpapmini addition are still some three orders of magnitude faster than those estimated for centriolar microtubules in cells ( kuriyama and borisy , 1981 , chretien et al . , 2013 ) , as well as post - translational modifications , could be important to further bolster cpap activity ; these features are not recapitulated by the cpapmini and cpaplong constructs used in our study . the reaction mix with or without cpapmini proteins ( mrb80 buffer supplemented with 15 m tubulin , 0.5 m rhodamine - tubulin , 50 mm kcl , 1 mm guanosine triphosphate , 0.2 mg / ml -casein , 0.1% methylcellulose , and oxygen scavenger mix [ 50 mm glucose , 400 g / ml glucose oxidase , 200 g / ml catalase , and 4 mm dtt ] ) was added to the flow chamber . intensity analysis for cpapmini along microtubules , single - molecule fluorescence intensity analysis of cpapmini , cpapmini molecule counting at microtubule tips , analysis of microtubule plus - end dynamics , and statistical analysis procedures are all described in full in supplemental experimental procedures .

schizophrenia is a complex mental illness characterized by positive ( hallucinations , paranoia , disorganized behavior ) and negative symptoms ( social withdrawal , anhedonia , flat affect ) as well as cognitive dysfunction ( deficits in attention , learning , and memory ) . current treatments , including typical and second - generation atypical antipsychotics , are based largely upon the dopaminergic hypothesis of schizophrenia , which targets overactivation of subcortical dopamine d2 receptors . both classes treat the positive symptoms ; however , neither class of antipsychotics has made a substantial impact on the negative and cognitive symptoms . after several decades of clinical use , a significant understanding of the risks and side effect profiles that plague both classes of antipsychotics has been gained . these include extrapyramidal side effects , sexual dysfunction , weight gain ( metabolic syndrome ) , agranulocytosis , increased cardiac risk , and poor patient compliance . the development of positive allosteric modulators ( pams ) of metabotropic glutamate receptor 5 ( mglu5 ) as a novel approach to test the glutamate or n - methyl - d - aspartate ( nmda ) receptor hypofunction hypothesis of schizophrenia has provided preclinical evidence for therapeutic potential in multiple psychosis and cognitive animal models , and , for more than a decade , industry and academic drug discovery groups have been in pursuit of brain penetrant small molecule mglu5 potentiators . a testament to the success of the field has been the identification of over eight reported chemotypes with in vivo efficacy in preclinical models , beginning with 3-cyano - n-(1,3-diphenyl-1h - pyrazol-5-yl)benzamide ( cdppb , 1 ) , piperidinyl 1,2,4-oxadiazoles from addex ( adx-47273 , 2 ) , and subsequently acetylenes n - methyl-5-(phenylethynyl)pyrimidin-2-amine ( mppa , 3 ) , vu0360172 ( 4 ) , lsn2463359 ( 8) , piperazines vu0364289 ( 5 ) and 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone ( cppz , 6 ) , ether vu0404251 ( 7 ) , and triazole lsn2814617 from lilly ( 9 ) . however , recent findings from merck - addex and at the vanderbilt center for neuroscience drug discovery ( vcndd ) have unveiled a target mediated cns adverse - effect ( ae ) liability , driven by excessive glutamate fold potentation or allosteric agonism , respectively ; suggesting that pams with lower functional cooperativity with glutamate ( e.g. , glutamate fold - shift or potentiation ) and devoid of allosteric agonism may be preferred for improved therapeutic index . more recently , we disclosed a phenoxy - based ether dihydrothiazolopyridone series , with the representative in vivo tool compound vu0408899 ( 10 ) ( figure 1 ) . ether 10 represents , in part , efforts to constrain amides of type 7 ; however , due to inherent instability in acidic media ( 20% hp--cd in water , ph = 4 ) leading to the corresponding 2-hydroxy - dihydrothiazolopyridone fragment , full exploration of the analogous system containing a benzyloxy ether linkage was not possible . activity relationships ( sar ) suggested a preference for the phenoxy - based linker within the dihydrothiazolopyridones ( two examples tested ) ; however , this trend was not found within monocyclic nicotinamides of type 7 . thus , it was unknown if alteration of the linker moiety within the context of a bicyclic system to more closely mimic 7 ( e.g. , dihydronaphthyridinone core structures 1217 , figure 2 ) , would be productive for mglu5 receptor potentiation . prior studies within a dihydronaphthyridinone class utilizing an acetylene - based linker , initially identified from an hts screen , led to pams with submicromolar potency , including dihydronaphthyridinones 11 , suggested that linkers employing either ch2o or och2 might be successful as an acetylene replacement . within acetylenic dihydronaphthyridinones 11 via simple amide congeners led to fundamental changes in the mode of pharmacology ( e.g. , from pams to negative allosteric modulators or nams ) . the pursuit of ethers 1220 were undertaken as part of a larger comprehensive study aimed to target four key structure activity relationships : ( 1 ) endocyclic versus exocyclic amide , ( 2 ) phenoxy versus benzyloxy linker , ( 3 ) central ring pyridine isomers , and ( 4 ) site of the pendant group attachment to central heterocycle . it was unclear at the outset if ethers 1220 would exhibit subtle changes in the mode of pharmacology similar to 11(32 ) and other chemotypes or would maintain a higher fidelity for potentiation similar to cdppb ( 1),n-[5-chloro-2-[(1,3-dioxoisoindolin-2-yl)methyl]phenyl]-2-hydroxybenzamide ( cppha ) , piperazines ( 56 ) , and glycine sulfonamide ( ml332 ) based mglu5 pams . herein we describe the synthesis and pharmacological profile of a series of tetrahydronaphthyridine and dihydronaphthyridinone ethers ( 1220 ) as positive allosteric modulators of mglu5 . low cooperativity pam 12c ( vu0405372 ) was ultimately identified and found to demonstrate a suitable balance of in vitro and in vivo properties for proof - of - concept studies in an amphetamine hyperlocomotor challenge model . in contrast to previously reported and related monocyclic ether series , these bicyclic modulator scaffolds were overall found to exhibit narrow sar , low efficacy , and have a higher propensity for pharmacological mode switching upon chemical modification , demonstrating an sar profile reminiscent of related acetylenic pams . mglu5 receptor pams with reported efficacy in preclinical models of schizophrenia and cognition . evolution of tetrahydronaphthyridine and dihydronaphthyridinone ether based mglu5 allosteric modulators ( 1220 ) from nicotinamide 7(26 ) and acetylenic modulator scaffold 11 . to prepare bicyclic analogues that most closely mimic the structural features found in 11 , we initially prepared a series of dihydro-1,6-naphthyridin-5(6h)-ones ( 12a12 t , scheme 1 ) . starting from known compound 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 21a , supporting information ) , substitution under basic conditions in the presence of various alcohols with heating afforded 12a12h , 12q , and 12s in low to moderate yield . a subset was treated with alkylating agents under basic conditions , or with various aryl and heteroaryl halides using a copper iodide diamine ligand protocol to effect a modified ullman lactam cross - coupling to give the respective alkyl ( 12i12n , 12s12 t ) and n - aryl ( 12o12p , 12r ) lactam products . reagents and conditions : ( a ) 21a , rch2oh , kot - bu , dmf , 100 c , 416 h , 2363% ; ( b ) lactam , rch2br or rch2cl , kot - bu , dmf , 60 c , 24 h , 4075% ; ( c ) lactam , cui ( 0.2 equiv ) , ar / hetbr , n , n - dimethylethylene diamine , k2co3 , toluene , 120 c , 18 h , 3559% . evaluation of the alternate ether linkage based upon an aryloxy methyl dihydronaphthyridinone , series 13 , began synthetically from 21a as shown in scheme 2 . protection with para - methoxybenzyl chloride ( pmbcl ) afforded 21b in good yield . miyaura coupling under molander s conditions using vinyl potassium trifluoroborate salt gave vinyl dihydronaphthyridinone intermediate 22 . mitsunobu alkylation in the presence of either phenol or 3-fluorophenol provided 23a23b in 3540% yield over two steps from 22 . removal of the pmb protecting group using cerium ammonium nitrate proceeded smoothly to give 13a13b . remaining analogues 13c13i were prepared similarly as demonstrated for series 12 , using alkylating conditions or copper - based arylation conditions to give alkyl and n - aryl lactam products 13c13e and 13f13i , respectively . reagents and conditions : ( a ) 21a , pmbcl , kot - bu , dmf , 60 c , 2 h , 66% ; ( b ) vinyl potassium trifluoroborate , cs2co3 , pd(pph3)4 , etoh , 80 c , 2 h , 74% ; ( c ) o3 , ch2cl2:meoh , 78 c , 40 min , nabh4 , 0 c ; ( d ) roh , diad , pph3 , thf , 0 c , 16 h , 3540% ( over two steps ) ; ( e ) can , ch3cn : h2o , rt , 50 min , 6065% ; ( f ) 13a b , rch2br or rch2cl , kot - bu , dmf , 60 c , 24 h , 4468% ; ( g ) 13a b , cui ( 0.2 equiv ) , ar / hetbr , n , n - dimethylethylene diamine , k2co3 , toluene , 120 c , 18 h , 2589% . within a third class of lactams 14 ( scheme 3 ) , a benzyloxy linkage was retained and an alternate dihydro-1,7-naphthyridinone ring system was explored , placing the pyridine nitrogen at the position ortho to the lactam amide . synthesis of series 14 began with borane reduction of 5,6-dichloronicotinic acid 24 and subsequent cyanation using pd(0 ) catalysis to give 25 in moderate yield . treatment of 25 under mitsunobu conditions using phenol and di - tert - butyl azodicarboxylate ( dbab ) , followed by hydrolysis and ester formation , afforded chloroester 27 . allylation of 27 using stille conditions with allyl tributyltin , followed by osmium tetroxide sodium periodate mediated cleavage , provided key aldehyde intermediate 28 in good yield for the two steps . at this stage , installation of the lactam could be accomplished using a variety of amines in a one - pot two - step reductive alkylation , ring - cyclization reaction using sodium triacetoxyborohydride in thf reagents and conditions : ( a ) 24 , bh3 thf , 0 c , 16 h , 68% ; ( b ) zn(cn)2 , cs2co3 , pd(pph3)4 , dmf , 100 c , 3 h , 56% ; ( c ) dbab , phenol , pph3 , thf , 0120 c , 40 min , 80% ; ( d ) naoh , 115 c , 3 h , quant ; ( e ) ch3i , k2co3 , dmf , rt , 16 h , 73% ; ( f ) bu3sn(allyl ) , pd(pph3)4 , toluene , 125 c , 16 h , 56% ; ( g ) naio4 , oso4 , thf , 0 c rt , 2 h , 74% ; ( h ) rnh2 , thf - meoh , nabh(oac)3 , rt , 16 h , 1525% . synthesis of phenoxymethyl - dihydro-2,7-naphthyridinones 15 was accomplished using a seven - step sequence from methyl-6-methylnicotinate ( 29 ) as shown in scheme 4 and represented the fourth class of dihydronaphthyridinones investigated . chlorination followed by pyridine n - oxide formation using catalytic methyltrioxorhenium(vii ) in the presence of hydrogen peroxide gave intermediate 30 . installation of the vinyl moiety using stille conditions provided key pyridine intermediate 33 in good yield . vinylpyridine 33 smoothly underwent one - pot michael addition and lactam cyclization using ammonia or several alkyl and aryl amines to give 15a15d in moderate to excellent yield . reagents and conditions : ( a ) 29 , pocl3 , 16 h , 95 c , 68% ; ( b ) cat . ch3reo3 , h2o2 , ch2cl2 , 16 h , rt , quant ; ( c ) ac2o , 120 c , 45 min , 11% ; ( d ) k2co3 , meoh , rt , 15 min , 92% ; ( e ) dbab , phenol , pph3 , thf , 60 c , 16 h , 73% ; ( f ) bu3(vinyl)sn , pd(pph3)4 , toluene dioxane , 90 c , 16 h , 76% ; ( g ) amine / ammonia in thf or meoh , sealed tube , 100 c , 16 h , 3993% . alternative tetrahydronaphthyridines wherein the ether linkage is appended at the 3-position as a benzyloxy with an exocyclic amide were achieved according to scheme 5 . starting bromide 34 ullman - based etherification using cui and 1,10-phenanthroline in toluene with heat afforded targets 20d20 g . the related aryloxymethyl targets retaining a similar substitution pattern and acyl - tetrahydronaphthyridine were prepared according to scheme 6 . introduction of 4-fluoro - benzamide provided intermediate 35c , followed by stille cross - coupling to give vinyl 36 . subsequent ozonolysis and reductive work - up gave alcohol 37 , which was elaborated via mitsunobu alkylation to provide 20a20c in low to moderate yields . reagents and conditions : ( a ) 34 , hatu , dipea , dmf , rt , 16 h , 6090% ; ( b ) 35 , arch2oh , cs2co3 , 1,10-phen , cui , toluene , 110 c , 4 h , 2035% . reagents and conditions : ( a ) 34 , 4-fluorobenzoic acid , hatu , dipea , dmf , rt , 16 h , 96% ; ( b ) bu3(vinyl)sn , pd(pph3)4 , toluene , 120 c , 1 h , 82% ; ( c ) o3 , ch2cl2:meoh , 78 c , 30 min , nabh4 , 0 c ; ( d ) roh , diad , polystyrene pph3 , thf , rt , 16 h , 2540% ( over two steps ) . as reported previously , compounds were profiled in a rat mglu5 low receptor expression cell line using a triple add protocol allowing for detection of agonism as well as positive and negative allosteric modulation simultaneously . importantly , we have shown that detection of allosteric agonism observed using the low receptor expressing cell line correlates with functional response observed in native systems and modulators with an allosteric agonist profile have been shown to be associated with epileptiform activity and behavioral convulsions in rodents . in contrast , an allosteric modulator devoid of agonism in these low - expressing mglu5 cells was found not to display a seizure liability in vivo . triple add protocol provided an important initial in vitro assessment of both agonist and potentiation activity at mglu5 , which could be utilized to prioritize analogues for further progression . sar are shown in tables 14 ( 1215 , 20 ) and figures 46 ( 1619 ) and summarize in vitro potency optimization efforts from 10 distinct subseries . initial efforts within tables 12 were focused on further expanding our previously utilized pharmacophore noted within several chemotypes , including acetylene 4 , ethers 7 and 10 , which retain a pyridyl nitrogen and an amide oxygen as potential hydrogen bond accepting groups in an optimal spatial arrangement as illustrated in figure 3 . as can be seen in table 1 , in the case of 2-benzyloxy derivatives , half of the analogues were active as mglu5 pams . among actives , the parent lactams ( r = h , 12a12h , 12q ) showed a range of potency from 62 to 1171 nm with low to moderate maximal efficacy ( glutamate max 4368% ) . notably , no evidence for allosteric agonism was observed for actives in the series . sar on the r group demonstrated toleration for meta - substituted fluorine and chlorine congeners 12c and 12e , respectively , representing two of the more interesting actives from this subseries on the basis of overall potency and efficacy ( ec50 < 250 nm , glu max > n - butyloxy derivatives 12q and 12r , reminiscent of a previously disclosed non - mpep ether pam , were not successful . the 2 and 3-pyridyl congeners 12 g and 12h were also not tolerated at r. additionally , eastern alkyl derivatives 12i12n containing linear , branched , cyclic , phenyl , and heterocyclic systems were either inactive ( a < 10% increase in the baseline ec20 glutamate fluorescence response in calcium assays at the highest concentration tested of 30 m is defined as inactive ) or had moderate potency and low efficacy ( ec50 > 300 nm , pam glu max < 40% , 12l12n , 12s12 t ) . n - aryl congeners 12o and 12p demonstrated comparable potency below 250 nm , with 12p slightly more efficacious ( glu max 50% vs 38% ) . both the western fluoro and the eastern amide n - aryl sar parallel the recently reported thiazolyl core structure system ; however , interestingly , n - alkyl derivatives were significantly better tolerated in the thiazolyl ring system . calcium mobilization assay using hek293 cells stably expressing rat mglu5 receptors ; values are the average of three or more independent determinations ; inactive , less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response using 30 m test compound ( percentage of maximal response versus 100 m glutamate ) . heterocycle , amide , linker pharmacophore relationships , and relative profile for endocyclic series 1215 dihydronaphthyridinones and hypothetical benzyloxy lactam core regioisomers of 1415 . studies by astrazeneca pharmaceuticals and within the dihydrothiazolopyridone series 10(31 ) have demonstrated successful utilization of the both benzyloxy- and phenoxymethyl - based spacers as replacements for the acetylene moiety , with varied potency depending on the nature of the heterocycle scaffold . interestingly , in contrast to the unsubstituted benzyloxy lactams 12a12b , alkoxymethyl- dihydronaphthyridinones 13a13b were weak pams ( a weak pam results in a response that is greater than a 10% increase compared to the submaximal glutamate addition ( ec20 ) but does not potentiate the overall glutamate ec20 response by at least 2-fold ) . similarly , n - alkyl derivatives were not productive as pams , leading to compounds categorized as weak or inactive ( 13c13e , 13i ) . direct n - aryl derivatives were more beneficial , with the western 3-fluorophenyl congener 13 g bearing an n-4-fluoro - phenyl proving to be potent ( ec50 = 97 nm ) and efficacious ( glu max = 72% ) ; the 2-pyridyl derivative 13h was 5-fold less active ( ec50 = 595 nm ) and less efficacious . fortunately , introduction of a 3-fluoro moiety on the distal phenyl consistently boosted potency ( 23-fold ) and proved transferrable among the subseries ( e.g. , 13f vs 13 g ) . the potency for 13g13h was overall similar to the benzyloxy direct comparators 12o12p ; however , phenoxymethyl - based pams 13g13h have a 2030% elevated glu max response . despite modification of the linker , calcium mobilization assay using hek293 cells stably expressing rat mglu5 receptors ; values are the average of three or more independent determinations ; inactive , less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response using 30 m test compound ( percentage of maximal response versus 100 m glutamate ) . in parallel , we evaluated isomeric dihydronaphthyridinones 1415 ( table 3 ) , which retain the linkage site for the distal phenyl para to the lactam amide ; however , the pyridine nitrogen is adjacent to the lactam amide in a position reminiscent of a hydrogen bond accepting ( hba ) pharmacophore proposed in a related ether series by varnes and co - workers . within both series 14 ( y = n ) and series 15 ( x = n ) nh lactams ( 14a , 15a ) , n - methyl analogues ( 14b , 15b ) , and chiral ( r)-3,3-dimethylbutan-2-yl substitute analogues , inspired by monocyclic ether 7 , all proved to be inactive . the lack of activity in the calcium assay for parent lactams 14a and 15a versus the benzyloxy comparator 12a , in addition to western 3-fluoro congeners 13b ( inactive ) versus 12c ( ec50 = 225 nm ) , suggests that for series 1215 wherein the distal phenyl is maintained para to the lactam amide , the heteroatom arrangement between the linker oxygen and the pyridine nitrogen within series 12 is more preferred . however , n - aryl analogues have a unique sar , suggesting that in this context the central tetrahydronaphthyridine scaffold is more critical for mglu5 potentiation than the nature of the linker , as 12 and 13 are equally tolerated in the context of an n - aryl substituent . n-4-fluorophenyl pams 14d and 15d , which differ in the location of the central pyridine nitrogen , have comparable potency with dissimilar efficacy , with pyridine isomer 14d being one of the most efficacious pams reported across the tetrahydronaphthyridine subseries described . the related congener 13f was 2.5-fold more potent ( ec50 = 198 nm ) with 68% glutamate max , and 13 g was 2-fold more potent than 13f . relative to series 12 , 12o is equipotent to 13 g ; however , 13 g revealed a 2-fold higher maximal glutamate response ( 72% vs 38% ) . although a more comprehensive survey was undertaken for series 1213 relative to 1415 , on the basis of direct comparisons , general trends were noted between the series . neither molecular switches nor allosteric agonism proved to be issues for 1215 and sar was generally steep . active compounds within 1213 have capacity for high potency ( ec50 200 nm ) and moderate to high efficacy ( % glu max 50% ) , whereas actives within series 1415 , although more limited , exhibited moderate potency and variable efficacy ( % glu max 30% and 81% ) . subtle changes in structure efficacy / cooperativity relationships are best addressed via glutamate fold - shift experiments ; however , glutamate max trends were consistent within series 1215 , and series 12 shows variable efficacies that encompass the full range reported for all subseries ( figure 3 ) . in general , phenoxy linkages show improved efficacy over benzyloxy linkages for n - arylated analogues . the emerging pharmacophore for 1213 suggested these dihydronaphthyridinone scaffolds interact at the allosteric site in a manner distinct than that proposed by varnes and co - workers and likely more similar to a monocyclic series of nicotinamides previously described . although formally benzyloxy variants of 14 and 15 ( figure 3 ) remained as potential mglu5 pams , the template was not pursued . this decision was made in part on the basis of the overall efficacy and potency observed within subseries 13 , thus phenoxy - based systems remained higher priority for the remaining survey . calcium mobilization assay using hek293 cells stably expressing rat mglu5 receptors ; values are the average of three or more independent determinations ; inactive , less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response using 30 m test compound ( percentage of maximal response versus 100 m glutamate ) . we next pursued alternative sites for linker attachment of the pendant aryl examining 3-substituted dihydronaphthyridinones based on either a benzyloxy ( 16 ) or phenoxy - based ( 17 ) system ( figure 4 ) . brief surveys of both n - alkyl and n - aryl and heteroaryl substituents , including several congeners related to those previously described ( table 13 ) , were unsuccessful in identifying favorable starting points and led to modulators which were either inactive or weak pams ( ec50 > 10 m ) . interestingly , a related more rigid , acetylene tetralone scaffold recently reported by merz pharmaceuticals gmbh displays a similar array of hba moieties and was reported to have excellent pam activity ( ec50 < 100 nm ) in a recombinant cho cell line expressing human mglu5 . inspired in part by the addex piperidine chemotype 2 , we postulated that perhaps a more extended pharmacophore placing the amide carbonyl moiety exocyclic of the tetrahydronaphthyridine scaffold as found within the addex chemotype might prove viable ( figure 5 ) . pam 12c served as a precursor ( scheme 1 ) and allowed for facile access to representative benzyloxy targets 18a d in two steps via borane reduction and subsequent amide coupling . intriguingly , all proved to be weak to moderate antagonists of mglu5 ( figure 5 ) . at this juncture , in light of the unexpected switch in the mode of pharmacology for subseries 18 , as well as the general challenges of multidimensional combinations of linker , core , attachment site , and lactam versus exocyclic amide ( minimum of 24 formal combinations / scaffolds ) , we employed a ligand - based computational tool previously reported to prioritize and potentially streamline medicinal chemistry efforts and ligand design . using this approach , we assessed ligand - based conformational ensembles of known highly efficacious pams , including addex ligand 2(52 ) and other lead molecules , with pam activity below 150 nm and robust glu max values above 70% . hypotheses or reference pams for a mutual flexible low energy shape - based alignment of proposed dihydronaphthyridinone and tetrahydronaphthyridines using the surflex - sim algorithm as implemented in sybyl . the molecular similarity and alignment optimization algorithms used in surflex - sim docking utilize a functional term to minimize the volume of molecular superpositions to construct an objective function for scoring superpositions of multiple molecules . the objective function can then be used as targets for virtual screening , or in this case , for scaffold hopping prioritization . the results from these studies using 2 are summarized in figure 6 with a depicted overlay of the lowest energy structures for 2 versus 2-phenoxy methyl model amide 19 and 3-phenoxy methyl model amide 20 . a surflex - sim score ( ss ) that approaches the reference hypothesis , arbitrarily set to 10 , is indicative of a template that more closely mimics the reference conformer ensemble . using 2 as a template , series 20 , containing a 3-phenoxymethyl , was predicted to have greater molecular similarity ( ss = 9.43 ) than the 2-phenoxymethyl prototype 19 ( ss = 8.90 ) . unlike 2-benzyloxy derivatives within series 18 , compounds within series 19 were not antagonists and proved inactive in the primary calcium assay ( nine analogues were prepared and tested from a 3-fluoro 1-(2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)ethanone scaffold , see supporting information ) ; however , at this time , it is unclear if compounds within series 19 have neutral cooperativity and thus retained affinity for mglu5 ( a silent allosteric modulator or sam ) . with this caveat in mind , it is also recognized that the pam ec50 is better described as a composite of four parameters encompassing affinity of the modulator for the receptor , allosteric effects on binding and signaling of the orthosteric ligand and potential intrinsic agonist efficacy of the modulator . thus , although within closely related series , the surflex - sim approach appears to facilitate scaffold prioritization based on pam ec50 activity at of glu5 , the power of the surflex - sim methodology warrants a broader more rigorous retrospective analysis with these additional facets of modulator activity taken into consideration . relationship between 2 and 1820 and resulting first - generation exocyclic amides 18 utilizing 2-benzyloxy linkage . example of flexible alignment of model 2 and 3-phenoxymethyl - tetrahydronaphthyridines 19 and 20 ( green surface ) vs 2 ( purple surface ) . with some insight that perhaps 3-substituted tetrahyrdonaphthyridines 20 were preferred from ligand - based modeling , additional analogues were prioritized . selected derivatives 20a20c , which retains a 3-phenoxy methyl linker , and 3-benzyloxy variants 20d20 g , are shown in table 4 . within benzyloxy derivatives 20d20 g , compounds covered a spectrum of profiles ranging from inactive ( 20d , 20f ) , to weak pams ( 20e ) , to weak partial antagonists ( 20 g ) ; the latter effect driven by subtle changes in fluorine substitution patterns , a phenomenon not uncommon among certain mglu5 modulator chemotypes . within the phenoxy subseries analogues 20a20c , success was found with 4-fluorobenzamide derivatives ; in particular , the western phenyl and 3-fluoro phenyl derivatives 20a and 20b have statistically comparable potency and efficacy , with ec50s of 640 and 1090 nm , respectively , and a glu max of 69% . . three significant findings are evident among these lactam and exocyclic amide templates examined : ( 1 ) parent dihydronaphthyridinone 12c remains a confounding singleton relative to regioisomeric and positional congeners 13b , 14a , 15a , 16 , and 17 , ( 2 ) relative to earlier acetylenic dihyronaphthyridinones 11 , no molecular switches were noted within dihydronaphthyridinone systems , however , steep sar was evident , and ( 3 ) in contrast to dihydronaphthyridinones , exocyclic acyl - tetrahydronaphthyridines were highly sensitive to the linker attachment site , as 2-substituted based ethers 18 and 19 , proved to be either weak antagonists or functionally inactive in the calcium assay . in addition , within the exocyclic acyl - tetrahydronaphthyridines , phenoxymethyl - based ethers were better tolerated and less susceptible to changes in the mode of pharmacology ( e.g. , 20a versus 20 g ) ; however , overall potency was generally diminished relative to series 1213 . calcium mobilization assay using hek293 cells stably expressing rat mglu5 receptors ; values are the average of three or more independent determinations ; inactive , less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response using 30 m test compound ( percentage of maximal response versus 100 m glutamate ) . weak antagonist , data represents plc50/ic50 from ec80 window and % glu represents emin not emax . in light of the overall mglu5 pam potency ( ec50 < 250 nm ) and efficacy ( glu max > 50% ) observed for compounds 12c , 12p , 13f , and 13 g , these pams were selected for further characterization ( table 5 ) . in addition , the moderately potent pam 14d ( ec50 = 538 nm ) was considered for further evaluation based upon its enhanced efficacy ( glu max = 81% ) . for clint , intrinsic clearance ; clhep , predicted hepatic clearance , human and rat ( h , r ) , ml / min / kg . further examined , 12p , 13f , 13 g , and 14d have similar clogp and molecular weight ( mw ) by virtue of their common lactam n - aryl substituent , while the simple nh lactam 12c stands out as a low mw modulator ( mw = 272 ) with excellent calculated ligand efficiency metrics ( lipe and lelp , see table 5 ) . pam 12c maintains the highest calculated lipe and lowest lelp among the set , suggesting a highly favorable enthalpy driven effect on cooperativity . despite the less favorable physicochemical and le properties for 12p , 13f , 13 g , and 14d relative to 12c , intrinsic clearance in human and rat microsomes suggested moderate predicted hepatic clearance and 110% fraction unbound . subsequent selectivity profiling against mglu14,68 using 12c , 13 g , and 14d revealed that n - aryl congeners 13 g and 14d were also active as mglu3 negative allosteric modulators ( nams ) with negative cooperativity fold - shift values between 0.2 and 0.3 , while 12c was fully selective for mglu5 ( mglu14,68 ec50 > 10 m , see supporting information ) . in light of the unexpected lack of selectivity for 13 g and 14d which maintain a similar n - aryl endocyclic amide pharmacophore found within 12p and 13f , we elected to continue to profile 12c in more depth based on its overall potency , selectivity , and excellent physicochemical and ligand efficiency parameters ( see figure 7 for overall profile ) . positive allosteric modulator activity was investigated by fold - shift experiments to determine the degree of cooperativity with glutamate . as shown in figure 8 , progressive fold - shift experiments utilizing the rat mglu5 receptor revealed a small leftward shift in the glutamate concentration response curve ( crc ) with increasing concentration of 12c ( 55 nm to 30 m ) , with no significant effect on the maximal response . a 2.2-fold leftward shift in the glutamate ec50 was observed in the presence of 30 m 12c , and a predicted allosteric modulator affinity of 2.17 m and an efficacy cooperativity factor ( log ) between glutamate and indicated allosteric modulator of 0.23 ( cooperativity 1.71 ) was defined as calculated using the operational model of allosterism . these results support a positive allosteric interaction between glutamate and 12c and were confirmed in a cell line expressing the human mglu5 receptor , where 10 m 12c produced a 3.3-fold leftward shift in the glutamate crc and a pam crc potency of 370 nm ( 71% glu max ) . in comparison to recently reported pams from our lab that have been profiled in glutamate progressive fold - shift studies up to 30 m , including acetylenic picolinamides ( e.g. , ml254 , maximum rat mglu5 fold - shift 3.0 ) and dihydrothiazolopyridone 10 ( maximum rat mglu5 fold - shift 5.4),12c represents one of the weakest mglu5 allosteric modulators reported in terms of measurable positive cooperativity . profile summary of 12c . rat brain homogenate binding to assess fraction unbound in brain revealed a reasonable brain fu of 2.0% for 12c . inhibition of the major human cytochrome p450 ( cyp ) enzymes ( 2c9 , 2d6 , 3a4 , 1a2 ) was measured in a cocktail assay using human liver microsomes and known substrates . pam 12c was found to display weak cyp inhibitory activity at 1a2 ( ic50 = 25.7 m ) , while no activity was observed against the other cyps tested ( ic50 > 30 m ) . in kinetic aqueous solubility assays , 12c displayed moderate to good solubility ( ph 2 , 4 , 7.4 buffer : 38 , 29 , and 28 m ) and moderate solubility in fasted simulated intestinal fluid ( fassif , ph = 7.2 ) of 31 g / ml ( 114 m ) . in a nontoxic vehicle screen using 20% hp -cyclodextrin , 12c was a solution at 1 mg / ml and a microsuspension at 35 mg / ml ( ph 4.0 ) . rat pharmacokinetic studies ( 1 mg / kg iv , 10 mg / kg po ) revealed a high plasma clearance ( clp ) of 75 ml / min / kg and a short half - life ( t1/2 ) of 0.3 h. after oral administration ( 10 mg / kg , 0.5% methyl cellulose , 1 mg / ml , noncrossover ) to male sprague dawley rats , 12c reached an average maximal concentration ( cmax ) of 3.36 m with a corresponding time to reach cmax ( tmax ) of 0.5 h and an auc024h of 6.2 m - h , thus affording an estimated % f of 75 . we performed a single dose in vivo screen to study the ability of 12c to reverse amphetamine - induced hyperlocomotion in rats ( po , 56.6 mg / kg 20% hp -cyclodextrin ) . robust reversal of the hyperlocomotion response was noted at this dose ( > 30% , data not shown ) and high brain exposure was observed at 1.5 h ( cbrain = 16.6 m , 332 nm unbound ) , producing an average brain - to - plasma ( b : p ) ratio of 1.67 and unbound brain level above the rat in vitro ec50 . furthermore , in vivo b : p and in vitro fu , plasma / fu , brain ( 1.7 ) measures were identical , with a calculated kp , uu of 1.1 , suggesting passive cns penetration for 12c in rat . thus , 12c continued to demonstrate promising properties to warrant further evaluation in vivo . with respect to ancillary pharmacology , in a broad panel selectivity screen against 68 gpcrs , ion channels and transporters using 10 m 12c , no significant off - target activity was noted ( eurofins inc . ) . encouraged by its overall profile as a low fold - shift pam , 12c was evaluated across a range of doses for its ability to reverse amphetamine - induced hyperlocomotion ( ahl ) , an established model of antipsychotic activity ( figure 8) . as seen in figure 9 , 12c dosed orally showed robust dose - dependent effects in reversal of hyperlocomotion , with a maximal effect ( 55% ) observed at the highest dose tested of 100 mg / kg and an average terminal unbound brain concentration of 1.5 m . the lowest active dose of 30 mg / kg afforded an estimated average terminal unbound brain concentration of 712 nm , which represents a 3-fold multiple of the rat in vitro potency and according to the progressive fold - shift studies ( figure 8) , represents a left - ward shift in the glutamate crc in the 1.21.4 fold range . despite the overall low positive cooperativity , 12c retains efficacy in ahl at unbound brain exposures that are a multiple of the in vitro ec50 and thus illustrates that high cooperativity with glutamate is not fully required for an mglu5 pam to maintain activity in the ahl model . this observation appears to be in contrast to data reported recently for low fold - shift pams 89 reported by lilly , wherein little effect was noted in a similar amphetamine - induced hyperlocomotor activity assay using male lister hooded rats . rotarod studies were performed using 12c at a doses of 30 , 56.6 , and 100 mg / kg po ( 20% hp--cd in water ) to assess balance and motor coordination ( see supporting information ) . pam 12c showed no statistically significant effect on general motor output ( 120 s cutoff ) . in a modified irwin neurological test battery in rats , 12c , when dosed at 100 mg / kg po ( 20% hp--cd in water ) , had no performance deficits on any autonomic or somatomotor nervous system functions out to 4 h. collectively , these results highlight that pam 12c is free from overt neurological side effects and motor behavior deficits at doses that produce maximal efficacy in reversal of amphetamine - induced hyperlocomotion ( 100 mg / kg po ) . dose - dependent effect and calculated terminal unbound brain of 12c on the reversal of amphetamine - induced hyperlocomotion in rats . diverse mglu5 modulator pharmacological profiles were attained within a series of tetrahydronaphthyridine and dihydronaphthyridinone scaffolds through modifications of heteroatoms within the linker , core structure , and location of the pendant linkage moiety . up to 10 diverse subseries were examined , and five of these series , 1215 and 20 , were described in detail , leading to preferred endocyclic series 12 and 13 which , although free from allosteric agonism and molecular switches , tended to have steep sar . n - aryl dihydronaphthyridinone congeners were nonselective and displayed negative cooperativity at mglu3 and thus were not further progressed . parent benzyloxy lactam 12c emerged as a suitable tool compound with good potency , excellent selectivity , and pharmacokinetic properties suitable for acute studies . despite behaving in vitro as an ultralow cooperativity pam in recombinant systems , 12c showed robust , dose - dependent effects in the reversal of amphetamine - induced hyperlocomotion , with a lowest active dose of 30 mg / kg po , that produced an estimated brain unbound level 3-fold above its in vitro potency . maximal efficacy of 12c was observed at a dose of 100 mg / kg with no significant motor impairment or overt neurological side effects . 12c provides another tool compound to complement the available mglu5 pams with well characterized cooperativity profiles to better enable comparative studies with other chemotypes in native systems and in preclinical animal models . all reagents purchased from commercial suppliers were used without purification . unless noted , all solvents used were anhydrous and all reactions were carried out under argon atmosphere . microwave assisted reactions were performed in a single - mode reactor : emrys optimizer microwave reactor ( biotage ) . analytical thin layer chromatography was performed on analtech silica gel gf 250 m plates and on silica gel 60 f254 plates ( merck ) under standard techniques . unless otherwise specified , preparative reverse - phase high performance liquid chromatography ( rp - hplc ) purification was performed on a gilson inc . preparative uv - based system using a phenomenex luna c18 column ( 50 mm 30 mm i.d . , 5 m ) , with an acetonitrile ( unmodified)0.1% trifluoroacetic acid in water gradient . normal - phase silica gel preparative purification was performed using an automated combi - flash rf from isco using either isco or merck ready - to - connect cartridges . analytical lc / ms was performed using the following instruments : ( a ) agilent 1200 series with uv detection at 215 and 254 nm and elsd detection ( polymer laboratories pl - els 2100 ) , utilizing an accucore c18 2.6 , 2.1 mm 30 mm column , a 1.1 min gradient , 7% [ ch3cn/0.1%tfa]95% [ ch3cn/0.1%tfa ] and a g6130 single quadrupole mass spectrometer or ( b ) ultra performance liquid chromatography ( uplc ) measurement performed using an acquity uplc ( waters ) system comprising a sampler organizer , a binary pump with degasser , a four column s oven , a diode array detector ( dad ) , and a beh - c18 column ( 1.7 m , 2.1 mm 50 mm ) from waters , with a flow rate of 1.0 ml / min at 50 c without split to the ms detector . the gradient conditions consisted of 95% a ( 0.5 g / l ammonium acetate solution + 5% acetonitrile ) , 5% b ( acetonitrile ) , to 40% a , 60% b in 3.8 min , to 5% a , 95% b in 4.6 min , kept until 5.0 min without split to the ms detector . the ms detector was configured with an esci dual ionization source ( electrospray combined with atmospheric pressure chemical ionization ) . low - resolution mass spectra ( single quadrupole , sqd detector ) were acquired by scanning from 100 to 1000 in 0.1 s using an interchannel delay of 0.08 s. the capillary needle voltage was 3 kv . the cone voltage was 25 v for positive ionization mode and 30 v for negative ionization mode . gc / ms measurement was performed using a 6890 series gas chromatograph ( agilent technologies ) system comprising a 7683 series injector and autosampler , coupled to a 5973n msd mass selective detector ( single quadrupole , agilent technologies ) . the ms detector was configured with an electronic impact ionization source / chemical ionization source ( ei / ci ) . ei low - resolution mass spectra were acquired by scanning from 50 to 550 at a rate of 14.29 scans / s . gc / ms was carried out on a j&w hp-5ms column ( 20 m 0.18 mm , 0.18 m ) from agilent technologies , with a flow rate of 0.7 ml / min . the temperature gradient applied was : initial temperature 50 c , hold for 2.0 min , then a 50 c / min ramp applied for 5.0 min until 300 c and hold for 3.0 min in a 10 min run . split injection mode was used , 0.2 l injection volume , with a 50/1 ratio into the gc / ms system . hrms were obtained using a micromass ( waters ) q - tof api - us calibrated and verified with sodium iodide . the samples were diluted with a 50:50 0.1% formic acid ( in milli - q):acetonitrile solution , directly infused using leucine enkephalin ( m + h = 556.2771 ) as a lockmass . scan range was from 100 to 1000 da , using a scan time of one second . the [ m + h ] or [ m + na ] ion was observed . nmr spectra were recorded on either a bruker dpx-400 or a bruker av-500 mhz spectrometer with standard pulse sequences . h chemical shifts are reported as values in cdcl3 or dmso - d6 . data are reported as follows : chemical shift , integration , multiplicity ( s = singlet , bs = broad singlet , d = doublet , t = triplet , q = quartet , p = pentet , hex = hextet , sep = septet , dd = doublet of doublets , dt = doublet of triplets , dq = doublet of quartets , m = multiplet ) , coupling constant j reported in hz . c chemical shifts are reported in values in cdcl3 as follows : chemical shift , c f coupling constants ( jc f ) reported in hz . for a number of compounds , melting points ( mp ) were determined in open capillary tubes on a fp62 or on a fp81htfp90 apparatus ( mettler ) . melting points were measured with a temperature gradient of 10 c / min ( maximum temperature 300 c ) , and the melting point was read from a digital display . melting point values are peak values and were obtained with experimental uncertainties that are commonly associated with this analytical method . experimental procedures , analytical data , hrms , and nmr spectra can be found within supporting information . benzyl alcohol ( 85 l , 0.82 mmol ) was dissolved into dmf ( 1.6 ml ) and treated with kot - bu ( 184 mg , 1.64 mmol ) and stirred for 30 min . the mixture was treated with 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 20 , 100 mg , 0.55 mmol , see supporting information ) and heated to 100 c for 4 h. the mixture was cooled to rt , acidified with 6 n hcl , and extracted with etoac ( 3 ) . the combined organic layers were washed with brine , dried over na2so4 , and evaporated to dryness . the crude product mixture was purified by rp - hplc ( eluting with 4090% mecn / h2o with 0.1% tfa modifier ) to afford the title compound ( 73 mg , 53% ) . h nmr ( 400 mhz , cdcl3 ) 8.20 ( 1h , d , j = 8.6 hz ) , 7.46 ( 2h , m ) , 7.36 ( 3h , m ) , 6.75 ( 1h , d , j = 8.6 hz ) , 5.85 ( 1h , bs ) , 5.43 ( 2h , s ) , 3.62 ( 2h , t , j = 6.8 hz ) , 3.09 ( 2h , t , j = 6.8 hz ) . c nmr ( 100 mhz , cdcl3 ) 166.4 , 165.2 , 158.0 , 138.3 , 136.7 , 128.4 , 128.0 , 127.9 , 118.3 , 109.8 , 68.1 , 39.2 , 30.7 . hrms ( es+ , m + h ) calcd for c15h15n2o2 , 255.1134 , found , 255.1130 . 3-fluorobenzyl alcohol ( 1.66 g , 13.14 mmol ) was dissolved into dmf ( 25 ml ) and treated with kot - bu ( 3.2 g , 26.29 mmol ) and stirred for 30 min . the mixture was treated with 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 20 , 1.49 g , 8.82 mmol , see supporting information ) and heated to 100 c overnight . the mixture was adsorbed onto silica and purified ( 0 to 80% etoac / hexanes ) to give a semipure solid . this material was further purified on rp - hplc ( eluting with 4090% mecn / h2o with 0.1% tfa modifier ) to give 840 mg of the title compound ( 35% ) . h nmr ( 400 mhz , cdcl3 ) 8.44 ( 1h , d , j = 7.6 hz ) , 7.38 ( 1h , dd , j = 14.6 , 7.6 hz ) , 7.24 ( 1h , d , j = 7.5 hz ) , 7.22 ( 1h , d , j = 9.6 hz ) , 7.04 ( 1h , t , j = 8 hz ) , 6.79 ( 1h , d , j = 8.4 hz ) , 5.79 ( 1h , br s ) , 5.45 ( 2h , s ) , 3.64 ( 2h , t , j = 6.4 hz ) , 3.10 ( 2h , t , j = 6.4 hz ) . lcms tr = 0.736 min , > 98% at 215 and 254 nm , m / z = 273.1 [ m + h ] . hrms ( es+ , m + h ) calcd for c15h14fn2o2 , 273.0961 ; found , 273.0963 . starting from 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 100 mg , 0.55 mmol ) and following the general procedure outlined in 12a and 12c , rp - hplc afforded the title compound as a powder ( 72 mg , 60% ) . h nmr ( 400 mhz , cdcl3 ) 8.17 ( 1h , d , j = 8.6 hz ) , 6.67 ( 1h , d , j = 8.6 hz ) , 5.84 ( 1h , bs ) , 4.33 ( 2h , t , j = 6.6 hz ) , 3.61 ( 2h , t , j = 6.8 hz ) , 3.06 ( 2h , t , j = 6.8 hz ) , 1.77 ( 2h , p , j = 7.9 hz ) , 1.48 ( 2h , hex , j = 7.5 hz ) , 0.98 ( 3h , t , j = 7.4 hz ) . c nmr ( 100 mhz , cdcl3 ) 166.6 , 165.7 , 158.1 , 138.1 , 117.9 , 109.4 , 66.3 , 39.2 , 30.9 , 30.8 , 19.1 , 13.7 . hrms ( es+ , m + h ) calcd for c12h17n2o2 , 221.1290 ; found , 221.1290 . 2-butoxy-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 12q , 50 mg , 0.23 mmol ) , 1-bromo-2,4-difluorobenzene ( 52 l , 0.46 mmol ) , cui ( 9.1 mg , 0.047 mmol ) , n , n - dimethylethane-1,2-diamine ( 14 l , 0.129 mmol ) , and potassium carbonate ( 64 mg , 0.46 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . degassed toluene was added and the mixture heated at 110 c for 6 h. the mixture was cooled to rt , filtered over celite , and the filtrate concentrated to dryness . rp - hplc purification afforded the title compound as a powder ( 45 mg , 59% ) . h nmr ( 400 mhz , cdcl3 ) 8.22 ( 1h , d , j = 8.6 hz ) , 7.32 ( 1h , m ) , 6.92 ( 2h , m ) , 6.68 ( 1h , d , j = 8.6 hz ) , 4.35 ( 2h , t , j = 6.6 hz ) , 3.91 ( 2h , t , j = 6.7 hz ) , 3.19 ( 2h , t , j = 6.6 hz ) , 1.77 ( 2h , p , j = 7.9 hz ) , 1.48 ( 2h , hex , j = 7.6 hz ) , 0.98 ( 3h , t , j = 7.4 hz ) . c nmr ( 100 mhz , cdcl3 ) 165.9 , 163.9 , 161.4(dd , jc f = 247.6 , 11.3 hz ) , 157.8 ( dd , jc f = 250.8 , 12.3 hz ) , 157.66 , 139.00 , 129.61 ( dd , jc f = 9.8 , 2.6 hz ) , 126.42 , ( dd , jc f = 12.8 , 3.9 hz ) , 118.0 , 111.55 ( dd , jc f = 22.2 , 3.6 hz ) , 109.7 , 104.9 ( dd , jc f = 26.0 , 24.0 hz ) , 66.4 , 48.5 , 31.2 , 31.0 , 19.2 , 13.8 . hrms ( es+ , m + h ) calcd for c18h19f2n2o2 , 333.1415 ; found , 333.1413 . ( 19 l , 0.158 mmol ) were dissolved into dmf ( 0.5 ml ) and kot - bu added ( 16 mg , 0.143 mmol ) . the mixture was stirred for 3 h at 60 c and then cooled to rt . water ( 1.0 ml ) was added to the mixture and extracted with etoac ( 3 ) . the combined organic layers were evaporated to dryness and the crude product mixture purified by rp - hplc to afford 16 mg of the desired product ( 40% ) . h nmr ( 400 mhz , cdcl3 ) 8.28 ( 1h , d , j = 8.6 hz ) , 7.45 ( 2h , m ) , 7.33 ( 8h , m ) , 6.76 ( 1h , d , j = 8.6 hz ) , 5.41 ( 2h , s ) , 4.77 ( 2h , s ) , 3.53 ( 2h , t , j = 6.9 hz ) , 3.02 ( 2h , t , j = 6.9 hz ) . c nmr ( 100 mhz , cdcl3 ) 165.1 , 164.1 , 156.9 , 139.0 , 137.3 , 136.8 , 128.7 , 128.5 , 128.1 , 128.0 , 127.9 , 127.5 , 118.7 , 109.9 , 68.1 , 50.2 , 44.5 , 30.7 . hrms ( es+ , m + h ) calcd for c22h21n2o2 , 345.1603 ; found , 345.1600 . ( 40 mg , 0.158 mmol ) were dissolved into dmf ( 0.5 ml ) and kot - bu added ( 36 mg , 1.35 mmol ) . the mixture was stirred for 3 h at 60 c and then cooled to rt . water ( 1.0 ml ) the combined organic layers were evaporated to dryness and the crude product mixture purified by rp - hplc to afford 16.2 mg of 12 t ( 40% ) . h nmr ( 400 mhz , cdcl3 ) 0.8.55 ( 1h , d , j = 4.6 hz ) , 8.25 ( 1h , d , j = 8.6 hz ) , 7.66 ( 1h , dt , j = 7.6 , 1.7 hz ) , 7.45 ( 2h , m ) , 7.35 ( 4h , m ) , 7.2 ( 1h , m ) , 6.74 ( 1h , d , j = 8.6 hz ) , 5.41 ( 2h , s ) , 4.88 ( 2h , s ) , 3.70 ( 2h , t , j = 6.9 hz ) , 3.07 ( 2h , t , j = 6.9 hz ) . c nmr ( 100 mhz , cdcl3 ) 165.1 , 164.2 , 157.3 , 157.2 , 149.2 , 140.0 , 136.9 , 136.8 , 128.5 , 128.1 , 128.0 , 122.4 , 122.3 , 118.6 , 109.8 , 68.1 , 52.4 , 45.6 , 30.7 . hrms ( es+ , m + h ) calcd for c21h20n3o2 , 346.1556 ; found , 346.1553 . [ 2-(phenoxy)methyl)-6-(4-methoxybenzyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 14 mg , 0.0.037 mmol ) was dissolved in ch3cn ( 0.25 ml ) and water ( 0.1 ml ) added . cerium ammonium nitrate ( 24.3 mg , 0.0.04 mmol ) was added , and after 15 min , the reaction was partitioned between etoac ( 5 ml ) and brine ( 1 ml ) . the mixture was extracted with etoac ( 2 ) , and the organic layers combined , concentrated , and purified by rp - hplc to give title compound ( 2.6 mg , 28% ) . h nmr ( 400 mhz , cdcl3 ) 8.32 ( 1h , d , j = 8.0 hz ) , 7.49 ( 1h , d , j = 8.0 hz ) , 7.25 ( 2h , m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 6.49 ( 1h , bs ) , 5.10 ( 2h , s ) , 3.65 ( 2h , dt , j = 6.9 , 2.5 hz ) , 3.20 ( 2h , t , j = 6.7 hz ) . lcms tr = 0.732 min , > 98% at 215 and 254 nm , m / z = 255.1 [ m + h ] . hrms ( es+ , m + h ) calcd for c15h14n2o2 , 255.1055 ; found , 255.1057 . [ 2-((3-fluorophenoxy)methyl)-6-(4-methoxybenzyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 23b , 300 mg , 0.77 mmol ) was dissolved in ch3cn ( 5 ml ) and water ( 2 ml ) added . cerium ammonium nitrate ( 419 mg , 0.77 mmol ) was added , and after 15 min , the reaction was partitioned between etoac ( 10 ml ) and brine ( 10 ml ) . the organic layers were concentrated and purified by rp - hplc to give title compound as a solid ( 74 mg , 76% ) . h nmr ( 400 mhz , cdcl3 ) 8.36 ( 1h , d , j = 8.0 hz ) , 7.53 ( 1h , d , j = 8.0 hz ) , 7.25 ( 1h , m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 6.49 ( 1h , bs ) , 5.20 ( 2h , s ) , 3.68 ( 2h , dt , j = 6.7 , 2.5 hz ) , 3.21 ( 2h , t , j = 6.7 hz ) . c nmr ( 100 mhz , cdcl3 ) 165.3 , 163.6 ( d , jc f = 10.9 hz ) , 158.3 , 136.8 , 130.4 ( d , jc f = 9.8 hz ) , 123.9 , 119.9 , 110.5 ( d , jc f = 2.9 hz ) , 108.3 ( d , jc f = 21.1 hz ) , 102.7 ( d , jc f = 24.9 hz ) , 70.5 , 39.4 , 30.9 . hrms ( es+ , m + h ) calcd for c15h14fn2o2 , 273.1039 ; found , 273.1037 . 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 10.0 mg , 0.04 mmol ) was dissolved in dmf ( 0.5 ml ) . nah ( 2.0 mg , 0.08 mmol ) was added into the solution and stirred at rt . after 30 min , mei ( 5.0 l ) was added into the mixture and allowed to stir for 1 h. the mixture was then quenched with h2o ( 2.0 ml ) and extracted with etoac ( 3 ) . the organic layers were combined and concentrated to afford product as a white solid ( 9.2 mg , 87% ) . lcms tr = 0.81 min , > 98% at 215 and 254 nm , m / z = 287.2 [ m + h ] . h nmr ( 400 mhz , cdcl3 ) 8.30 ( 1h , d , j = 8.0 hz ) , 7.45 ( 1h , d , j = 8.0 hz ) , 7.25 ( 2h , m ) , 6.76 ( 1h , m ) , 6.62 ( 2h , m ) , 5.10 ( 2h , s ) , 3.65 ( 2h , dt , j = 6.9 , 2.5 hz ) , 3.20 ( 2h , t , j = 6.7 hz ) , 3.16 ( s , 3 h ) . 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) was dissolved in dmf ( 2.0 ml ) . kot - bu ( 55 mg , 0.5 mmol ) was added followed by 1-bromo-2-methylpropane ( 70 l , 0.5 mmol ) . the mixture was heated to 60 c for 4 h. the mixture was then quenched with h2o ( 2.0 ml ) and extracted with etoac ( 3 ) . the resulting crude mixture was purified by rp - hplc to give title compound as a powder ( 43 mg , 44% ) . h nmr ( 400 mhz , cdcl3 ) 8.37 ( 1h , d , j = 8.0 hz ) , 7.51 , ( 1h , d , j = 8.0 hz ) , 7.22 ( 1h , m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 5.19 ( 2h , s ) , 3.65 ( 2h , t , j = 6.8 hz ) , 3.40 ( 2h , d , j = 7.5 hz ) , 3.19 ( 2h , t , j = 6.7 hz ) , 2.05 ( 1h , sep , j = 6.8 hz ) , 0.97 ( 6h , d , j = 6.7 hz ) . f = 10.7 hz ) , 159.1 , 157.4 , 137.1 , 130.4 ( d , jc f = 10.0 hz ) , 124.5 , 119.9 , 110.5 ( d , jc f = 2.9 hz ) , 108.2 ( d , jc f = 21.2 hz ) , 102.7 ( d , jc f = 24.9 hz ) , 70.5 , 54.7 , 45.8 , 30.9 , 27.2 , 20.1 . hrms ( es+ , m + h ) calcd for c19h22fn2o2 , 329.1665 ; found , 329.1662 . starting from 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) and ( bromomethyl)cyclopropane ( 50 l , 0.50 mmol ) , the general procedure outlined in 13d was performed . rp - hplc afforded the title compound as a powder ( 64 mg , 66% ) . h nmr ( 400 mhz , cdcl3 ) 8.36 ( 1h , d , j = 8.0 hz ) , 7.50 ( 1h , d , j = 8.0 hz ) , 7.23 ( 1h , m ) , 6.75 ( 1h , m ) , 6.68 ( 2h , m ) , 5.18 ( 2h , s ) , 3.75 ( 2h , t , j = 6.7 hz ) , 3.47 ( 2h , d , j = 7.0 hz ) , 3.21 ( 2h , t , j = 6.7 hz ) , 1.07 ( 1h , m ) , 0.55 ( 2h , m ) , 0.31 ( 2h , m ) . c nmr ( 100 mhz , cdcl3 ) 163.6 ( d , jc f = 244.2 . f = 10.8 hz ) , 159.1 , 157.5 , 137.0 , 130.3 ( d , jc f = 9.9 hz ) , 124,4 , 119.8 , 110.5 ( d , jc f = 2.8 hz ) , 108.2 ( d , jc f = 21.2 hz ) , 102.7 ( d , jc f = 24.8 hz ) , 70.5 , 51.4 , 45.2 , 30.8 , 9.5 , 3.5 . hrms ( es+ , m + h ) calcd for c19h20fn2o2 , 327.1509 ; found , 327.1508 . 2-(phenoxymethyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13a , 50 mg , 0.19 mmol ) , 1-bromo-2,4-difluorobenzene ( 42 l , 0.38 mmol ) , cui ( 7.2 mg , 0.038 mmol ) , and potassium carbonate ( 52 mg , 0.38 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . degassed toluene was added followed by n , n - dimethylethane-1,2-diamine ( 12 l , 0.110 mmol ) and the mixture heated at 120 c for 16 h. the mixture was cooled to rt , filtered over celite , and the filtrate concentrated to dryness . rp - hplc purification afforded the title compound as a powder ( 16 mg , 31% ) . h nmr ( 400 mhz , cdcl3 ) 8.43 ( 1h , d , j = 8.0 hz ) , 7.60 ( 1h , d , j = 8.0 hz ) , 7.33 ( 4h , m ) , 7.12 ( 2h , m ) , 6.99 ( 3h , m ) , 5.25 ( 2h , m ) , 4.06 ( 2h , t , j = 6.7 hz ) , 3.36 ( 2h , t , j = 6.6 hz ) . c nmr ( 100 mhz , cdcl3 ) 163.3 , 160.9 ( d , jc f = 245.0 hz ) , 160.7 , 158.1 , 157.5 , 138.4 ( d , jc f = 3.2 hz ) , 137.5 , 129.6 , 127.0 ( d , jc f = 8.5 hz ) , 124.3 , 121.4 , 120.1 , 115.9 ( d , jc f = 22.5 hz ) , 114.8 , 70.1 , 48.7 , 31.2 . hrms ( es+ , m + h ) calcd for c21h18fn2o2 , 349.1352 ; found , 349.1351 . 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b , 26 mg , 0.095 mmol ) , 2-bromo-5-fluoropyridine ( 33 mg , 0.19 mmol ) , cui ( 3.8 mg , 0.02 mmol ) , and potassium carbonate ( 26 mg , 0.19 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . degassed toluene ( 1.0 ml ) was added followed by n , n - dimethylethane-1,2-diamine ( 5.8 l , 0.053 mmol ) and the mixture heated at 120 c for 16 h. the mixture was cooled to rt and filtered over celite . the filtrate was diluted with h2o and extracted with etoac ( 3 ) . rp - hplc purification afforded the title compound as a powder ( 31 mg , 89% ) . h nmr ( 400 mhz , cdcl3 ) 8.46 ( 1h , d , j = 8.0 hz ) , 8.29 ( 1h , d , j = 3.0 hz ) , 8.01 ( 1h , dd , j = 9.1 , 4.0 hz ) , 7.57 ( 1h , d , j = 8.1 hz ) , 7.47 ( 1h , m ) , 7.23 ( 1h , m ) , 6.77 ( 1h , m ) , 6.71 ( 2h , m ) , 5.23 ( 2h , s ) , 4.38 ( 2h , t , j = 6.7 hz ) , 3.32 ( 2h , t , j = 6.6 hz ) . c nmr ( 100 mhz , cdcl3 ) 163.6 ( d , jc f = 244.4 hz ) , 163.6 , 160.4 , 159.4 ( d , jc f = 10.8 hz ) , 158.4 , 156.8 ( d , jc f = 252.0 hz ) , 149.5 ( d , jc f = 25.2 hz ) , 130.4 ( d , jc f = 9.9 hz ) , 124.4 , 124.3 ( d , jc f = 2.9 hz ) , 108.3 ( d , jc f = 21.1 hz ) , 102.7 ( d , jc f = 24.9 hz ) , 70.5 , 45.0 , 31.2 . hrms ( es+ , m + h ) calcd for c20h16f2n3o2 , 368.1211 ; found , 368.1210 . starting from 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) and ( bromomethyl)benzene ( 67 l , 0.55 mmol ) , the general procedure outlined in 13d was performed . rp - hplc afforded the title compound as a powder ( 74 mg , 66% ) . h nmr ( 400 mhz , cdcl3 ) 8.44 ( 1h , d , j = 8.0 hz ) , 7.53 ( 1h , d , j = 8.0 hz ) , 7.32 ( 5h , m ) , 7.24 ( 1h , m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 5.19 ( 2h , s ) , 4.80 ( 2h , s ) , 3.59 ( 2h , t , j = 6.8 hz ) , 3.15 ( 2h , t , j = 6.8 hz ) . f = 244.3 hz ) , 163.4 , 159.4 ( d , jc f = 10.6 hz ) , 159.3 , 157.5 , 137.2 , 136.9 , 130.4 ( d , jc f = 10.0 hz ) , 128.8 , 128.1 , 127.7 , 124.2 , 119.9 , 110.5 ( d , jc f = 2.9 hz ) , 108.2 ( d , jc f = 21.4 hz ) , 102.7 ( d , jc f = 24.9 hz ) , 70.5 , 50.4 , 44.5 , 30.7 . hrms ( es+ , m + h ) calcd for c22h20fn2o2 , 363.1509 ; found , 363.1507 . ring - closure step : starting from 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic acid methyl ester ( 128 mg , 0.45 mmol ) and benzylamine ( 0.059 ml , 0.54 mmol ) in ch2cl2 ( 8 ml ) , the mixture was stirred at rt for 1 h and sodium triacetoxyborohydride ( 95 mg , 0.45 mmol ) was added and the mixture was stirred at rt for 16 h. the mixture was diluted with ch2cl2 and washed with a saturated solution of nahco3 and brine . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in vacuo . the crude product was purified by flash column chromatography ( silica ; 7n solution of ammonia in methanol in ch2cl2 0/100 to 4/96 ) . the desired fractions were collected and concentrated to yield , 7-benzyl-3-phenoxymethyl-6,7-dihydro-5h - naphthyridin-8-one as an oil ( 41 mg , 23% ) . lcms tr = 2.83 min , > 90% at 215 and 254 nm , m / z = 255.1 [ m + h ] . debenzylation step : trifluoromethanesulfonic acid 0.020 ml , 0.23 mmol ) was added to a solution of 7-benzyl-3-phenoxymethyl-6,7-dihydro-5h - naphthyridin-8-one ( 20 mg , 0.058 mmol ) in toluene ( 0.5 ml ) in a sealed tube . dowex 1 2100 ( strongly basic anion exchanger ) ( 300 mg ) was added , followed by meoh ( 1 ml ) . the mixture was shaken 4 h at rt , and the resin was filtered and washed with ch2cl2 ( 1 ml ) , meoh ( 1 ml ) , ch2cl2 ( 1 ml ) , and meoh ( 1 ml ) . the crude product was purified by flash column chromatography ( silica , 7 m ammonia in methanol in ch2cl2 0/100 to 5/95 ) . the desired fractions were collected and the solvents evaporated in vacuo to yield an impure fraction which was purified by rp - hplc on ( c18 xbridge 19 mm 100 mm 5 m ) . mobile phase ( gradient from 80% 0.1% nh4co3h / nh4oh ph 9 solution in water , 20% ch3cn to 0% 0.1% nh4co3h / nh4oh ph 9 solution in water , 100% ch3cn ) to yield 14a as a white solid ( 6.26 mg , 42% yield ) . lcms tr = 1.36 min , > 98% at 215 and 254 nm , m / z = 255.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 8.74 ( d , j = 1.4 hz , 1 h ) , 7.73 ( br . s , 1 h ) , 7.397.29 ( m , 2 h ) , 7.24 ( br . s , 1 h ) , 7.056.94 ( m , 3 h ) , 5.14 ( s , 2 h ) , 3.64 ( td , j = 6.6 , 2.9 hz , 2 h ) , 3.09 ( t , j = 6.6 hz , 2 h ) . methylamine ( 2.0 m in thf ) ( 2 ml ) was added to a stirred solution of 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic acid methyl ester ( 40 mg , 0.14 mmol ) in ch2cl2 ( 2 ml ) . the mixture was stirred at rt for 1 h. then , sodium triacetoxyborohydride ( 45 mg , 0.21 mmol ) was added and the mixture was stirred at rt for 16 h. the mixture was diluted with ch2cl2 and washed with a saturated solution of nahco3 and brine . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in vacuo . the crude product was purified by flash column chromatography ( silica ; 7n solution of ammonia in methanol in ch2cl2 0/100 to 4/96 ) . the desired fractions were collected and the solvents evaporated in vacuo to yield an impure fraction which was repurified by rp - hplc on ( c18 xbridge 19 mm mm 100 5 m ) . mobile phase ( gradient from 80% 0.1% nh4co3h / nh4oh ph 9 solution in water , 20% ch3cn to 0% 0.1% nh4co3h / nh4oh ph 9 solution in water , 100% ch3cn ) to yield 14b as a white solid ( 5.8 mg , 15% ) . lcms tr = 1.59 min , > 98% at 215 and 254 nm , m / z = 269.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 8.73 ( d , j = 1.7 hz , 1 h ) , 7.68 ( s , 1 h ) , 7.367.28 ( m , 2 h ) , 7.086.90 ( m , 3 h ) , 5.13 ( s , 2 h ) , 3.62 ( t , j = 6.6 hz , 2 h ) , 3.22 ( s , 3 h ) , 3.08 ( t , j = 6.8 hz , 2 h ) . . starting from 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic acid methyl ester ( 40 mg , 0.14 mmol ) and ( r)-(+)-3,3-dimethyl 2-aminobutane 0.039 ml , 0.34 mmol ) following the procedure described for compound 14a b , compound 14c was obtained as a beige solid ( 24 mg , 25% ) . lcms tr = 2.82 min , > 98% at 215 and 254 nm , m / z = 339.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 8.73 ( d , j = 1.7 hz , 1 h ) , 7.68 ( s , 1 h ) , 7.287.37 ( m , 2 h ) , 6.927.04 ( m , 3 h ) , 5.13 ( s , 2 h ) , 4.92 ( q , j = 7.2 hz , 1 h ) , 3.623.46 ( m , 2 h ) , 3.102.98 ( m , 1 h ) , 2.982.84 ( m , 1 h ) , 1.22 ( d , j = 7.2 hz , 3 h ) , 1.00 ( s , 9 h ) ; d = 15.7 ( c = 0.55 w / v% , meoh ) . 4-fluoroaniline ( 0.032 ml , 0.34 mmol ) was added to a stirred solution of 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic acid methyl ester ( 80 mg , 0.28 mmol ) in ch2cl2 ( 4 ml ) . the mixture was stirred at rt for 1 h. then , sodium triacetoxyborohydride ( 89 mg , 0.42 mmol ) was added and the mixture was stirred at rt for 16 h. then , acetic acid ( 0.040 ml ) was added and the mixture was stirred at rt for additional 20 h. the mixture was diluted with ch2cl2 and washed with a saturated solution of nahco3 and brine . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in vacuo . the crude product was purified by flash column chromatography ( silica ; 7n solution of ammonia in methanol in ch2cl2 0/100 to 4/96 ) . the desired fractions were collected and the solvents evaporated in vacuo to yield an impure fraction which was repurified by rp - hplc on ( c18 xbridge 19 mm 100 mm , 5 m ) . mobile phase ( gradient from 80% 0.1% nh4co3h / nh4oh ph 9 solution in water , 20% ch3cn to 0% 0.1% nh4co3h / nh4oh ph 9 solution in water , 100% ch3cn ) , to yield 14d as a white solid ( 15 mg , 15% ) . lcms tr = 2.00 min , > 98% at 215 and 254 nm , m / z = 349.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 8.78 ( d , j = 1.7 hz , 1 h ) , 7.76 ( br . s , 1 h ) , 7.427.36 ( m , 2 h ) , 7.357.29 ( m , 2 h ) , 7.167.07 ( m , 2 h ) , 7.01 ( t , j = 7.5 hz , 1 h ) , 6.98 ( d , j = 7.8 hz , 2 h ) , 5.17 ( s , 2 h ) , 4.01 ( t , j = 6.4 hz , 2 h ) , 3.22 ( t , j = 6.4 hz , 2 h ) . a 7n solution of ammonia in methanol ( 5.5 ml ) was added to 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester ( 33 , 55 mg , 0.2 mmol ) . the mixture was stirred at 100 c for 16 h. the solvents were evaporated in vacuo . the crude product was purified by flash chromatography ( silica ; acoet in ch2cl2 0/100 to 100/0 ) . the desired fractions were collected and the solvents evaporated in vacuo to yield an impure fraction which was triturated with et2o and repurified by rp - hplc on ( c18 xbridge 30 mm 100 mm , 5 m ) . mobile phase ( gradient from 80% 0.1% nh4co3h / nh4oh ph 9 solution in water , 20% ch3cn to 0% 0.1% nh4co3h / nh4oh ph 9 solution in water , 100% ch3cn ) to yield 15a as a white solid ( 20 mg , 39% ) ; mp 193 c . lcms tr = 1.47 min , > 98% at 215 and 254 nm , m / z = 255.1 [ m + h ] . h nmr ( 400 mhz , cdcl3 ) 9.18 ( s , 1 h ) , 7.43 ( d , j = 0.5 hz , 1 h ) , 7.367.28 ( m , 2 h ) , 7.066.94 ( m , 3 h ) , 6.26 ( br s , 1 h ) , 5.24 ( s , 2 h ) , 3.61 ( td , j = 6.6 , 2.9 hz , 2 h ) , 3.03 ( t , j = 6.7 hz , 2 h ) .. starting from 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester ( 33 , 55 mg , 0.2 mmol ) and 2 m methylamine in thf ( 5.5 ml ) following the procedure described for compound 15a , compound 15b was obtained as a white solid ( 51 mg , 93% ) ; mp 134 c . lcms tr = 1.72 min , > 98% at 215 and 254 nm , m / z = 269.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 9.18 ( s , 1 h ) , 7.38 ( s , 1 h ) , 7.31 ( s , 2 h ) , 7.046.95 ( m , 3 h ) , 5.23 ( s , 2 h ) , 3.59 ( t , j = 6.6 hz , 2 h ) , 3.16 ( s , 3 h ) , 3.03 ( t , j = 6.6 hz , 2 h ) .. acetic acid ( 0.006 ml , 0.11 mmol ) was added to a solution of 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester ( 33 , 29 mg , 0.11 mmol ) and ( r)-(+)-3,3-dimethyl 2-aminobutane ( 0.030 ml , 0.225 mmol ) in meoh ( 1 ml ) . the mixture was stirred at 100 c for 16 h. then ( r)-(+)-3,3-dimethyl 2-aminobutane ( 0.030 ml , 0.225 mmol ) and acetic acid ( 0.006 ml , 0.11 mmol ) were added , and the mixture was heated at 100 c for 2 days . the solvents were evaporated in vacuo and the residue diluted with ch2cl2 and extracted with a saturated solution of nahco3 . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in vacuo . the crude product was purified by flash chromatography ( silica ; acoet in ch2cl2 0/100 to 30/70 ) . the desired fractions were collected and the solvents evaporated in vacuo to yield compound 15c as a colorless oil which solidified upon standing at room temperature ( 29 mg , 79% ) . lcms tr = 3.10 min , > 98% at 215 and 254 nm , m / z = 339.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 9.19 ( s , 1 h ) , 7.38 ( s , 1 h ) , 7.31 ( t , j = 8.1 hz , 2 h ) , 7.036.92 ( m , 3 h ) , 5.24 ( s , 2 h ) , 4.83 ( q , j = 7.2 hz , 1 h ) , 3.593.43 ( m , 2 h ) , 3.062.93 ( m , 1 h ) , 2.932.81 ( m , 1 h ) , 1.21 ( d , j = 6.9 hz , 3 h ) , 0.99 ( s , 9 h ) . starting from 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester ( 33 , 30 mg , 0.11 mmol ) and 4-fluoroaniline ( 0.044 ml , 0.47 mmol ) following the procedure described for compound 15c , title compound 15d was obtained as a white solid ( 24 mg , 62% ) ; mp 198.8 c . lcms tr = 2.66 min , > 98% at 215 and 254 nm , m / z = 349.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 ) 9.24 ( s , 1 h ) , 7.46 ( s , 1 h ) , 7.397.29 ( m , 4 h ) , 7.177.08 ( m , 2 h ) , 7.046.96 ( m , 3 h ) , 5.27 ( s , 2 h ) , 3.99 ( t , j = 6.5 hz , 2 h ) , 3.17 ( t , j = 6.4 hz , 2 h ) .. 3-(3-bromo-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)(cyclopropyl)methanone ( 35a , 35 mg , 0.125 mmol ) , 3-fluorobenzyl alcohol ( 15 mg , 0.125 mmol ) , cesium carbonate ( 59 mg , 0.18 mmol ) , cui ( 3 mg , 0.01 mmol ) , and 1,10-phenanthroline ( 3.9 mg , 0.02 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . degassed toluene was added and the mixture heated at 110 c for 4 h. the mixture was cooled to rt , filtered over celite , and the filtrate concentrated to dryness . rp - hplc purification afforded 20d as an off - white powder ( 15 mg , 33% ) . lcms tr = 0.79 min , > 98% at 215 and 254 nm , m / z = 328.1 [ m + h ] . hrms ( es+ , m + h ) calcd for c22h20fn2o2 , 327.1509 ; found , 327.1512 . 3-bromo-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)(4-fluorophenyl)methanone ( 35c , 28 mg , 0.09 mmol ) , benzyl alcohol ( 9.6 mg , 0.09 mmol ) , cesium carbonate ( 43 mg , 0.13 mmol ) , cui ( 2 mg , 0.009 mmol ) , and 1,10-phenanthroline ( 3.2 mg , 0.018 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . degassed toluene was added and the mixture heated at 110 c for 4 h. the mixture was cooled to rt , filtered over celite , and the filtrate concentrated to dryness . rp - hplc purification afforded the 20 g as an off - white powder ( 6.9 mg , 22% ) . lcms tr = 0.85 min , > 98% at 215 and 254 nm , m / z = 363.1 [ m + h ] . hrms ( es+ , m + h ) calcd for c22h22fn2o2 , 363.1509 ; found , 363.1503 . ( 4-fluorophenyl)(3-(hydroxymethyl)-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)methanone ( 39 , 40 mg , 0.14 mmol ) , phenol ( 40 mg , 0.43 mmol ) , and polystyrene supported triphenylphosphine ( 150 mg , 0.45 mmol ) were combined in thf ( 6 ml ) and diisopropyl azodicarboxylate ( 42 mg , 0.21 mmol ) was added , and the reaction was allowed to stir overnight at rt . the residue was purified via rp - hplc ( 1090% gradient of ch3cn in water with 0.1% tfa modifier ) to afford title compound as an off - white powder ( 35 mg , 69% ) . lcms tr = 0.75 min , > 98% at 215 and 254 nm , m / z = 363.1 [ m + h ] . h nmr ( 400 mhz , cdcl3 ) 8.77 ( s , 1h ) , 8.06 ( s , 1h ) , 7.537.49 ( m , 3h ) , 7.36 ( t , j = 8.0 , 2h ) , 7.18 ( t , j = 8.0 , 2h ) , 7.06 ( t , j = 8.2 , 1h ) , 6.87 ( d , j = 8.0 , 1h ) , 5.18 ( s , 2h ) , 4.98 ( s , 2h ) , 3.92 ( s , 2h ) , 3.36 ( t , j = 6.0 , 2h ) . hrms ( es+ , m + h ) calcd for c22h19fn2o2 , 362.1431 ; found , 362.1434 . for measurement of compound - evoked increases in intracellular calcium , hek293 cells stably expressing rat mglu5 were plated in 384-well , poly - d - lysine coated , black - walled , clear - bottomed plates in 20 l of assay medium ( dmem supplemented with 10% dialyzed fetal bovine serum , 20 mm hepes , and 1 mm sodium pyruvate ) at a density of 15000 cells / well . the next day , medium was removed and cells were incubated with 20 l / well of 1 m fluo-4am ( invitrogen , carlsbad , california ) prepared as a 2.3 mm stock in dimethyl sulfoxide ( dmso ) and mixed in a 1:1 ratio with 10% ( w / v ) pluronic acid f-127 and diluted in calcium assay buffer ( hank s balanced salt solution ( hbss ; invitrogen , carlsbad , ca ) supplemented with 20 mm hepes and 2.5 mm probenecid , ph 7.4 ) for 50 min at 37 c . dye loading solution was then removed and replaced with 20 l / well of assay buffer . for pam potency curves , mglu5 compounds were diluted in calcium assay buffer and added to the cells , followed by the addition of an ec20 concentration of glutamate 140 s later and then an ec80 concentration of glutamate 90120 s later . for fold - shift experiments , either a single concentration ( 10 m ) or multiple fixed concentrations ( 55 nm to 30 m ) of mglu5 compound or vehicle was added , followed by the addition of a concentration calcium flux was measured over time as an increase in fluorescence using a functional drug screening system 6000 ( fdss 6000 , hamamatsu , japan ) . the change in relative fluorescence over basal was calculated before normalization to the maximal response to glutamate . a 2-fold enhancement over basal response at the maximum concentration was sufficient criteria for weak pam activity ( e.g. , 40% glu max at an ec20 add of glutamate ; the basal ec20 response range allowed was 1030% ) . to assess the effect of test compounds at mglu1 , ca mobilization assays were performed as described previously . briefly , hek293 cells stably expressing rat mglu1 were plated in black - walled , clear - bottomed , poly - d - lysine coated 384-well plates ( greiner bio - one , monroe , nc ) in assay medium at a density of 20000 cells / well . calcium flux was measured over time as an increase in fluorescence of the ca indicator dye , fluo-4am using a fdss 6000 . either vehicle or a fixed concentration of test compound ( 10 m , final concentration ) was added , followed 140 s later by a crc of glutamate . the functional activity of the compounds of interest was assessed at the rat group ii and iii mglu receptors by measuring thallium flux through girk channels as previously described . briefly , hek293-girk cells expressing mglu subtypes 2 , 3 , 4 , 6 , 7 , or 8 were plated into 384-well , black - walled , clear - bottom poly - d - lysine coated plates at a density of 15000 cells / well in assay medium . a single concentration of test compound ( 10 m ) or vehicle was added , followed 140 s later by a crc of glutamate ( or l - ap4 for mglu7 ) diluted in thallium buffer ( 125 mm nahco3 , 1 mm mgso4 , 1.8 mm caso4 , 5 mm glucose , 12 mm thallium sulfate , 10 mm hepes ) , and fluorescence was measured using a fdss 6000 . animals were housed in the animal care facility certified by the american association for the accreditation of laboratory animal care ( aaalac ) under a 12 h light / dark cycle ( lights on , 7 a.m. ; lights off , 7 p.m. ) and had free access to food and water . the animals used in these experiments were food - deprived the evening before experimentation for oral administration of test compound . the experimental protocols performed during the light cycle were approved by the institutional animals care and use committee of vanderbilt university and conformed to the guidelines established by the national research council guide for the care and use of laboratory animals . 12c was formulated in volumes specific to the number of animals dosed each day . the solutions were formulated so that animals were injected with a maximal dosing volume of 10 ml / kg . the appropriate amount according to the dosage was mixed into a 20% 2-(hydroxypropyl)--cyclodextrin in sterile water ( hp--cd ; sigma , catalogue no . c0926 - 10 g ) solution . each mixture was ultrahomogenized on ice for 23 min using a hand - held tissue homogenizer . next , the ph of all solutions was checked using 014 emd ph strips and adjusted to a ph of 67 if necessary with 1n naoh . the mixtures were then vortexed and stored in a sonication bath at 40 c until time of injection . d - amphetamine hemisulfate ( amp ) was obtained from sigma ( catalogue no . a5880 - 1 g ; st . salt - correction was used to determine the correct amount of the d - amphetamine hemisulfate form in mg to add to sterile water in order to yield a 1 mg / ml solution , injected with a maximal dosing volume of 10 ml / kg . studies were conducted using smart open field activity chambers ( 27 cm 27 cm 20 cm ) ( kinder scientific , poway , ca ) equipped with 16 horizontal ( x- and y - axes ) infrared photobeams . changes in locomotor activity were measured as the number of photobeam breaks over time and were recorded with a pentium i computer equipped with rat activity monitoring system software ( motor monitor , kinder scientific , poway , ca ) . male harlan sprague dawley rats ( harlan laboratories , indianapolis , in ) weighing 250375 g were used . animals were next pretreated for an additional 30 min with either vehicle or a dose of 12c po , followed by a subcutaneous injection of 1 mg / kg amphetamine or vehicle and monitored for an additional 60 min . dose group 1 , vamp = 20% -cd ( 12c vehicle ) , po + 1 mg / kg amp , sc ( n = 8) . dose group 2 , 3amp = 3 mg / kg 12c , po + 1 mg / kg amp , sc ( n = 6 ) . dose group 3 , 10amp = 10 mg / kg 12c , po + 1 mg / kg amp , sc ( n = 7 ) . dose group 4 , 30amp = 30 mg / kg 12c , po + 1 mg / kg amp , sc ( n = 8) . mg / kg 12c , po + 1 mg / kg amp , sc ( n = 8) . mg / kg 12c , po + 1 mg / kg amp , sc ( n = 8) . dose group 7 , 100v = 100 mg / kg 12c , po + sterile water ( amp vehicle ) , sc ( n = 8) . data were expressed as changes in ambulation defined as the total number of photobeam breaks per 5 min interval . at the end of this behavioral study , each animal was euthanized , then decapitated , and the plasma and brain tissues were collected for the evaluation of exposure levels of 12c . behavioral data were analyzed using a one - way anova with main effects of treatment and time . post hoc analyses were performed using a dunnett s t - test with all treatment groups compared to the vamp group using jmp 8.0 ( sas institute , cary , nc ) statistical software . data were graphed using sigmaplot for windows version 11.0 ( saugua , ma ) . a probability of p 0.05 was taken as the level of statistical significance . percent effect and reversal calculations for the 3amp , 10amp , 30amp , 56.6amp , and 100amp treatment groups were performed with the following formula relative to the vamp treatment group : ( 1 ) total number of photobeam breaks in the time interval from t = 60 to t = 120 was calculated for each rat in each treatment group , ( 2 ) mean total number of photobeam breaks in the time interval from t = 60 to t = 120 was calculated for the vamp group , ( 3 ) percent effect = ratio of the total number of photobeam breaks for each rat in each treatment group divided by the mean total number of photobeam breaks in the time interval from t = 60 to t = 120 of the vamp group multiplied by 100 , ( 4 ) percent reversal = 100 the percent effect for each rat in each treatment group , ( 5 ) finally , the mean se percent reversal for each treatment group was calculated from the individual percent reversal values . percent effect and change calculations for the vamp and 100 v treatment groups relative to the vv treatment group were also performed with the following formula : ( 1 ) total number of photobeam breaks in the time interval from t = 60 to t = 120 was calculated for each rat in each treatment group , ( 2 ) mean total number of photobeam breaks in the time interval from t = 60 to t = 120 was calculated for the vv group ; ( 3 ) percent effect = ratio of the total number of photobeam breaks for each rat in each treatment group divided by the mean total number of photobeam breaks in the time interval from t = 60 to t = 120 of the vv group multiplied by 100 , ( 4 ) percent change = the percent effect for each rat in each treatment group 100 , ( 5 ) finally , the mean se percent change for each treatment group was calculated from the individual percent change values . brain samples were homogenized in 3 ml of 70:30 2-propanol : water and then centrifuged at 3500 rpm for 5 min . resulting brain sample supernatant and plasma samples were transferred into a 96-well plate containing plasma blanks , plasma double blank , a standard curve of the test article , and quality control samples for subsequent lc - ms / ms analysis . each sample was diluted with acetonitrile containing 50 nm carbamazepine ( internal standard ) , centrifuged at 3500 rpm , and the resulting supernatant was transferred to a new 96-well plate . after an equal volume of water was added to each sample ( to provide 50:50 acetonitrile : water solution ) , the plate was analyzed via esi on a triple - quadrupole mass spectrometer ( ab sciex api-4000 ) coupled with an autosampling liquid chromatography system ( shimadzu lc-10ad pumps , leap technologies ctc pal autosampler ) . analyte ( test article ) was separated by gradient elution using a c18 3.0 mm 50 mm , 3 m column ( fortis technologies ) thermostated at 40 c . hplc mobile phase a was 0.1% formic acid in water ( ph unadjusted ) , mobile phase b was 0.1% formic acid in acetonitrile ( ph unadjusted ) , and the gradient used was 3090% mobile phase b with 0.2 min hold at 30% b , linear increase to 90% b over 0.8 min , hold at 90% b for 0.6 min , and a return to 30% b over 0.1 min followed by a re - equilibration ( 0.5 min ) to provide a 2.0 min total run time per sample . hplc flow rate was 0.5 ml / min , source temperature was 500 c , and mass spectral analyses were performed using the following mrm transitions : 353.5 259.2 and 353.5 123.2 m / z . esi was achieved by a turbo - ionspray source in positive ionization mode ( 5.0 kv spray voltage ) . the raw plasma and brain concentration data obtained by lc - ms / ms were analyzed by analyst software ( ab sciex , version 1.5.1 ) , which used the ratio of peak area responses of drug relative to internal standard to construct a standard curve with a dynamic range covering the concentrations found in the samples . free plasma and brain concentrations were calculated by multiplication of the total concentration values by fu , plasma and fu , brain , respectively , based on data from in vitro rat plasma protein and rat brain homogenate binding experiments . the effects of 12c on motor performance were evaluated using an automated rotarod setup with a rotarod ( 7.0 cm in diameter ) rotating at a constant speed of 20 rotations / min ( medassociates , inc . , male harlan sprague dawley rats ( harlan laboratories , indianapolis , in ) weighing 250300 g were used . animals were given two training trials of 120 s on the rotarod with a 10 min interval between trials , followed by baseline assessment of performance with a 120 s trial , and any animals that did not reach a performance criteria of 85 s were excluded from the study . animals were next pretreated for 30 min with vehicle ( 20% hp--cd ) or a dose of 12c ( n = 6 each group ) and then tested on the rotarod using a 120 s trial . the amount of time in seconds that each animal remained on the rotarod was recorded ; animals not falling off of the rotarod were given a maximal score of 120 s. data were expressed as the mean latency to fall off the rotarod in seconds for each treatment group . behavioral data were analyzed using a one - way anova with main effects of treatment . post hoc analyses were performed using a dunnett s t test with all treatment groups compared to the vehicle group using jmp 8.0 ( sas institute , cary , nc ) statistical software . data were graphed using sigmaplot for windows version 11.0 ( saugua , ma ) . a probability of p 0.05 was taken as the level of statistical significance . male harlan sprague dawley rats ( harlan laboratories , indianapolis , in ) weighing 250300 g were used . animals were evaluated in the modified irwin neurological test battery at t = 0 to provide a baseline measurement across each functional end point . animals were next administered either vehicle ( 20% hp--cd ) or a 100 mg / kg po dose of 12c ( n = 6 each group ) and then assessed after a 30 min , 1 h , and 4 h pretreatment interval in the modified irwin neurological test battery . changes in the different functional end points of the irwin test battery were given a score of 0 , 1 or 2 , with 0 = no effect , 1 = moderate effect , and 2 = robust full effect . autonomic nervous system functions : ptosis , exophthalmos , miosis , mydriasis , corneal reflex , pinna reflex , piloerection , respiratory rate , writhing , tail erection , lacrimation , salivation , vasodilation , skin color , irritability , and rectal temperature . somatomotor nervous system functions : motor activity , ataxia , arch / roll , tremors , leg weakness , rigid stance , spraddle , placing loss , grasping loss , righting loss , catalepsy , tail pinch reaction , escape loss , and physical appearance . mean change values for each functional end point in the irwin test battery of each treatment group were calculated using microsoft excel . behavioral data were then analyzed using a one - way anova with main effect of treatment . post hoc analyses were performed using a dunnett s t test with all treatment groups compared to the vehicle group using jmp 8.0 ( sas institute , cary , nc ) statistical software . data were graphed using sigmaplot for windows version 11.0 ( saugua , ma ) . a probability of p 0.05 was taken as the level of statistical significance .

positive allosteric modulators ( pams ) of metabotropic glutamate receptor 5 ( mglu5 ) represent a promising therapeutic strategy for the treatment of schizophrenia . starting from an acetylene - based lead from high throughput screening , an evolved bicyclic dihydronaphthyridinone was identified . we describe further refinements leading to both dihydronaphthyridinone and tetrahydronaphthyridine mglu5 pams containing an alkoxy - based linkage as an acetylene replacement . exploration of several structural features including western pyridine ring isomers , positional amides , linker connectivity / position , and combinations thereof , reveal that these bicyclic modulators generally exhibit steep sar and within specific subseries display a propensity for pharmacological mode switching at mglu5 as well as antagonist activity at mglu3 . structure activity relationships within a dihydronaphthyridinone subseries uncovered 12c ( vu0405372 ) , a selective mglu5 pam with good in vitro potency , low glutamate fold - shift , acceptable dmpk properties , and in vivo efficacy in an amphetamine - based model of psychosis .

Introduction Results and Discussion Conclusions Experimental Section

schizophrenia is a complex mental illness characterized by positive ( hallucinations , paranoia , disorganized behavior ) and negative symptoms ( social withdrawal , anhedonia , flat affect ) as well as cognitive dysfunction ( deficits in attention , learning , and memory ) . these include extrapyramidal side effects , sexual dysfunction , weight gain ( metabolic syndrome ) , agranulocytosis , increased cardiac risk , and poor patient compliance . the development of positive allosteric modulators ( pams ) of metabotropic glutamate receptor 5 ( mglu5 ) as a novel approach to test the glutamate or n - methyl - d - aspartate ( nmda ) receptor hypofunction hypothesis of schizophrenia has provided preclinical evidence for therapeutic potential in multiple psychosis and cognitive animal models , and , for more than a decade , industry and academic drug discovery groups have been in pursuit of brain penetrant small molecule mglu5 potentiators . a testament to the success of the field has been the identification of over eight reported chemotypes with in vivo efficacy in preclinical models , beginning with 3-cyano - n-(1,3-diphenyl-1h - pyrazol-5-yl)benzamide ( cdppb , 1 ) , piperidinyl 1,2,4-oxadiazoles from addex ( adx-47273 , 2 ) , and subsequently acetylenes n - methyl-5-(phenylethynyl)pyrimidin-2-amine ( mppa , 3 ) , vu0360172 ( 4 ) , lsn2463359 ( 8) , piperazines vu0364289 ( 5 ) and 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone ( cppz , 6 ) , ether vu0404251 ( 7 ) , and triazole lsn2814617 from lilly ( 9 ) . , glutamate fold - shift or potentiation ) and devoid of allosteric agonism may be preferred for improved therapeutic index . activity relationships ( sar ) suggested a preference for the phenoxy - based linker within the dihydrothiazolopyridones ( two examples tested ) ; however , this trend was not found within monocyclic nicotinamides of type 7 . prior studies within a dihydronaphthyridinone class utilizing an acetylene - based linker , initially identified from an hts screen , led to pams with submicromolar potency , including dihydronaphthyridinones 11 , suggested that linkers employing either ch2o or och2 might be successful as an acetylene replacement . the pursuit of ethers 1220 were undertaken as part of a larger comprehensive study aimed to target four key structure activity relationships : ( 1 ) endocyclic versus exocyclic amide , ( 2 ) phenoxy versus benzyloxy linker , ( 3 ) central ring pyridine isomers , and ( 4 ) site of the pendant group attachment to central heterocycle . it was unclear at the outset if ethers 1220 would exhibit subtle changes in the mode of pharmacology similar to 11(32 ) and other chemotypes or would maintain a higher fidelity for potentiation similar to cdppb ( 1),n-[5-chloro-2-[(1,3-dioxoisoindolin-2-yl)methyl]phenyl]-2-hydroxybenzamide ( cppha ) , piperazines ( 56 ) , and glycine sulfonamide ( ml332 ) based mglu5 pams . herein we describe the synthesis and pharmacological profile of a series of tetrahydronaphthyridine and dihydronaphthyridinone ethers ( 1220 ) as positive allosteric modulators of mglu5 . low cooperativity pam 12c ( vu0405372 ) was ultimately identified and found to demonstrate a suitable balance of in vitro and in vivo properties for proof - of - concept studies in an amphetamine hyperlocomotor challenge model . in contrast to previously reported and related monocyclic ether series , these bicyclic modulator scaffolds were overall found to exhibit narrow sar , low efficacy , and have a higher propensity for pharmacological mode switching upon chemical modification , demonstrating an sar profile reminiscent of related acetylenic pams . mglu5 receptor pams with reported efficacy in preclinical models of schizophrenia and cognition . starting from known compound 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 21a , supporting information ) , substitution under basic conditions in the presence of various alcohols with heating afforded 12a12h , 12q , and 12s in low to moderate yield . within a third class of lactams 14 ( scheme 3 ) , a benzyloxy linkage was retained and an alternate dihydro-1,7-naphthyridinone ring system was explored , placing the pyridine nitrogen at the position ortho to the lactam amide . treatment of 25 under mitsunobu conditions using phenol and di - tert - butyl azodicarboxylate ( dbab ) , followed by hydrolysis and ester formation , afforded chloroester 27 . as reported previously , compounds were profiled in a rat mglu5 low receptor expression cell line using a triple add protocol allowing for detection of agonism as well as positive and negative allosteric modulation simultaneously . in contrast , an allosteric modulator devoid of agonism in these low - expressing mglu5 cells was found not to display a seizure liability in vivo . triple add protocol provided an important initial in vitro assessment of both agonist and potentiation activity at mglu5 , which could be utilized to prioritize analogues for further progression . sar are shown in tables 14 ( 1215 , 20 ) and figures 46 ( 1619 ) and summarize in vitro potency optimization efforts from 10 distinct subseries . studies by astrazeneca pharmaceuticals and within the dihydrothiazolopyridone series 10(31 ) have demonstrated successful utilization of the both benzyloxy- and phenoxymethyl - based spacers as replacements for the acetylene moiety , with varied potency depending on the nature of the heterocycle scaffold . subtle changes in structure efficacy / cooperativity relationships are best addressed via glutamate fold - shift experiments ; however , glutamate max trends were consistent within series 1215 , and series 12 shows variable efficacies that encompass the full range reported for all subseries ( figure 3 ) . at this juncture , in light of the unexpected switch in the mode of pharmacology for subseries 18 , as well as the general challenges of multidimensional combinations of linker , core , attachment site , and lactam versus exocyclic amide ( minimum of 24 formal combinations / scaffolds ) , we employed a ligand - based computational tool previously reported to prioritize and potentially streamline medicinal chemistry efforts and ligand design . hypotheses or reference pams for a mutual flexible low energy shape - based alignment of proposed dihydronaphthyridinone and tetrahydronaphthyridines using the surflex - sim algorithm as implemented in sybyl . subsequent selectivity profiling against mglu14,68 using 12c , 13 g , and 14d revealed that n - aryl congeners 13 g and 14d were also active as mglu3 negative allosteric modulators ( nams ) with negative cooperativity fold - shift values between 0.2 and 0.3 , while 12c was fully selective for mglu5 ( mglu14,68 ec50 > 10 m , see supporting information ) . positive allosteric modulator activity was investigated by fold - shift experiments to determine the degree of cooperativity with glutamate . as shown in figure 8 , progressive fold - shift experiments utilizing the rat mglu5 receptor revealed a small leftward shift in the glutamate concentration response curve ( crc ) with increasing concentration of 12c ( 55 nm to 30 m ) , with no significant effect on the maximal response . , ml254 , maximum rat mglu5 fold - shift 3.0 ) and dihydrothiazolopyridone 10 ( maximum rat mglu5 fold - shift 5.4),12c represents one of the weakest mglu5 allosteric modulators reported in terms of measurable positive cooperativity . furthermore , in vivo b : p and in vitro fu , plasma / fu , brain ( 1.7 ) measures were identical , with a calculated kp , uu of 1.1 , suggesting passive cns penetration for 12c in rat . encouraged by its overall profile as a low fold - shift pam , 12c was evaluated across a range of doses for its ability to reverse amphetamine - induced hyperlocomotion ( ahl ) , an established model of antipsychotic activity ( figure 8) . the lowest active dose of 30 mg / kg afforded an estimated average terminal unbound brain concentration of 712 nm , which represents a 3-fold multiple of the rat in vitro potency and according to the progressive fold - shift studies ( figure 8) , represents a left - ward shift in the glutamate crc in the 1.21.4 fold range . despite the overall low positive cooperativity , 12c retains efficacy in ahl at unbound brain exposures that are a multiple of the in vitro ec50 and thus illustrates that high cooperativity with glutamate is not fully required for an mglu5 pam to maintain activity in the ahl model . this observation appears to be in contrast to data reported recently for low fold - shift pams 89 reported by lilly , wherein little effect was noted in a similar amphetamine - induced hyperlocomotor activity assay using male lister hooded rats . in a modified irwin neurological test battery in rats , 12c , when dosed at 100 mg / kg po ( 20% hp--cd in water ) , had no performance deficits on any autonomic or somatomotor nervous system functions out to 4 h. collectively , these results highlight that pam 12c is free from overt neurological side effects and motor behavior deficits at doses that produce maximal efficacy in reversal of amphetamine - induced hyperlocomotion ( 100 mg / kg po ) . diverse mglu5 modulator pharmacological profiles were attained within a series of tetrahydronaphthyridine and dihydronaphthyridinone scaffolds through modifications of heteroatoms within the linker , core structure , and location of the pendant linkage moiety . up to 10 diverse subseries were examined , and five of these series , 1215 and 20 , were described in detail , leading to preferred endocyclic series 12 and 13 which , although free from allosteric agonism and molecular switches , tended to have steep sar . parent benzyloxy lactam 12c emerged as a suitable tool compound with good potency , excellent selectivity , and pharmacokinetic properties suitable for acute studies . despite behaving in vitro as an ultralow cooperativity pam in recombinant systems , 12c showed robust , dose - dependent effects in the reversal of amphetamine - induced hyperlocomotion , with a lowest active dose of 30 mg / kg po , that produced an estimated brain unbound level 3-fold above its in vitro potency . analytical lc / ms was performed using the following instruments : ( a ) agilent 1200 series with uv detection at 215 and 254 nm and elsd detection ( polymer laboratories pl - els 2100 ) , utilizing an accucore c18 2.6 , 2.1 mm 30 mm column , a 1.1 min gradient , 7% [ ch3cn/0.1%tfa]95% [ ch3cn/0.1%tfa ] and a g6130 single quadrupole mass spectrometer or ( b ) ultra performance liquid chromatography ( uplc ) measurement performed using an acquity uplc ( waters ) system comprising a sampler organizer , a binary pump with degasser , a four column s oven , a diode array detector ( dad ) , and a beh - c18 column ( 1.7 m , 2.1 mm 50 mm ) from waters , with a flow rate of 1.0 ml / min at 50 c without split to the ms detector . starting from 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) and ( bromomethyl)cyclopropane ( 50 l , 0.50 mmol ) , the general procedure outlined in 13d was performed . c nmr ( 100 mhz , cdcl3 ) 163.6 ( d , jc f = 244.4 hz ) , 163.6 , 160.4 , 159.4 ( d , jc f = 10.8 hz ) , 158.4 , 156.8 ( d , jc f = 252.0 hz ) , 149.5 ( d , jc f = 25.2 hz ) , 130.4 ( d , jc f = 9.9 hz ) , 124.4 , 124.3 ( d , jc f = 2.9 hz ) , 108.3 ( d , jc f = 21.1 hz ) , 102.7 ( d , jc f = 24.9 hz ) , 70.5 , 45.0 , 31.2 . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in vacuo . h nmr ( 400 mhz , cdcl3 ) 9.18 ( s , 1 h ) , 7.43 ( d , j = 0.5 hz , 1 h ) , 7.367.28 ( m , 2 h ) , 7.066.94 ( m , 3 h ) , 6.26 ( br s , 1 h ) , 5.24 ( s , 2 h ) , 3.61 ( td , j = 6.6 , 2.9 hz , 2 h ) , 3.03 ( t , j = 6.7 hz , 2 h ) .. starting from 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester ( 33 , 55 mg , 0.2 mmol ) and 2 m methylamine in thf ( 5.5 ml ) following the procedure described for compound 15a , compound 15b was obtained as a white solid ( 51 mg , 93% ) ; mp 134 c . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in vacuo . h nmr ( 500 mhz , cdcl3 ) 9.24 ( s , 1 h ) , 7.46 ( s , 1 h ) , 7.397.29 ( m , 4 h ) , 7.177.08 ( m , 2 h ) , 7.046.96 ( m , 3 h ) , 5.27 ( s , 2 h ) , 3.99 ( t , j = 6.5 hz , 2 h ) , 3.17 ( t , j = 6.4 hz , 2 h ) .. 3-(3-bromo-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)(cyclopropyl)methanone ( 35a , 35 mg , 0.125 mmol ) , 3-fluorobenzyl alcohol ( 15 mg , 0.125 mmol ) , cesium carbonate ( 59 mg , 0.18 mmol ) , cui ( 3 mg , 0.01 mmol ) , and 1,10-phenanthroline ( 3.9 mg , 0.02 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . 3-bromo-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)(4-fluorophenyl)methanone ( 35c , 28 mg , 0.09 mmol ) , benzyl alcohol ( 9.6 mg , 0.09 mmol ) , cesium carbonate ( 43 mg , 0.13 mmol ) , cui ( 2 mg , 0.009 mmol ) , and 1,10-phenanthroline ( 3.2 mg , 0.018 mmol ) were combined in a 2.0 ml microwave vial and placed under an argon atmosphere . for fold - shift experiments , either a single concentration ( 10 m ) or multiple fixed concentrations ( 55 nm to 30 m ) of mglu5 compound or vehicle was added , followed by the addition of a concentration calcium flux was measured over time as an increase in fluorescence using a functional drug screening system 6000 ( fdss 6000 , hamamatsu , japan ) . a single concentration of test compound ( 10 m ) or vehicle was added , followed 140 s later by a crc of glutamate ( or l - ap4 for mglu7 ) diluted in thallium buffer ( 125 mm nahco3 , 1 mm mgso4 , 1.8 mm caso4 , 5 mm glucose , 12 mm thallium sulfate , 10 mm hepes ) , and fluorescence was measured using a fdss 6000 . animals were next administered either vehicle ( 20% hp--cd ) or a 100 mg / kg po dose of 12c ( n = 6 each group ) and then assessed after a 30 min , 1 h , and 4 h pretreatment interval in the modified irwin neurological test battery .

chronically ill employees themselves state that , apart from work accommodations , they need acceptance of having a disease , coping strategies and support from their supervisor in order to stay at work ( detaille et al . 2003 ) . this suggests that vocational rehabilitation aimed at changing personal attitudes and improving personal skills , including communication skills , is needed . we developed a theory - driven group training programme for employees with chronic disease who experience work - related problems . the programme provided participants with knowledge , skills and insight regarding their values and needs , and we called it an empowerment programme ( feste and anderson 1995 ) . it focused on solving work - related problems and aimed at job retention and maintenance and an increase in job satisfaction . in this article , we present a process evaluation of eight training courses with a total of 64 participants . a systematic process evaluation can tell us whether the intervention was feasible and describe potential barriers to its implementation . furthermore , it may clarify how the intervention works and gives insight into factors that influence its effectiveness ( swanborn 2004 ; baranowski and stables 2000 ; saunders et al . 2005 ; jonkers et al . the research questions for the process evaluation are : did the recruitment go as planned?was the target group reached?did participants follow the programme as it was intended?was the programme administered as intended?was the programme tailored to the group of participants?were participants satisfied with the program?was the programme perceived as effective?the medical ethics committee of the academic medical center in amsterdam approved of the study idea , but deemed ethical review unnecessary because they did not perceive the study to be medical research . the effectiveness of this intervention is studied with a randomized controlled trial ( rct ) design . did participants follow the programme as it was intended ? was the programme administered as intended ? was the programme tailored to the group of participants ? were participants satisfied with the program ? was the programme perceived as effective ? the training programme consisted of six three - hour sessions every 2 weeks and a seventh session 2 months after the sixth session . , there was an actor present for practicing role - playing . to discuss personal problems and progress at more length , three individual consultations also took place , one at the beginning , one halfway through the training and one after the sixth session . the trainers were experienced in working with groups , had psychotherapeutic knowledge of the principles of rational emotive therapy ( ret ) and occupational psychology , and a basic understanding of chronic disease and its consequences . the pilot version was adapted based on the trainers experiences , the researcher s observations , a pre- and post - test questionnaire and interviews with the participants by telephone . the programme had a stepwise approach : first , exploring and clarifying work - related problems ; second , a focus on communication at work ; and third , developing and realizing solutions . work - related problems were clarified with the help of the quality of work model , which emphasizes the energizing or distressing influences of work tasks , social relationships at work , working conditions and work - home interference . a seventy - page course book accompanied the training , and the sessions consisted of four to seven components , including discussion of the homework and preparations for the next session . each session focused on one theme : exploration and clarification of practical and psychosocial work - related problems with the help of the model quality of work;insight into feelings and thoughts about having a chronic disease and how these may influence communication;communication in daily work situations : theory and role play with an actor;practical matters : the occupational physician , the employment expert , legislation and facilities for disabled employees;communication and assertiveness : theory and role play with an actor;a smart plan to solve problems ; andfollow - up : what works and what does not.participants were eligible for the intervention if they had a chronic physical disease , had a paid job , experienced problems at work and feared losing their job or job satisfaction . workers with predominant psychiatric conditions were excluded ; people with a chronic physical disease in combination with depression were not excluded . workers on long - term full sick leave that was expected to extend into the following months were excluded . the candidates themselves had to apply to the programme by telephone , also when they were referred by medical professionals . exploration and clarification of practical and psychosocial work - related problems with the help of the model quality of work; insight into feelings and thoughts about having a chronic disease and how these may influence communication ; communication in daily work situations : theory and role play with an actor ; practical matters : the occupational physician , the employment expert , legislation and facilities for disabled employees ; communication and assertiveness : theory and role play with an actor ; a smart plan to solve problems ; and follow - up : what works and what does not . a comprehensive description of the set - up and contents of the training programme , its development and theoretical framework is published elsewhere ( varekamp et al . 2008 ) , as well as a systematic review of interventions of the same kind ( varekamp et al . the various elements of the process evaluation , their operationalisation and measurement are presented in table 1 ( baranowski and stables 2000 ; jonkers et al . specific barriers or other observations could be noted on the form , in addition to the structured items . participants filled in a questionnaire at baseline , and again after 4 , 8 , 12 and 24 months . opinions about the importance of several themes , satisfaction with various methods used in the training and overall satisfaction were measured on a 110 scale . participant opinions of the trainers capacities were assessed with an adapted version of the satisfaction with occupation rehabilitation ( 2004 ) , consisting of six subscales : expertise ( 2 items ) , advice given ( 4 items ) , friendly treatment ( 3 ) , personal attention ( 3 ) , usefulness of programme ( 2 ) and information about programme ( 2 ) . 24 mn.recruitment of participantskind of agencies approached and ways and frequency of approachxreach of target populationcharacteristics of participantsxparticipation in programmefrequency and reasons for dropoutxxfrequency and reasons for not attending meetingsxextent to which the programme was implementedextent to which all components were administered as plannedxdose received or fit with the participants needs and capabilitiestrainers opinion on participants ability to cognitively and emotionally grasp the components of the programxtrainers opinion on commitment and goal attainment of components of the programxsatisfaction of participants with the training programme and the traineroverall opinion on programme ( 110)xxxopinion on various themes ( 110)xopinion on various methods ( 110)xxopinion on various skills of the trainerxeffectiveness as perceived by participantsopinion on effectiveness with regard to three phasesxopinion on effectiveness of consultation with supervisor with regard to problem solvingxpersonal goal and opinion on goal attainmentxxopinion on effectiveness with regard to attitudes , skills , work accommodations and situation at homexopinion on permanence of effectxnotes res . notes of the researcher about recruitment and on drop outs after contact with trainers and participantsproc . questionnaire filled in by participants in advance , after 4 , 8 , 12 and 24 months elements of process evaluation notes res . notes of the researcher about recruitment and on drop outs after contact with trainers and participants proc . questionnaire filled in by participants in advance , after 4 , 8 , 12 and 24 months the medical ethics committee of academic medical center in amsterdam approved the study design and deemed ethical review unnecessary due to the non - medical nature of the research . participants were recruited for the training programme and study from late spring 2006 to january 2008 . participants were recruited via outpatient clinics , occupational health services , patient organizations , companies and so on . presentations were given to patient organizations , doctors , nurses and social workers in outpatient clinics , professionals at occupational health centres and to a national conference on chronic diseases . in addition , mailings were sent to several large companies and one patient organization sent a recruitment mailing to their members . advertisements were published in patient organization magazines , electronic newsletters and/or websites , in staff magazines at large companies and in magazines from an occupational health centre . about 3,500 paper leaflets were distributed via outpatient clinics , an occupational health centre and a patient information centre . it is difficult to assess the relative success of the various recruitment strategies , as we had no reports of the actions of medical professionals after hearing our presentations or reading about the project . presentations at outpatient clinics were seldom successful ; when they were , it was due to interested nurses who advised patients to contact us . contacts with companies were successful if they paid attention to the project in the staff magazine . table 2 presents figures on the sources of information about the project that the participants encountered ( control group included ) . recruitment took considerably more time than expected ; we estimate roughly that it took 810 months of full - time effort for one person to complete . one of the reasons for recruitment problems , according to some professionals of outpatient clinics and occupational health services , was that these professionals felt restrained from referring persons to the project because of the possibility of randomization to the control group ( personal communications to iv ) . another possible reason was that occupational physicians were afraid to lose patients when they referred them to the training programme ( personal communication to fvd).table 2source of information about the training programme for the participants of the study ( training participants and controls)sources of information%patient organization : magazine , presentation , website , mailing34companies : house organ or supervisor21occupational health service20outpatient clinic13conference on chronic diseases : magazine or presentation7other10more than one answer was possible ( n = 122 ) source of information about the training programme for the participants of the study ( training participants and controls ) more than one answer was possible ( n = 122 ) the personal , work and medical characteristics of the participants of the programme are presented in table 3 . mean age was 46 years , most participants were women , and highly educated people were over - represented . mean disease duration was 10 years and almost half had more than one chronic disease . fourteen per cent had categories of diseases , such as renal failure , poor eyesight , hiv and chronic fatigue syndrome . the great majority of the participants worked in the commercial or non - commercial service sector , for 30 h weekly , on average.table 3personal , medical and work characteristics of the training programme participants ( n = 64)mean ( sd ) or % age46.1 ( 8.8)women 83living alone ( not with partner , children or parents ) 33education lower3 middle36 higher61chronic disease icd classification 1 . diseases of the musculoskeletal system and connective tissue28 2 . diseases of the nervous system20 3 . diseases not otherwise specified14disease duration in years10.2 ( 9.6)an additional chronic disease % ( co morbidity)48branch of industry agriculture and fishing0 industry and building industry0 commercial services27 non - commercial services73appointment hours per week30 ( 8.6 ) personal , medical and work characteristics of the training programme participants ( n = 64 ) from november 2006 to march 2008 , eight training courses took place , including three trainers and 64 participants in total . three participants withdrew halfway , one due to medical treatment that interfered and two because they were not satisfied with the programme . overall , there were 55 missed sessions , but in the majority of cases , participants called to say they were unable to attend . forty - eight per cent participated in the training programme during working hours , 31% used days off and 20% combined these . generally , all the planned components of the sessions were discussed , although some only briefly because of lack of time . quality of work, which emphasizes energizing and fatiguing or distressing factors , took too much time . another trainer observed that the participants preferred to have time to exchange experiences with each other rather than listen to theoretical explanations , which they felt they could read in the course book . when discussing homework , it was often not possible to discuss each participant s work . when discussing work - related problems in the group using the quality of work model , only one instead of the planned two participants was often discussed . it was often impossible to have all participants practice role - playing in one session . one of the reasons was that discussing role - playing afterwards took a lot of time . according to the trainers , participants had rarely cognitive difficulties with understanding the various components of the training . one thing that some people found difficult to grasp was reflection on their work in terms of subjective perceptions instead of objective facts . slight or more severe emotional difficulties were met when discussing the consequences of having a chronic disease , feelings and thoughts on having a chronic disease and practical matters . one homework exercise presented difficulties for several participants ; they were asked twice in the course of the programme to arrange a consultation with their supervisor . the first session was intended to be a discussion of how the supervisor judged their work performance , the second to discuss work - related problems and solutions . pointless, because of their supervisor s attitude , or they wanted to practice such a consultation beforehand in order to be prepared ( see also last paragraph of the results section ) . the participants were asked to score how important the sessions themes were for them on a 110 scale ( table 4 ) . the themes insight into feelings and thoughts about having a chronic disease ( session 2 ) and communication and assertiveness ( sessions 3 and 5 ) , were valued highest , with a mean score of 8.0 . the theme exploration of practical and psychosocial work - related problems, which included the explanation of the model quality of work ( session 1 ) , scored 7.6 . practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees was evaluated as lowest , with a mean score of 7.0 , and a high standard deviation . the training programme as a whole was evaluated with a mean score of 8.1 immediately after completion ; this dropped 0.2 points 8 months later and 0.3 points 24 months later.table 4opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64)rating ( 110 ) mean ( sd)overall training programme opinion after 4 months8.1 ( 1.1 ) opinion after 12 months7.9 ( 1.1 ) opinion after 24 months7.8 ( 1.3)themes exploration and clarification of practical and psychosocial problems ; quality of work model ( session 1)7.6 ( 1.7 ) insight into feelings and thoughts about having a chronic disease ( session 2)8.0 ( 1.4 ) communication in daily work situations and standing up for oneself ( sessions 3 and 5)8.0 ( 1.4 ) practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees ( session 4)7.0 ( 2.0 ) a smart plan to solve problems ( session 6)7.5 ( 1.7 ) the course book7.9 ( 1.2)methods theory explanation7.2 ( 1.6 ) exchanging experiences8.3 ( 1.4 ) filling in and discussing quality of work model7.5 ( 1.2 ) discussing others quality of work model7.7 ( 1.5 ) role play with actor8.1 ( 1.6 ) questioning occupational physician and employment expert7.1 ( 1.7 ) having a consultation with the supervisor ( homework)7.2 ( 1.9 ) having a consultation with an occupational physician ( homework)6.7 ( 2.2 ) individual consultation with trainer halfway7.9 ( 1.4 ) individual consultation with trainer at the end7.9 ( 1.2)including opinion of three persons that dropped out halfwaylow response , n = 57low response , n = 49 opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64 ) including opinion of three persons that dropped out halfway eighty - six per cent of the participants always read the short introductions in the course book to prepare for the group sessions , whereas 95% had read the entire course book at the end of the training course . most valued were the chapters on communication and assertiveness , and on feelings and thoughts about having a chronic disease . lowest valued , with the highest standard deviation , was the chapter on legislation and work accommodations . a variety of methods was used in the training programme : theoretical explanation , exchange of experiences , role - playing , and homework , such as completing the model quality of work , or arranging a consultation with a supervisor and occupational physician . role - playing and seeing and discussing others role - playing was also highly appreciated , as were the individual consultations with the trainers . non - response on these two questionnaire items was high , 7 and 15 , respectively , which indicates that these arrangements not always took place . the expertise of the trainers was overall judged very positively ( mean score 68 on a 1680 scale ) , and the advice given by the trainers was felt to be helpful . the participants felt well - treated and felt that they received personal attention during the programme . they considered introductory information to be sufficient , although this could have been better for a minority . satisfaction with the trainers was not lower in the three groups in which the trainers acted for the first time , when compared to the five groups for which trainers were more experienced . the training programme used a stepwise approach : first exploring and clarifying work - related problems , then focusing on communication at work , and finally working on developing and realizing solutions . eight months after the start , 84% of the participants found that the first phase worked well , while 69% found that the second phase and 65% found that the third phase worked well ( table 5).table 5success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64)not successful at all % a little successful % amply successful % completely successful % 1clarifying bottlenecks ( model quality of work)016.455.727.92discussing bottlenecks at work3.327.945.923.03developing and realizing solutions6.728.345.020.0 success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64 ) the majority of the participants , 53 persons , had , as part of the training , discussed matters with their supervisor in order to find a solution for work - related problems . fifty - three per cent of them felt this contributed a great deal to solving problems , 40% said that it contributed somewhat , whereas 6% said that it did not contribute and 2% felt these discussions had worked negatively . table 6 presents the effects of the programme on various aspects of working with a chronic disease , as perceived at 12 months follow - up . the participants noticed positive effects most often with regard to how they experienced and dealt with disease and work . this was followed by how matters at work were discussed and how they dealt with the supervisor and colleagues . after 24 months , 79% perceived a lasting effect of the training programme , 10% perceived an effect that had faded away , 3% were not sure whether it had lasted , and 8% perceived none or only a limited effect.table 6effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64)effect training on large negative effectsmall negative effectno effectsmall positive effectlarge positive effecthow i experience my disease and my work03.311.748.336.7how i deal with my disease and my work03.38.345.043.3how i discuss matters at work01.726.741.730.0how i deal with my supervisor0023.351.725.0how i deal with my colleagues0028.356.715.0how my supervisor deals with me0038.343.318.3how my colleagues deal with me0041.738.320.0the situation at home0043.330.026.7accommodations of my workplace or work tasks1.71.753.326.716.7 effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64 ) in the course of the programme , the participants formulated a plan of action with one or more personal goals . these goals related to work - home interference ( 78% ) , feelings and thoughts about having a chronic disease ( 59% ) , communication at the workplace ( 44% ) , leisure time ( 33% ) , work accommodations ( 29% ) or other topics ( 18% ) . one year after the start of the programme , 6 per cent felt that they had not reached the goal that they set in the course of the programme , 38% reached it participants were recruited for the training programme and study from late spring 2006 to january 2008 . participants were recruited via outpatient clinics , occupational health services , patient organizations , companies and so on . presentations were given to patient organizations , doctors , nurses and social workers in outpatient clinics , professionals at occupational health centres and to a national conference on chronic diseases . in addition , mailings were sent to several large companies and one patient organization sent a recruitment mailing to their members . advertisements were published in patient organization magazines , electronic newsletters and/or websites , in staff magazines at large companies and in magazines from an occupational health centre . about 3,500 paper leaflets were distributed via outpatient clinics , an occupational health centre and a patient information centre . it is difficult to assess the relative success of the various recruitment strategies , as we had no reports of the actions of medical professionals after hearing our presentations or reading about the project . presentations at outpatient clinics were seldom successful ; when they were , it was due to interested nurses who advised patients to contact us . contacts with companies were successful if they paid attention to the project in the staff magazine . table 2 presents figures on the sources of information about the project that the participants encountered ( control group included ) . recruitment took considerably more time than expected ; we estimate roughly that it took 810 months of full - time effort for one person to complete . one of the reasons for recruitment problems , according to some professionals of outpatient clinics and occupational health services , was that these professionals felt restrained from referring persons to the project because of the possibility of randomization to the control group ( personal communications to iv ) . another possible reason was that occupational physicians were afraid to lose patients when they referred them to the training programme ( personal communication to fvd).table 2source of information about the training programme for the participants of the study ( training participants and controls)sources of information%patient organization : magazine , presentation , website , mailing34companies : house organ or supervisor21occupational health service20outpatient clinic13conference on chronic diseases : magazine or presentation7other10more than one answer was possible ( n = 122 ) source of information about the training programme for the participants of the study ( training participants and controls ) more than one answer was possible ( n = 122 ) the personal , work and medical characteristics of the participants of the programme are presented in table 3 . mean age was 46 years , most participants were women , and highly educated people were over - represented . mean disease duration was 10 years and almost half had more than one chronic disease . fourteen per cent had categories of diseases , such as renal failure , poor eyesight , hiv and chronic fatigue syndrome . the great majority of the participants worked in the commercial or non - commercial service sector , for 30 h weekly , on average.table 3personal , medical and work characteristics of the training programme participants ( n = 64)mean ( sd ) or % age46.1 ( 8.8)women 83living alone ( not with partner , children or parents ) 33education lower3 middle36 higher61chronic disease icd classification 1 . diseases not otherwise specified14disease duration in years10.2 ( 9.6)an additional chronic disease % ( co morbidity)48branch of industry agriculture and fishing0 industry and building industry0 commercial services27 non - commercial services73appointment hours per week30 ( 8.6 ) personal , medical and work characteristics of the training programme participants ( n = 64 ) from november 2006 to march 2008 , eight training courses took place , including three trainers and 64 participants in total . three participants withdrew halfway , one due to medical treatment that interfered and two because they were not satisfied with the programme . overall , there were 55 missed sessions , but in the majority of cases , participants called to say they were unable to attend . forty - eight per cent participated in the training programme during working hours , 31% used days off and 20% combined these . generally , all the planned components of the sessions were discussed , although some only briefly because of lack of time . one trainer mentioned that explaining the model quality of work, which emphasizes energizing and fatiguing or distressing factors , took too much time . another trainer observed that the participants preferred to have time to exchange experiences with each other rather than listen to theoretical explanations , which they felt they could read in the course book . when discussing homework , it was often not possible to discuss each participant s work . when discussing work - related problems in the group using the quality of work model , only one instead of the planned two participants was often discussed it was often impossible to have all participants practice role - playing in one session . one of the reasons was that discussing role - playing afterwards took a lot of time . according to the trainers , participants had rarely cognitive difficulties with understanding the various components of the training . one thing that some people found difficult to grasp was reflection on their work in terms of subjective perceptions instead of objective facts . slight or more severe emotional difficulties were met when discussing the consequences of having a chronic disease , feelings and thoughts on having a chronic disease and practical matters . one homework exercise presented difficulties for several participants ; they were asked twice in the course of the programme to arrange a consultation with their supervisor . the first session was intended to be a discussion of how the supervisor judged their work performance , the second to discuss work - related problems and solutions . pointless, because of their supervisor s attitude , or they wanted to practice such a consultation beforehand in order to be prepared ( see also last paragraph of the results section ) . the participants were asked to score how important the sessions themes were for them on a 110 scale ( table 4 ) . the themes insight into feelings and thoughts about having a chronic disease ( session 2 ) and communication and assertiveness ( sessions 3 and 5 ) , were valued highest , with a mean score of 8.0 . the theme exploration of practical and psychosocial work - related problems, which included the explanation of the model quality of work ( session 1 ) , scored 7.6 . smart personal plan , scored 7.5 . practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees was evaluated as lowest , with a mean score of 7.0 , and a high standard deviation . the training programme as a whole was evaluated with a mean score of 8.1 immediately after completion ; this dropped 0.2 points 8 months later and 0.3 points 24 months later.table 4opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64)rating ( 110 ) mean ( sd)overall training programme opinion after 4 months8.1 ( 1.1 ) opinion after 12 months7.9 ( 1.1 ) opinion after 24 months7.8 ( 1.3)themes exploration and clarification of practical and psychosocial problems ; quality of work model ( session 1)7.6 ( 1.7 ) insight into feelings and thoughts about having a chronic disease ( session 2)8.0 ( 1.4 ) communication in daily work situations and standing up for oneself ( sessions 3 and 5)8.0 ( 1.4 ) practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees ( session 4)7.0 ( 2.0 ) a smart plan to solve problems ( session 6)7.5 ( 1.7 ) the course book7.9 ( 1.2)methods theory explanation7.2 ( 1.6 ) exchanging experiences8.3 ( 1.4 ) filling in and discussing quality of work model7.5 ( 1.2 ) discussing others quality of work model7.7 ( 1.5 ) role play with actor8.1 ( 1.6 ) questioning occupational physician and employment expert7.1 ( 1.7 ) having a consultation with the supervisor ( homework)7.2 ( 1.9 ) having a consultation with an occupational physician ( homework)6.7 ( 2.2 ) individual consultation with trainer halfway7.9 ( 1.4 ) individual consultation with trainer at the end7.9 ( 1.2)including opinion of three persons that dropped out halfwaylow response , n = 57low response , n = 49 opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64 ) including opinion of three persons that dropped out halfway eighty - six per cent of the participants always read the short introductions in the course book to prepare for the group sessions , whereas 95% had read the entire course book at the end of the training course . most valued were the chapters on communication and assertiveness , and on feelings and thoughts about having a chronic disease . lowest valued , with the highest standard deviation , was the chapter on legislation and work accommodations . a variety of methods was used in the training programme : theoretical explanation , exchange of experiences , role - playing , and homework , such as completing the model quality of work , or arranging a consultation with a supervisor and occupational physician . role - playing and seeing and discussing others role - playing was also highly appreciated , as were the individual consultations with the trainers . non - response on these two questionnaire items was high , 7 and 15 , respectively , which indicates that these arrangements not always took place . the expertise of the trainers was overall judged very positively ( mean score 68 on a 1680 scale ) , and the advice given by the trainers was felt to be helpful . the participants felt well - treated and felt that they received personal attention during the programme . they considered introductory information to be sufficient , although this could have been better for a minority . satisfaction with the trainers was not lower in the three groups in which the trainers acted for the first time , when compared to the five groups for which trainers were more experienced . the training programme used a stepwise approach : first exploring and clarifying work - related problems , then focusing on communication at work , and finally working on developing and realizing solutions . eight months after the start , 84% of the participants found that the first phase worked well , while 69% found that the second phase and 65% found that the third phase worked well ( table 5).table 5success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64)not successful at all % a little successful % amply successful % completely successful % 1clarifying bottlenecks ( model quality of work)016.455.727.92discussing bottlenecks at work3.327.945.923.03developing and realizing solutions6.728.345.020.0 success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64 ) the majority of the participants , 53 persons , had , as part of the training , discussed matters with their supervisor in order to find a solution for work - related problems . fifty - three per cent of them felt this contributed a great deal to solving problems , 40% said that it contributed somewhat , whereas 6% said that it did not contribute and 2% felt these discussions had worked negatively . table 6 presents the effects of the programme on various aspects of working with a chronic disease , as perceived at 12 months follow - up . the participants noticed positive effects most often with regard to how they experienced and dealt with disease and work . this was followed by how matters at work were discussed and how they dealt with the supervisor and colleagues . after 24 months , 79% perceived a lasting effect of the training programme , 10% perceived an effect that had faded away , 3% were not sure whether it had lasted , and 8% perceived none or only a limited effect.table 6effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64)effect training on large negative effectsmall negative effectno effectsmall positive effectlarge positive effecthow i experience my disease and my work03.311.748.336.7how i deal with my disease and my work03.38.345.043.3how i discuss matters at work01.726.741.730.0how i deal with my supervisor0023.351.725.0how i deal with my colleagues0028.356.715.0how my supervisor deals with me0038.343.318.3how my colleagues deal with me0041.738.320.0the situation at home0043.330.026.7accommodations of my workplace or work tasks1.71.753.326.716.7 effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64 ) in the course of the programme , the participants formulated a plan of action with one or more personal goals . these goals related to work - home interference ( 78% ) , feelings and thoughts about having a chronic disease ( 59% ) , communication at the workplace ( 44% ) , leisure time ( 33% ) , work accommodations ( 29% ) or other topics ( 18% ) . one year after the start of the programme , 6 per cent felt that they had not reached the goal that they set in the course of the programme , 38% reached it a little, 36% reached it amply and 20% completely . the recruitment for this intervention yielded enough participants but was time - consuming . we enrolled a sample in which higher - educated women working in the service sector are over - represented . the majority of the participants were satisfied with the programme , and only a few dropouts were noted . for the most part , the programme was administered as planned , although some components took too much time . quality of work models and/or homework were not always discussed and not everybody had the opportunity to do role - playing as planned . the participants had no or only minor difficulties with understanding the materials discussed , but were more often emotionally upset , particularly when consequences of disease or feelings and thoughts were discussed , or during role - playing . generally , the participants completed their homework , but when asked to organize a consultation with their supervisor , many hesitated to do so ; a minority did not complete this assignment . among those who completed these consultations , the perceived effectiveness of the training programme was highest in how it shaped participants personal attitudes and lowest in matters that are more practical . the forms were completed by the trainers themselves and were likely correct as far as objective facts are concerned . the validity of some answers may be questionable , however , as trainers gave subjective judgments on whether the programme s components were tailored to the participants . furthermore , they give an overall response for the whole group , rather than individuals . however , the forms are of special value when the three trainers showed consensus on less positive aspects or when they noted barriers . for instance , there was consensus on the lack of time for some components , all three observed that some components are likely to raise emotional difficulties and all noted that consultations with the supervisor are often met with resistance . another weakness of this study is that we do not know what proportion of the target group was reached . , information about the project was disseminated through various channels and potential participants had to contact researchers to participate . the consequence is that we do not know how many employees who experience serious work - related problems were not interested in our programme or did not enrol for other reasons . we do know that the group we reached was a selected group in terms of socio - demographic characteristics . we know that our programme is implementable , although we have to keep in mind that the majority in this study was highly educated . at some sessions the time to discuss personal experiences is not an option . because participants have three individual consultations with a trainer , and because lack of personal attention appeared not to be a problem , it is presumed to be better to accept this programme design but to indicate at the beginning of the sessions that not everyone may receive equal attention in all components of the programme . we found in the pilot phase that participants with a variety of chronic physical diseases could be put together in the same group . people experience the general aspects of chronic diseases as more important than the disease specifics . the trainers observed that many of the components raised emotional feelings , and it is interesting to note that these components were often highly valued . apparently , many participants realized that going through a phase of mourning and learning to accept having a chronic disease is difficult , but it assists in learning to cope . this brings us to our assumption that participants needed to pass through three phases : clarifying , communicating and solving problems . we understood the earlier phases as necessary to accomplish the last essential phase and understood this final phase implicitly as organizing work accommodations . however , it appears that organizing work accommodations may be the primary problem for some persons ; for others , the main problem is in the earlier phases of accepting the chronic disease and learning to communicate about it and/or in maintaining an enjoyable life outside work . another remarkable phenomenon was that many participants showed resistance to a consultation with their supervisor , but in the end , the majority felt that it helped in solving problems . 2003 ; post et al . 2005 ) , that a good relationship with the supervisor is very important . for future use , it is important to discuss with the participants that this consultation often is successful in the end , the more so when it is practiced beforehand with role - playing . one reason is the randomization procedure , but the fact that the majority of the participants needed to use days of might have played a part as well . recruitment through professionals in outpatient clinics was problematic compared to recruitment with the help of patient organizations . disseminating this kind of programme through normal health care channels appears not to work ; lack of interest in work - related problems among many health care professionals is a primary reason ( van weel et al . physicians and nurses should be encouraged in the course of their education and by post - graduate courses to pay attention to the working life of their patients ; there is little chance for referral of patients to vocational rehabilitation programmes without conversations about these matters . it is positive that practice guidelines for physicians increasingly pay attention to work - related problems of patients . maybe incentives like co - authorship of a scientific article may help to raise interest in this kind of research and development projects . in addition , focus on specialized nurses as collaborating partners may prove beneficial , as these professionals concentrate more on the social consequences of chronic disease . working together more intensively with outpatient clinics in the future would have the added advantage of contact with a more diverse group of potential participants . heavy manual work and low education are prognostic factors for work disability among employees with chronic disease ( detaille et al . we do not know why we had only a few participants working in industry , and fewer men and less - educated people than expected . research into whether similar communication - focused programmes are attuned to the culture and working conditions outside of the service sector is necessary . we need to know why less - educated people seldom applied for the study , as well as whether and how more men can be convinced to participate in empowerment programmes , which focus on sometimes emotionally disturbing topics . several vocational rehabilitation approaches aimed at job retention for people with chronic or longstanding disease have recently been developed , varying widely in approach . multidisciplinary rehabilitation has been developed for patients with rheumatoid arthritis ( de buck et al . this is an outpatient clinic - based intervention where medical and psychosocial specialists combine their expertise in advising the patient and his or her occupational physician on aspects of work . this focuses on the employee and supervisor and aims to improve their ability to solve work - related problems with the help of a mediator . more research is needed on what kind of rehabilitation method best suits a particular employee and circumstances . the extent to which employers are willing to accommodate the workplace to employees with a chronic disease or handicap also needs research . we may conclude that empowering employees with a chronic disease with help of a group training programme is feasible and highly valued . for that reason , it should be offered in occupational health care or other health care settings .

purposeemployees with a chronic disease may experience work - related problems that contribute to the risk of job loss . we developed a group - based intervention programme aimed at clarifying problems , making these a subject of discussion at work , and realizing solutions . this process evaluation investigates the intervention s feasibility and the satisfaction of 64 participants in eight groups.methodsdata were collected through process evaluation forms and self - report questionnaires.resultsthe recruitment of participants was time - consuming . highly educated women working in the service sector were overrepresented . the programme was administered as planned , although components were sometimes only discussed briefly , due to lack of time . satisfaction with the overall programme among participants was high ; it was perceived as effective and there were only three dropouts . in particular , the focus on feelings and thoughts about having a chronic disease was highly valued , as were the exchange of experiences and role - playing directed at more assertive communication.conclusionsa vocational rehabilitation programme aimed at job retention is feasible and is perceived to be effective . such a programme should address psychosocial aspects of working with a chronic disease beside practical problems . the recruitment of participants is time - consuming . cooperation with outpatient clinics is necessary in order to reach all groups of employees with a chronic disease that might benefit from job retention programmes . trial registration : isrctn77240155

Introduction Set-up and contents of the training programme Methods Results Recruitment of participants Reach of target population Participation in the programme Extent to which the programme was implemented Dose received or fit with the participants capabilities and needs Satisfaction of the participants with the programme Effectiveness as perceived by the participants Discussion and conclusion

chronically ill employees themselves state that , apart from work accommodations , they need acceptance of having a disease , coping strategies and support from their supervisor in order to stay at work ( detaille et al . we developed a theory - driven group training programme for employees with chronic disease who experience work - related problems . it focused on solving work - related problems and aimed at job retention and maintenance and an increase in job satisfaction . in this article , we present a process evaluation of eight training courses with a total of 64 participants . the research questions for the process evaluation are : did the recruitment go as planned?was the target group reached?did participants follow the programme as it was intended?was the programme administered as intended?was the programme tailored to the group of participants?were participants satisfied with the program?was the programme perceived as effective?the medical ethics committee of the academic medical center in amsterdam approved of the study idea , but deemed ethical review unnecessary because they did not perceive the study to be medical research . the programme had a stepwise approach : first , exploring and clarifying work - related problems ; second , a focus on communication at work ; and third , developing and realizing solutions . work - related problems were clarified with the help of the quality of work model , which emphasizes the energizing or distressing influences of work tasks , social relationships at work , working conditions and work - home interference . each session focused on one theme : exploration and clarification of practical and psychosocial work - related problems with the help of the model quality of work;insight into feelings and thoughts about having a chronic disease and how these may influence communication;communication in daily work situations : theory and role play with an actor;practical matters : the occupational physician , the employment expert , legislation and facilities for disabled employees;communication and assertiveness : theory and role play with an actor;a smart plan to solve problems ; andfollow - up : what works and what does not.participants were eligible for the intervention if they had a chronic physical disease , had a paid job , experienced problems at work and feared losing their job or job satisfaction . exploration and clarification of practical and psychosocial work - related problems with the help of the model quality of work; insight into feelings and thoughts about having a chronic disease and how these may influence communication ; communication in daily work situations : theory and role play with an actor ; practical matters : the occupational physician , the employment expert , legislation and facilities for disabled employees ; communication and assertiveness : theory and role play with an actor ; a smart plan to solve problems ; and follow - up : what works and what does not . 24 mn.recruitment of participantskind of agencies approached and ways and frequency of approachxreach of target populationcharacteristics of participantsxparticipation in programmefrequency and reasons for dropoutxxfrequency and reasons for not attending meetingsxextent to which the programme was implementedextent to which all components were administered as plannedxdose received or fit with the participants needs and capabilitiestrainers opinion on participants ability to cognitively and emotionally grasp the components of the programxtrainers opinion on commitment and goal attainment of components of the programxsatisfaction of participants with the training programme and the traineroverall opinion on programme ( 110)xxxopinion on various themes ( 110)xopinion on various methods ( 110)xxopinion on various skills of the trainerxeffectiveness as perceived by participantsopinion on effectiveness with regard to three phasesxopinion on effectiveness of consultation with supervisor with regard to problem solvingxpersonal goal and opinion on goal attainmentxxopinion on effectiveness with regard to attitudes , skills , work accommodations and situation at homexopinion on permanence of effectxnotes res . when discussing work - related problems in the group using the quality of work model , only one instead of the planned two participants was often discussed . it was often impossible to have all participants practice role - playing in one session . slight or more severe emotional difficulties were met when discussing the consequences of having a chronic disease , feelings and thoughts on having a chronic disease and practical matters . the first session was intended to be a discussion of how the supervisor judged their work performance , the second to discuss work - related problems and solutions . pointless, because of their supervisor s attitude , or they wanted to practice such a consultation beforehand in order to be prepared ( see also last paragraph of the results section ) . the themes insight into feelings and thoughts about having a chronic disease ( session 2 ) and communication and assertiveness ( sessions 3 and 5 ) , were valued highest , with a mean score of 8.0 . the training programme as a whole was evaluated with a mean score of 8.1 immediately after completion ; this dropped 0.2 points 8 months later and 0.3 points 24 months later.table 4opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64)rating ( 110 ) mean ( sd)overall training programme opinion after 4 months8.1 ( 1.1 ) opinion after 12 months7.9 ( 1.1 ) opinion after 24 months7.8 ( 1.3)themes exploration and clarification of practical and psychosocial problems ; quality of work model ( session 1)7.6 ( 1.7 ) insight into feelings and thoughts about having a chronic disease ( session 2)8.0 ( 1.4 ) communication in daily work situations and standing up for oneself ( sessions 3 and 5)8.0 ( 1.4 ) practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees ( session 4)7.0 ( 2.0 ) a smart plan to solve problems ( session 6)7.5 ( 1.7 ) the course book7.9 ( 1.2)methods theory explanation7.2 ( 1.6 ) exchanging experiences8.3 ( 1.4 ) filling in and discussing quality of work model7.5 ( 1.2 ) discussing others quality of work model7.7 ( 1.5 ) role play with actor8.1 ( 1.6 ) questioning occupational physician and employment expert7.1 ( 1.7 ) having a consultation with the supervisor ( homework)7.2 ( 1.9 ) having a consultation with an occupational physician ( homework)6.7 ( 2.2 ) individual consultation with trainer halfway7.9 ( 1.4 ) individual consultation with trainer at the end7.9 ( 1.2)including opinion of three persons that dropped out halfwaylow response , n = 57low response , n = 49 opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64 ) including opinion of three persons that dropped out halfway eighty - six per cent of the participants always read the short introductions in the course book to prepare for the group sessions , whereas 95% had read the entire course book at the end of the training course . most valued were the chapters on communication and assertiveness , and on feelings and thoughts about having a chronic disease . a variety of methods was used in the training programme : theoretical explanation , exchange of experiences , role - playing , and homework , such as completing the model quality of work , or arranging a consultation with a supervisor and occupational physician . role - playing and seeing and discussing others role - playing was also highly appreciated , as were the individual consultations with the trainers . satisfaction with the trainers was not lower in the three groups in which the trainers acted for the first time , when compared to the five groups for which trainers were more experienced . the training programme used a stepwise approach : first exploring and clarifying work - related problems , then focusing on communication at work , and finally working on developing and realizing solutions . eight months after the start , 84% of the participants found that the first phase worked well , while 69% found that the second phase and 65% found that the third phase worked well ( table 5).table 5success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64)not successful at all % a little successful % amply successful % completely successful % 1clarifying bottlenecks ( model quality of work)016.455.727.92discussing bottlenecks at work3.327.945.923.03developing and realizing solutions6.728.345.020.0 success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64 ) the majority of the participants , 53 persons , had , as part of the training , discussed matters with their supervisor in order to find a solution for work - related problems . table 6 presents the effects of the programme on various aspects of working with a chronic disease , as perceived at 12 months follow - up . after 24 months , 79% perceived a lasting effect of the training programme , 10% perceived an effect that had faded away , 3% were not sure whether it had lasted , and 8% perceived none or only a limited effect.table 6effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64)effect training on large negative effectsmall negative effectno effectsmall positive effectlarge positive effecthow i experience my disease and my work03.311.748.336.7how i deal with my disease and my work03.38.345.043.3how i discuss matters at work01.726.741.730.0how i deal with my supervisor0023.351.725.0how i deal with my colleagues0028.356.715.0how my supervisor deals with me0038.343.318.3how my colleagues deal with me0041.738.320.0the situation at home0043.330.026.7accommodations of my workplace or work tasks1.71.753.326.716.7 effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64 ) in the course of the programme , the participants formulated a plan of action with one or more personal goals . these goals related to work - home interference ( 78% ) , feelings and thoughts about having a chronic disease ( 59% ) , communication at the workplace ( 44% ) , leisure time ( 33% ) , work accommodations ( 29% ) or other topics ( 18% ) . when discussing work - related problems in the group using the quality of work model , only one instead of the planned two participants was often discussed it was often impossible to have all participants practice role - playing in one session . slight or more severe emotional difficulties were met when discussing the consequences of having a chronic disease , feelings and thoughts on having a chronic disease and practical matters . the first session was intended to be a discussion of how the supervisor judged their work performance , the second to discuss work - related problems and solutions . pointless, because of their supervisor s attitude , or they wanted to practice such a consultation beforehand in order to be prepared ( see also last paragraph of the results section ) . the themes insight into feelings and thoughts about having a chronic disease ( session 2 ) and communication and assertiveness ( sessions 3 and 5 ) , were valued highest , with a mean score of 8.0 . practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees was evaluated as lowest , with a mean score of 7.0 , and a high standard deviation . the training programme as a whole was evaluated with a mean score of 8.1 immediately after completion ; this dropped 0.2 points 8 months later and 0.3 points 24 months later.table 4opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64)rating ( 110 ) mean ( sd)overall training programme opinion after 4 months8.1 ( 1.1 ) opinion after 12 months7.9 ( 1.1 ) opinion after 24 months7.8 ( 1.3)themes exploration and clarification of practical and psychosocial problems ; quality of work model ( session 1)7.6 ( 1.7 ) insight into feelings and thoughts about having a chronic disease ( session 2)8.0 ( 1.4 ) communication in daily work situations and standing up for oneself ( sessions 3 and 5)8.0 ( 1.4 ) practical matters ; the occupational physician , the employment expert , legislation and facilities for disabled employees ( session 4)7.0 ( 2.0 ) a smart plan to solve problems ( session 6)7.5 ( 1.7 ) the course book7.9 ( 1.2)methods theory explanation7.2 ( 1.6 ) exchanging experiences8.3 ( 1.4 ) filling in and discussing quality of work model7.5 ( 1.2 ) discussing others quality of work model7.7 ( 1.5 ) role play with actor8.1 ( 1.6 ) questioning occupational physician and employment expert7.1 ( 1.7 ) having a consultation with the supervisor ( homework)7.2 ( 1.9 ) having a consultation with an occupational physician ( homework)6.7 ( 2.2 ) individual consultation with trainer halfway7.9 ( 1.4 ) individual consultation with trainer at the end7.9 ( 1.2)including opinion of three persons that dropped out halfwaylow response , n = 57low response , n = 49 opinion of the training programme participants on the overall training programme , significance of themes , course book and methods ( n = 64 ) including opinion of three persons that dropped out halfway eighty - six per cent of the participants always read the short introductions in the course book to prepare for the group sessions , whereas 95% had read the entire course book at the end of the training course . most valued were the chapters on communication and assertiveness , and on feelings and thoughts about having a chronic disease . a variety of methods was used in the training programme : theoretical explanation , exchange of experiences , role - playing , and homework , such as completing the model quality of work , or arranging a consultation with a supervisor and occupational physician . role - playing and seeing and discussing others role - playing was also highly appreciated , as were the individual consultations with the trainers . satisfaction with the trainers was not lower in the three groups in which the trainers acted for the first time , when compared to the five groups for which trainers were more experienced . the training programme used a stepwise approach : first exploring and clarifying work - related problems , then focusing on communication at work , and finally working on developing and realizing solutions . eight months after the start , 84% of the participants found that the first phase worked well , while 69% found that the second phase and 65% found that the third phase worked well ( table 5).table 5success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64)not successful at all % a little successful % amply successful % completely successful % 1clarifying bottlenecks ( model quality of work)016.455.727.92discussing bottlenecks at work3.327.945.923.03developing and realizing solutions6.728.345.020.0 success of three steps of the training programme , as perceived by the training programme participants after 8 months ( n = 64 ) the majority of the participants , 53 persons , had , as part of the training , discussed matters with their supervisor in order to find a solution for work - related problems . table 6 presents the effects of the programme on various aspects of working with a chronic disease , as perceived at 12 months follow - up . after 24 months , 79% perceived a lasting effect of the training programme , 10% perceived an effect that had faded away , 3% were not sure whether it had lasted , and 8% perceived none or only a limited effect.table 6effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64)effect training on large negative effectsmall negative effectno effectsmall positive effectlarge positive effecthow i experience my disease and my work03.311.748.336.7how i deal with my disease and my work03.38.345.043.3how i discuss matters at work01.726.741.730.0how i deal with my supervisor0023.351.725.0how i deal with my colleagues0028.356.715.0how my supervisor deals with me0038.343.318.3how my colleagues deal with me0041.738.320.0the situation at home0043.330.026.7accommodations of my workplace or work tasks1.71.753.326.716.7 effect of training programme on work as perceived by the training programme participants after 12 months ( n = 64 ) in the course of the programme , the participants formulated a plan of action with one or more personal goals . these goals related to work - home interference ( 78% ) , feelings and thoughts about having a chronic disease ( 59% ) , communication at the workplace ( 44% ) , leisure time ( 33% ) , work accommodations ( 29% ) or other topics ( 18% ) . the recruitment for this intervention yielded enough participants but was time - consuming . we enrolled a sample in which higher - educated women working in the service sector are over - represented . for the most part , the programme was administered as planned , although some components took too much time . the participants had no or only minor difficulties with understanding the materials discussed , but were more often emotionally upset , particularly when consequences of disease or feelings and thoughts were discussed , or during role - playing . the validity of some answers may be questionable , however , as trainers gave subjective judgments on whether the programme s components were tailored to the participants . we know that our programme is implementable , although we have to keep in mind that the majority in this study was highly educated . because participants have three individual consultations with a trainer , and because lack of personal attention appeared not to be a problem , it is presumed to be better to accept this programme design but to indicate at the beginning of the sessions that not everyone may receive equal attention in all components of the programme . for future use , it is important to discuss with the participants that this consultation often is successful in the end , the more so when it is practiced beforehand with role - playing . disseminating this kind of programme through normal health care channels appears not to work ; lack of interest in work - related problems among many health care professionals is a primary reason ( van weel et al . working together more intensively with outpatient clinics in the future would have the added advantage of contact with a more diverse group of potential participants . research into whether similar communication - focused programmes are attuned to the culture and working conditions outside of the service sector is necessary . several vocational rehabilitation approaches aimed at job retention for people with chronic or longstanding disease have recently been developed , varying widely in approach . this focuses on the employee and supervisor and aims to improve their ability to solve work - related problems with the help of a mediator . the extent to which employers are willing to accommodate the workplace to employees with a chronic disease or handicap also needs research . we may conclude that empowering employees with a chronic disease with help of a group training programme is feasible and highly valued .

cone beam computerized tomography ( cbct ) uses a collimated and cone - shaped x - ray beam rather than a larger fan or cone beam , which allows for a scan range with a more restricted field of view ( fov ) . the beam s cylindrical fov may differ from small to large fields for dental imaging or other facial examinations ( 1 - 3 ) . some cbct units allow for the selection of different fovs to suit a specific purpose ( 3 , 4 ) . the advantages of cbct over ct scanning include lower costs , smaller size , and a lower dose of radiation ( 1 , 3 , 5 , 6 ) . recent technological improvements have made it more comfortable for patients to undergo a cbct examination as well as more convenient for dentists to acquire and analyze images ( 7 , 8) . since the technology is new and the scanners are provided by different manufacturers , little of the process is currently understood by most radiologists , and some concerns have arisen regarding the patient s radiation dose ( 9 - 14 ) . due to the hazards of x - ray irradiation , it is imperative to reduce the radiation dose given to the patient to the lowest possible amount ( 8 , 15 ) . the cbct radiation dose is lower than that of a helical ct scan ( 8 , 16 , 17 ) . however , the examination dose varies considerably when using different cbct scanners , or when using a single cbct unit that is configured to different settings ( 8 , 10 - 14 , 16 ) . if the dose can be changed considerably by selecting different exposure parameters , it is necessary to thoroughly understand the various options as well as their impact on radiation safety ( 8 , 18 ) . moreover , technological advancements are expected to improve the efficacy of each newly introduced device , while at the same time reducing the adverse effects . such a health precaution can be achieved via altering multiple variables such as different structures ( filtration , the source - to - object distances , etc . ) and different radiation protocols ( kvp , mas , and fovs ) ( 3 , 8 , 10 , 16 , 17 , 19 - 22 ) . hence , when marketing a new model of cbct scanner , it is necessary to document its radiation doses . various studies have evaluated human phantoms in order to assess the radiation doses received ( 3 , 10 , 16 , 17 , 19 - 22 ) . of course , it is difficult if not impossible to compare the results of these studies because of methodological differences such as the number of sensors used , their types , and their positioning ( 23 ) . however , to the best of our knowledge , there has been only one study involving the newtom vgi ( in 15 15 and 23 23 cm fovs ) ( 23 ) , while there has been no study concerning the radiation doses of the newtom 5 g . therefore , we conducted this study to document the radiation doses of the newtom vgi ( in 8 8 and 6 6 cm fovs , none of which were documented before ) and the newtom 5 g ( not assessed before at any configurations ) in comparison to the promax three dimensional ( 3d ) scanner . large fovs result in volumes adequate for covering the maxillofacial area , while medium and small fovs generate volumes more appropriate for dentoalveolar and localized imaging , respectively ( 1 - 3 , 23 ) . some organs have higher tissue weightings , which increases the damaging impact of radiation . in the head and neck area , the thyroid , salivary glands , calvarium , mandible ramus / trunk , and cervical vertebrae are examples of such organs . this study analyzed the absorbed and effective doses of these critical organs under irradiation from two different fovs of three cbct scanners . this in vitro experimental study was performed on the ten upper sections of an anthropometric phantom ( radiation analogue dosimetry system ( rando ) , nuclear associates , hicksville , ny , usa ) . this phantom is similar to an average - sized man in terms of tissue density and radiation absorption . the ethical protocols of this study ( in terms of the health of the operators ) were approved by the research committee of the university . all devices used in this study were properly calibrated and their quality was assessed and approved . a total of 20 white circular thermoluminescence dosimeters ( tld ) of 0.8 mm in height and 4.5 mm in diameter ( ( lif : mg , cu , p ) , tld gr 200a , conqueror electronics technology co. ltd , china ) were used in this study . the most common crystal is lif , which has a radiation absorption capacity similar to that of human soft tissue . each tld was sensitive to 1 gy - 10 gy doses , with a linear response curve . calibration was repeated prior to the examination of each of the cbct devices as detailed below . first , the dosimeters were shipped to the dosimetry laboratory of the atomic energy organization for calibration purposes . in order to reduce the error , the exposure to radiation for calibrating and ecc calculation was carried out in six separate phases at the secondary standard dosimetry laboratory ( ssdl ) of the atomic energy organization . the energy used for the calibration was similar for all of the tlds and it was determined based on routine energy usage in cbct devices . these formulae would be used again later to convert the tld output into the absorbed dose . seventeen calibrated and annealed tlds were simultaneously positioned within the phantom in positions representative of the thyroid gland , parotid gland , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus ( table 1 ) . the number of tlds placed in each bony area was based on the relative proportion of bone marrow distributed among the different bones in the human body ( table 1 ) ( 24 ) . the remaining three tlds were positioned in different areas of the radiology center in order to measure the background radiation . for each imaging procedure , underhill et al . ( 25 ) suggested embedding at least three dosimeters in the calvarium for the assessment of calvarial dosage . therefore , we used four sensors so as to improve the accuracy of our tests . the lower sections of the rando were not used , since the dose received by the lower phantom sections is not significant in oral radiography ( 22 ) . the phantom was transferred to three private radiology centers , each of which housed a cbct device . at each clinic , radiologists positioned the phantom in such a way to accurately resemble a human subject . the phantom was exposed at two different fields of view ( a large and a small fovs ) ten times ( i.e. , five times per fov ) . the exposed area was adjusted to cover the mandible in such a way that the inferior border of the fov was 1 cm lower than the mandibular inferior border . also , the anterior border of the fov was 5 mm anterior to the most prominent point of the chin . the device automatically configured mas based on tissue mass and density in a smart and undisclosed manner . the average mas was measured as 6.87 and 12.24 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . this device automatically configured the resolution at high ( voxel size = 100 m ) when choosing the smaller fov . this could not be manually reserved . however , the resolution was optional ( high or normal ) for the larger fov . in order to increase the variables verona , italy ) , with a constant kvp of 110 and automatically configured mas . the average mas was measured as 6.76 and 11.52 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . as with the above device , we manually configured the resolution at normal for the larger fov . 3 . promax 3d ( planmeca , helsinki , finland ) , configured at standard parameters for an average mature person ( kvp = 82 , ma = 10 , and time = 12 s ) . the large and small fovs were 8 8 cm and 4 5 cm , respectively . to render the conditions similar to those of the above two devices , the resolution of this device also , the resolution was manually configured at normal for the larger fov in order to foster similarity with the other two devices . after each of the 30 exposures , the phantom and dosimeters were shipped to the tld laboratory at the university for reading ( fimel , france ) . the time between each irradiation and each measurement was constant for all measurements ( 24 hours ) . once at the laboratory , the dosimeters were first extracted using a vacuum device ( ett , fimel , france ) . then , they were read ( tld reader , fimel , france ) , preheated , and heated , so that the absorbed x - ray dose could be emitted as visible light . this light was read and converted to the absorbed dose using the formula obtained from the abovementioned six - phase tld calibration and ecc estimation procedure . finally , using an annealing device ( ett , fimel , france ) , the dosimeters were reset and calibrated for the next exposure . since the effective dose is still considered to be the most appropriate radiation risk estimator for live tissues , it was calculated according to the formula e = ( wt ht ) , wherein wt is the tissue weighting factor , which according to icrp-2007 is 0.04 , 0.01 , and 0.12 for the thyroid , salivary glands , and red bone marrow , respectively . ht is the equivalent dose , calculated using the formula ht = ( wr dr ) , wherein dr is the absorbed dose and wr is the radiation weighting factor , which is defined as 1 for x - ray . the equivalent dose of red bone marrow was calculated based on the mandibular ( trunk and ramus ) doses , cervical vertebral doses , and calvarial doses ( 16 ) . the doses pertaining to the devices and the organs were compared using the kruskal - wallis test and a dunn post hoc test . the small and large fov doses were compared using the mann - whitney u test . the effective doses related to the large and small fovs were compared using the wilcoxon signed - rank test . a total of 20 white circular thermoluminescence dosimeters ( tld ) of 0.8 mm in height and 4.5 mm in diameter ( ( lif : mg , cu , p ) , tld gr 200a , conqueror electronics technology co. ltd , china ) were used in this study . the most common crystal is lif , which has a radiation absorption capacity similar to that of human soft tissue . each tld was sensitive to 1 gy - 10 gy doses , with a linear response curve . calibration was repeated prior to the examination of each of the cbct devices as detailed below . first , the dosimeters were shipped to the dosimetry laboratory of the atomic energy organization for calibration purposes . in order to reduce the error , the exposure to radiation for calibrating and ecc calculation was carried out in six separate phases at the secondary standard dosimetry laboratory ( ssdl ) of the atomic energy organization . the energy used for the calibration was similar for all of the tlds and it was determined based on routine energy usage in cbct devices . these formulae would be used again later to convert the tld output into the absorbed dose . seventeen calibrated and annealed tlds were simultaneously positioned within the phantom in positions representative of the thyroid gland , parotid gland , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus ( table 1 ) . the number of tlds placed in each bony area was based on the relative proportion of bone marrow distributed among the different bones in the human body ( table 1 ) ( 24 ) . the remaining three tlds were positioned in different areas of the radiology center in order to measure the background radiation . for each imaging procedure , underhill et al . ( 25 ) suggested embedding at least three dosimeters in the calvarium for the assessment of calvarial dosage . . the lower sections of the rando were not used , since the dose received by the lower phantom sections is not significant in oral radiography ( 22 ) . the phantom was transferred to three private radiology centers , each of which housed a cbct device . at each clinic , radiologists positioned the phantom in such a way to accurately resemble a human subject . the phantom was exposed at two different fields of view ( a large and a small fovs ) ten times ( i.e. , five times per fov ) . the exposed area was adjusted to cover the mandible in such a way that the inferior border of the fov was 1 cm lower than the mandibular inferior border . also , the anterior border of the fov was 5 mm anterior to the most prominent point of the chin . the device automatically configured mas based on tissue mass and density in a smart and undisclosed manner . the average mas was measured as 6.87 and 12.24 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . this device automatically configured the resolution at high ( voxel size = 100 m ) when choosing the smaller fov . however , the resolution was optional ( high or normal ) for the larger fov . in order to increase the variables , we chose the normal resolution for the larger fov . 2 . newtom 5 g ( newtom inc . , verona , italy ) , with a constant kvp of 110 and automatically configured mas . the average mas was measured as 6.76 and 11.52 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . as with the above device , we manually configured the resolution at normal for the larger fov . 3 . promax 3d ( planmeca , helsinki , finland ) , configured at standard parameters for an average mature person ( kvp = 82 , ma = 10 , and time = 12 s ) . the large and small fovs were 8 8 cm and 4 5 cm , respectively . to render the conditions similar to those of the above two devices , the resolution of this device also , the resolution was manually configured at normal for the larger fov in order to foster similarity with the other two devices . after each of the 30 exposures , the phantom and dosimeters were shipped to the tld laboratory at the university for reading ( fimel , france ) . the time between each irradiation and each measurement was constant for all measurements ( 24 hours ) . once at the laboratory , the dosimeters were first extracted using a vacuum device ( ett , fimel , france ) . then , they were read ( tld reader , fimel , france ) , preheated , and heated , so that the absorbed x - ray dose could be emitted as visible light . this light was read and converted to the absorbed dose using the formula obtained from the abovementioned six - phase tld calibration and ecc estimation procedure . finally , using an annealing device ( ett , fimel , france ) , the dosimeters were reset and calibrated for the next exposure . since the effective dose is still considered to be the most appropriate radiation risk estimator for live tissues , it was calculated according to the formula e = ( wt ht ) , wherein wt is the tissue weighting factor , which according to icrp-2007 is 0.04 , 0.01 , and 0.12 for the thyroid , salivary glands , and red bone marrow , respectively . ht is the equivalent dose , calculated using the formula ht = ( wr dr ) , wherein dr is the absorbed dose and wr is the radiation weighting factor , which is defined as 1 for x - ray . the equivalent dose of red bone marrow was calculated based on the mandibular ( trunk and ramus ) doses , cervical vertebral doses , and calvarial doses ( 16 ) . descriptive statistics were calculated for the absorbed and effective doses . the doses pertaining to the devices and the organs the small and large fov doses were compared using the mann - whitney u test . the effective doses related to the large and small fovs were compared using the wilcoxon signed - rank test . the average absorbed doses , respectively , for the large and small fovs were 17.19 and 28.89 mgy in the promax 3d , 19.25 and 35.46 mgy in the newtom vgi , and 18.85 and 30.63 mgy in the newtom 5 g ( figure 1 , table 2 ) . the highest variation between doses received by the different organs was seen in the newtom 5 g and promax 3d ( mainly with the small fov ; table 3 ) . the kruskal - wallis test indicated a significant difference between the organ dosages measured at both fovs together ( p < 0.001 ; figure 2 , table 4 ) . the dunn post hoc test indicated significant differences between the thyroid and the submandibular gland , the mandibular trunk , and its ramus , between the parotid and the submandibular glands , and finally between the calvarium and the submandibular gland , mandibular ramus , mandibular trunk , and bone marrow ( table 5 ) . when comparing the radiation doses absorbed by organs at the smaller fov , the kruskal - wallis test showed a significant difference among the organs ( p = 0.0015 ; table 4 ) . the dunn test only showed significant differences between the submandibular gland and the thyroid and calvarium , while the other comparisons were not significant ( table 5 ) . when comparing organ doses at the larger fov , a significant difference among the organs was detected by the kruskal - wallis test ( p = 0.0027 ; table 4 ) . the dunn test only showed significant differences between the submandibular gland and the thyroid and calvarium ( table 5 ) . abbreviations : ci , confidence interval ; cv , coefficient of variation ; fov , field of view ; sd , standard deviation ; min , minimum ; max , maximum . abbreviations : ci , confidence interval ; cv , coefficient of variation ; fov , field of view ; sd , standard deviation ; min , minimum ; max , maximum . . the ratios of the absorbed doses of the newtom vgi and newtom 5 g as compared to the promax 3d scanner are presented in table 6 . the kruskal - wallis test did not detect a significant difference between the doses of the devices when both of their fovs were combined ( three variables ) ( p = 0.8944 ) . when each fov of each device was considered as a separate variable ( six variables ) , the kruskal - wallis test showed a non - significant difference between them ( p = 0.834 ) . the dunn post hoc test also failed to show any difference between the two fovs of each device ( all p values > 0.05 ) or between each pair of two sets of device - fov variables ( all p values > 0.05 ) . the mann - whitney u test showed insignificant differences between the fovs of each device : promax 3d ( p = 0.550 ) , newtom vgi ( p = 0.291 ) , and newtom 5 g ( p = 0.535 ) . the difference between the fov doses was insignificant according to the mann - whitney u test ( p value = 0.1930 ) . the effective doses were all greater when the device was set at the smaller fov compared to the larger fov . this difference was statistically significant according to the wilcoxon test ( p = 0.0039 ; table 7 ) . abbreviations : s / l , the ratio of the smaller - to - larger fov effective doses . a ratio greater than 1.0 indicates a higher effective dose caused by the smaller fov . in most cases , the promax 3d scanner showed lower doses compared to the newtom vgi ( and compared to the newtom 5 g in the case of a few organs ) . the radiation generated by all three devices ( with a few exceptions ) was slightly lower when absorbed by the parotid and sublingual glands and the cervical vertebrae . the absorbed dose of the calvarium was similar between the large and small fovs when generated by the newtom 5 g . it was slightly lower and greater , respectively , when the promax 3d and newtom vgi were tested . the rest of the organs had much higher absorbed doses ( 1.5 to 5 times greater ) when the device , regardless of its brand / model , was configured to use the small fov . the non - parametric evaluation of the fovs also showed no significant differences between the doses caused by the large and small fovs . however , the effective doses were considerably affected by the fovs , with the smaller fovs causing greater effective doses . this surprising finding is unlikely to be an artifact , since the experiment settings were controlled very carefully . moreover , the trends of dose increase / decrease consistently changed from organ to organ in the case of all three devices . among the evaluated organs , the calvarium and submandibular gland received the lowest and highest absorbed doses , respectively . based on these results , it can be suggested that reducing the fov for the sake of lowering the patient s received dose , if it accompanies an automatic resolution increase , is not a very effective method . this necessitates protecting this particular organ during cbct scanning . by reducing the fov and increasing the resolution , the parotid radiation dose was reduced by about 20% in the case of the promax 3d scanner , 3% in the case of the newtom vgi , and 30% in the case of the newtom 5 g . this might be attributed to the location of the parotid and the fact that the focus of imaging was on the anterior mandible region , which might lead to a reduction in the parotid dose in line with the reduction of the fov and not an increase together with the increase in resolution . this did not occur in the other salivary glands , and all salivary glands together showed about a 1.5 fold increase when the fov was reduced and the resolution was increased . decreasing the fov and increasing the projections led to small changes in the calvarium dose in the promax 3d scanner ( 3% ) and the newtom vgi ( 1% ) , although there were no changes in the newtom 5 g . the reason for this might be the distance of the calvarium from the region of interest , which reduced both its received dose and the changes in it . the reason for the smaller calvarium dose received in the newtom 5 g compared to the other two scanners might be the position of the patient , since the patient stands during imaging in the newtom vgi and promax 3d , but acquires a supine position in the newtom 5 g . the latter may place the calvarium at a farther distance from the region of interest and so expose it to less reflected radiation . unlike the calvarium , the mandible ramus and its body showed a considerable increase in received dose after decreasing the fov and increasing the resolution . the mandible ramus showed the highest increase in the case of the newtom vgi scanner ( about 2.2 fold ) when compared to the other two ( about 1.5 to 1.6 fold ) . similar increases was observed in the case of the mandible body : the newtom vgi scanner showed a 2.4 fold increase , while other two showed 1.8 to 1.9 fold increases . similar to the parotid , which showed a decrease when decreasing the fov , the vertebrae also received about 10% to 30% less radiation when the fov was reduced . to the best of our knowledge , no study to date has statistically compared the radiation doses of different fovs and different organs . all of the previous studies have been limited to cataloguing the doses . in a study performed by palomo et al . in 2008 ( 14 ) , the doses received to the head and neck organs ( esophagus , midline thyroid , mandible body , submandibular , center c spine , midbrain , and orbital surface ) from the cbct ( cb mercuray ) scanner in different fovs of 6 , 9 and 12 inches were examined . the average dose absorbed by the thyroid gland , mandible body on the right and left sides , submandibular gland on the right and left , and the vertebrae were 40.8 , 70.6 , 30.6 , 60.7 , 30.7 , and 93.5 mgy , respectively . the average absorbed dose received by the thyroid gland from all three cbct devices investigated in this study when in the normal mode was greater , with doses of 0.335 , 0.376 , and 0.379 mgy , respectively , in the case of the promax 3d , new tom vgi , and new tom 5 g at fovs of 8 8 . the difference between studies might be related to differences in exposure configurations ( kvp = 120 and ma = 15 ) and fov sizes , with the fov being 12 inches in the previous study . also , palomo et al . concluded that alongside decreasing the fov , the absorbed dose reduces . they stated that the reduction in dose is greater if the organ is farther from the direct radiation beam , which is similar to our findings ( 14 ) . in a study conducted in 2008 by hirsch et al . ( 3 ) , the doses absorbed by the head and neck organs and generated by two different cbct scanners at different fovs and protocols were examined for the anterior region of the jaws . the average dose absorbed by the parotid gland from the vera view 3d scanner in the case of fovs of 4 8 and 4 4 were 2.49 and 2.22 mgy , respectively , while for the accuitomo scanner in the case of fovs of 6 6 and 4 4 , the absorbed doses were 2.24 and 1.26 mgy , respectively . the radiation absorbed by the parotid gland was about 2 to 4 times greater in the present study compared to those results ( 3 ) . hirsch et al . also evaluated the absorbed dose of bone marrow , which was lower than that observed in this study . the reason for the higher doses observed in this study compared to the study of hirsch et al . ( 3 ) could be the larger fovs and higher resolutions adopted in this research , in addition to the differences in their scanner settings ( kvp = 80 and ma = 5 ) ( 3 ) . ( 26 ) evaluated the absorbed dose of the head and neck organs in terms of four cbct scanners and two mdct scanners . their results pertaining to the promax 3d scanner at a fov of 8 5 were lower in the case of the submandibular and sublingual glands , calvarium , vertebrae , and mandible ramus and body ( 26 ) . the reason for the lower doses observed in the submandibular and sublingual glands in their study ( 26 ) might be their smaller fov . however , it was interesting that despite their smaller fov , the parotid dose observed was higher than that in our study . a probable reason for this finding might be the differences in the positioning of the tlds and their levels . indeed , as pauwels et al . ( 23 ) concluded , a slight change in exposure settings , size of fov , the location of the dosimeters and patient , and a slight shift in the fov of a few centimeters can notably alter the radiation received by the dosimeters ( 23 ) . it should be noted that comparing the performance of devices based solely on dosimetric studies is not possible . the purpose of dosimetry comparisons is not to determine a better device , and diagnostic needs dictate the extent of necessary doses . due to the availability of various fov sizes in dental cbct , as well as various positions of fovs within the head and neck region , each point around the main beam can show high variability on the basis of its position relative to the isocenter ( 23 ) . using the same phantom this technique has proved reproducible , although some dosimeter locations might have some degree of variation , such as those placed close to the cranial and caudal ends of the x - ray beam , as well as those close to the skin , thyroid , and back of the neck . hence , patient position can also alter the dose of the head and neck organs such as the thyroid . in order to reduce this , the use of smaller fovs is suggested , which might significantly reduce the dose ( 10 , 23 ) . since the absorbed dose is an average value , the only way to improve the accuracy of dosimetry is to use as many tlds as possible . in order to ensure the precision of the measurements in the present study , numerous tlds were placed throughout the head and neck area to cover the head and neck organs still , these results should be cautiously compared with those of other studies , since the number of tlds and their positions differ in all studies , especially as many prior studies have used too few tlds ( 3 , 10 , 11 , 16 , 19 , 21 , 23 ) . utilizing too few tlds might result in either the overestimation or underestimation of such positioning alterations , with variations of up to 80% . this can become more vivid in the case of certain tissues such as the red bone marrow , thyroid , and salivary glands ( 23 ) . such excessive variations in the doses received by each organ , as caused by different cone beam collimations and exposure factors , imply that the average effective doses should not be used for comparisons between different radiographic techniques . still , it seems that the cbct dose is higher than that involved in plain dental radiographic techniques , while still being below that of multi - slice ct methods ( 16 , 17 , 23 ) . this can be increased by increasing the mas and kv and using a larger fov ( 23 ) . depending on the collimation features , maximum fov , and the quality of the diagnostic image , cbct units could be applied for different purposes ( 3 , 9 , 16 , 17 , 23 ) . hence , an important factor when optimizing the radiation dose is to ensure the proper quality of the produced image by employing appropriate protocols such as the proper size and position of the fov ( 9 , 23 ) . the alara principle dictates the usage of strategies to lower the radiation dose to that which is reasonably achievable ( 14 , 19 , 27 ) by choosing the most appropriate settings , fov , and adequate lead protection ( 14 ) . decreasing the fov as a collimation method is one of the approaches suggested to reduce the radiation dose . the choice of fov should be the smallest option that would capture a given region of interest ( 8 , 14 ) . it is observed that reducing the size of the fov can reduce the radiation dose ( 8) . therefore , it is recommended to reduce the fov when the lesions are limited to one jaw in order to reduce the absorbed dose . however , in this study , reducing the fov did not reduce the absorbed dose , although it did increase the effective dose . both newtom scanners automatically adjust the resolution to a higher level when a smaller fov is selected , and they do not allow for manual correction of the resolution modification . we also manually simulated this reverse resolution fov association of the newtom devices on the promax 3d device . a higher resolution increases the radiation dose ( 8) . it is possible that the devices are designed this way in order to improve the signal - to - noise ratio with a higher resolution . still , this strategy contradicts the philosophy of reducing the fov for the sake of reducing the dose . usually , offering many options for the operator to manually change the device s settings is not practical and so it might actually be desirable to have fewer ( but less confusing ) options . however , manufacturers are also suggested to let the operator have a minimum of control over the configurations . if a greater resolution is needed for a particular diagnostic task , it is important that the signal - to - noise ratio is adequate for the task . the worst form of excess exposure is a level too low to provide adequate image quality , which necessitates a repeat . however , there might be instances where a smaller fov with a lower resolution suffices for adequate image quality . as we wanted to make the groups uniform , the promax 3d scanner , which allowed manual resolution adjustment , was manually configured at its high resolution option for the smaller fov . the high and normal resolutions employ the same exposure parameters , while the low resolution might reduce the effective dose to about 10% of the normal dose resolution . generally , a low dose leads to an image with a low signal - to - noise ratio ( 8) . these resolution increases might be the reason for the increases observed in the promax 3d group when the smaller fov was used . however , it should be noted that the number of tlds used in this study was more than the number used in many previous examinations . additionally , due to the limited number of tlds available as well as other technical difficulties , we were limited to disregarding some areas and focusing on more critical organs ( 16 ) . as another limitation , it might be argued that the failure to match the devices configurations might confound the results . it should be taken into account that it was impossible to match the devices , since their settings are determined by their manufacturers ; therefore , all previous studies faced this issue as well . these prior studies were limited to comparing the same - name settings of different devices , without attempting to match the exposure parameters ( 3 , 16 ) . furthermore , as another constraint , a cbct study should also evaluate the quality of the image together with the extent of the absorbed doses . according to lorenzoni et al . ( 22 ) , the important factors in cbct imaging are the size and position of the fov and the quality of the image . the latter was not evaluated in this study , although other studies were similarly limited by this factor . other limitations centered on the numerous technical difficulties must be recognized , including the severe rarity of rando phantoms and tlds , very high sensitivity of tlds , difficulty of their transportation due to their very high fragility , lack of adequate laboratory experts , which all made conducting this study very difficult . however , we re - performed most of the steps involved in this study from the scratch ( with each step consisting of all exposures plus all calibration steps ) in order to ensure the validity of the results . while recognizing the limitations of this study , it seems that the devices did not differ considerably in terms of the generated dose . decreasing the fov but increasing the resolution did not reduce the absorbed dose and might actually increase the effective dose . when reducing the fov for the sake of x - ray safety , the resolution should be taken into consideration . the risk of absorbed doses is higher in the submandibular gland , mandible trunk and ramus , and red bone marrow . in terms of the three evaluated devices , it seems that resolution might play a more important role than fov in determining the absorbed dose of organs inside the fov or close to it such as the thyroid gland . however , the doses absorbed by organs farther away from the fov seem to be more affected by the size of the fov than the resolution . the structures that are distant from the fov ( such as the calvarium ) seem less likely to be affected by changes in the size of the fov or resolution . it should be noted that without statistical comparisons , these suggestions should be considered as theorems and not as evidence . future studies should hence conduct more experiments and perform further statistical analyses to assess these suggested theorems ( 28 , 29 ) .

backgroundmarketing new radiography devices necessitates documenting their absorbed x - ray doses . since the current literature lacks studies on new devices , we assessed the doses of two new devices that had not previously been assessed.objectivesthe new devices were compared to the promax three dimensional ( 3d ) scanner at two fields of view ( fov ) in nine critical head and neck tissues and organs.materials and methodsseventeen thermoluminescence dosimeters positioned in an average - sized male rando phantom were used to determine the dosimetry of the three cone beam computerized tomography devices ( newtom vgi , newtom 5 g , and promax 3d ) at two field of views ( fovs ) , one small and one large . the exposure by each device per fov was performed five times ( 30 exposures ) . the absorbed and effective doses were calculated for the thyroid , parotid , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus . the doses pertaining to the different devices , the fovs , and the tissues were compared using the kruskal - wallis , mann - whitney u , and wilcoxon tests.resultsthe average absorbed doses , respectively , for the large and small fovs were 17.19 and 28.89 mgy in the promax 3d , 19.25 and 35.46 mgy in the newtom vgi , and 18.85 and 30.63 mgy in the newtom 5 g . the absorbed doses related to the fovs were not significantly different ( p value = 0.1930 ) . however , the effective doses were significantly greater at the smaller fovs / higher resolutions ( p = 0.0039 ) . the doses of the three devices were not significantly different ( p = 0.8944 ) . the difference among the nine organs / tissues was significant ( kruskal - wallis p=0.0000).conclusionthe absorbed doses pertaining to the devices and the fovs were not significantly different , although the organs / tissues absorbed considerably different doses .

1. Background 2. Objectives 3. Materials and Methods 3.1. Thermoluminescence Dosimeter (TLD) Calibration 3.2. TLD Loading in the Phantom 3.3. TLD Exposure 3.4. TLD Reading 3.5. Absorption and Effective Dose Estimation 3.6. Statistical Analysis 4. Results 5. Discussion

cone beam computerized tomography ( cbct ) uses a collimated and cone - shaped x - ray beam rather than a larger fan or cone beam , which allows for a scan range with a more restricted field of view ( fov ) . since the technology is new and the scanners are provided by different manufacturers , little of the process is currently understood by most radiologists , and some concerns have arisen regarding the patient s radiation dose ( 9 - 14 ) . due to the hazards of x - ray irradiation , it is imperative to reduce the radiation dose given to the patient to the lowest possible amount ( 8 , 15 ) . and different radiation protocols ( kvp , mas , and fovs ) ( 3 , 8 , 10 , 16 , 17 , 19 - 22 ) . however , to the best of our knowledge , there has been only one study involving the newtom vgi ( in 15 15 and 23 23 cm fovs ) ( 23 ) , while there has been no study concerning the radiation doses of the newtom 5 g . therefore , we conducted this study to document the radiation doses of the newtom vgi ( in 8 8 and 6 6 cm fovs , none of which were documented before ) and the newtom 5 g ( not assessed before at any configurations ) in comparison to the promax three dimensional ( 3d ) scanner . in the head and neck area , the thyroid , salivary glands , calvarium , mandible ramus / trunk , and cervical vertebrae are examples of such organs . this study analyzed the absorbed and effective doses of these critical organs under irradiation from two different fovs of three cbct scanners . first , the dosimeters were shipped to the dosimetry laboratory of the atomic energy organization for calibration purposes . in order to reduce the error , the exposure to radiation for calibrating and ecc calculation was carried out in six separate phases at the secondary standard dosimetry laboratory ( ssdl ) of the atomic energy organization . seventeen calibrated and annealed tlds were simultaneously positioned within the phantom in positions representative of the thyroid gland , parotid gland , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus ( table 1 ) . the number of tlds placed in each bony area was based on the relative proportion of bone marrow distributed among the different bones in the human body ( table 1 ) ( 24 ) . the phantom was exposed at two different fields of view ( a large and a small fovs ) ten times ( i.e. also , the anterior border of the fov was 5 mm anterior to the most prominent point of the chin . the average mas was measured as 6.87 and 12.24 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . however , the resolution was optional ( high or normal ) for the larger fov . the average mas was measured as 6.76 and 11.52 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . promax 3d ( planmeca , helsinki , finland ) , configured at standard parameters for an average mature person ( kvp = 82 , ma = 10 , and time = 12 s ) . the large and small fovs were 8 8 cm and 4 5 cm , respectively . to render the conditions similar to those of the above two devices , the resolution of this device also , the resolution was manually configured at normal for the larger fov in order to foster similarity with the other two devices . after each of the 30 exposures , the phantom and dosimeters were shipped to the tld laboratory at the university for reading ( fimel , france ) . then , they were read ( tld reader , fimel , france ) , preheated , and heated , so that the absorbed x - ray dose could be emitted as visible light . since the effective dose is still considered to be the most appropriate radiation risk estimator for live tissues , it was calculated according to the formula e = ( wt ht ) , wherein wt is the tissue weighting factor , which according to icrp-2007 is 0.04 , 0.01 , and 0.12 for the thyroid , salivary glands , and red bone marrow , respectively . ht is the equivalent dose , calculated using the formula ht = ( wr dr ) , wherein dr is the absorbed dose and wr is the radiation weighting factor , which is defined as 1 for x - ray . the doses pertaining to the devices and the organs were compared using the kruskal - wallis test and a dunn post hoc test . the small and large fov doses were compared using the mann - whitney u test . the effective doses related to the large and small fovs were compared using the wilcoxon signed - rank test . first , the dosimeters were shipped to the dosimetry laboratory of the atomic energy organization for calibration purposes . in order to reduce the error , the exposure to radiation for calibrating and ecc calculation was carried out in six separate phases at the secondary standard dosimetry laboratory ( ssdl ) of the atomic energy organization . seventeen calibrated and annealed tlds were simultaneously positioned within the phantom in positions representative of the thyroid gland , parotid gland , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus ( table 1 ) . the phantom was exposed at two different fields of view ( a large and a small fovs ) ten times ( i.e. also , the anterior border of the fov was 5 mm anterior to the most prominent point of the chin . the average mas was measured as 6.87 and 12.24 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . however , the resolution was optional ( high or normal ) for the larger fov . newtom 5 g ( newtom inc . the average mas was measured as 6.76 and 11.52 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . promax 3d ( planmeca , helsinki , finland ) , configured at standard parameters for an average mature person ( kvp = 82 , ma = 10 , and time = 12 s ) . the large and small fovs were 8 8 cm and 4 5 cm , respectively . to render the conditions similar to those of the above two devices , the resolution of this device also , the resolution was manually configured at normal for the larger fov in order to foster similarity with the other two devices . after each of the 30 exposures , the phantom and dosimeters were shipped to the tld laboratory at the university for reading ( fimel , france ) . then , they were read ( tld reader , fimel , france ) , preheated , and heated , so that the absorbed x - ray dose could be emitted as visible light . this light was read and converted to the absorbed dose using the formula obtained from the abovementioned six - phase tld calibration and ecc estimation procedure . since the effective dose is still considered to be the most appropriate radiation risk estimator for live tissues , it was calculated according to the formula e = ( wt ht ) , wherein wt is the tissue weighting factor , which according to icrp-2007 is 0.04 , 0.01 , and 0.12 for the thyroid , salivary glands , and red bone marrow , respectively . ht is the equivalent dose , calculated using the formula ht = ( wr dr ) , wherein dr is the absorbed dose and wr is the radiation weighting factor , which is defined as 1 for x - ray . descriptive statistics were calculated for the absorbed and effective doses . the doses pertaining to the devices and the organs the small and large fov doses were compared using the mann - whitney u test . the effective doses related to the large and small fovs were compared using the wilcoxon signed - rank test . the average absorbed doses , respectively , for the large and small fovs were 17.19 and 28.89 mgy in the promax 3d , 19.25 and 35.46 mgy in the newtom vgi , and 18.85 and 30.63 mgy in the newtom 5 g ( figure 1 , table 2 ) . the highest variation between doses received by the different organs was seen in the newtom 5 g and promax 3d ( mainly with the small fov ; table 3 ) . the kruskal - wallis test indicated a significant difference between the organ dosages measured at both fovs together ( p < 0.001 ; figure 2 , table 4 ) . the dunn post hoc test indicated significant differences between the thyroid and the submandibular gland , the mandibular trunk , and its ramus , between the parotid and the submandibular glands , and finally between the calvarium and the submandibular gland , mandibular ramus , mandibular trunk , and bone marrow ( table 5 ) . when comparing the radiation doses absorbed by organs at the smaller fov , the kruskal - wallis test showed a significant difference among the organs ( p = 0.0015 ; table 4 ) . the dunn test only showed significant differences between the submandibular gland and the thyroid and calvarium , while the other comparisons were not significant ( table 5 ) . when comparing organ doses at the larger fov , a significant difference among the organs was detected by the kruskal - wallis test ( p = 0.0027 ; table 4 ) . the dunn test only showed significant differences between the submandibular gland and the thyroid and calvarium ( table 5 ) . the ratios of the absorbed doses of the newtom vgi and newtom 5 g as compared to the promax 3d scanner are presented in table 6 . the kruskal - wallis test did not detect a significant difference between the doses of the devices when both of their fovs were combined ( three variables ) ( p = 0.8944 ) . when each fov of each device was considered as a separate variable ( six variables ) , the kruskal - wallis test showed a non - significant difference between them ( p = 0.834 ) . the mann - whitney u test showed insignificant differences between the fovs of each device : promax 3d ( p = 0.550 ) , newtom vgi ( p = 0.291 ) , and newtom 5 g ( p = 0.535 ) . the difference between the fov doses was insignificant according to the mann - whitney u test ( p value = 0.1930 ) . the effective doses were all greater when the device was set at the smaller fov compared to the larger fov . this difference was statistically significant according to the wilcoxon test ( p = 0.0039 ; table 7 ) . abbreviations : s / l , the ratio of the smaller - to - larger fov effective doses . in most cases , the promax 3d scanner showed lower doses compared to the newtom vgi ( and compared to the newtom 5 g in the case of a few organs ) . the radiation generated by all three devices ( with a few exceptions ) was slightly lower when absorbed by the parotid and sublingual glands and the cervical vertebrae . the absorbed dose of the calvarium was similar between the large and small fovs when generated by the newtom 5 g . it was slightly lower and greater , respectively , when the promax 3d and newtom vgi were tested . the non - parametric evaluation of the fovs also showed no significant differences between the doses caused by the large and small fovs . however , the effective doses were considerably affected by the fovs , with the smaller fovs causing greater effective doses . moreover , the trends of dose increase / decrease consistently changed from organ to organ in the case of all three devices . among the evaluated organs , the calvarium and submandibular gland received the lowest and highest absorbed doses , respectively . by reducing the fov and increasing the resolution , the parotid radiation dose was reduced by about 20% in the case of the promax 3d scanner , 3% in the case of the newtom vgi , and 30% in the case of the newtom 5 g . this might be attributed to the location of the parotid and the fact that the focus of imaging was on the anterior mandible region , which might lead to a reduction in the parotid dose in line with the reduction of the fov and not an increase together with the increase in resolution . this did not occur in the other salivary glands , and all salivary glands together showed about a 1.5 fold increase when the fov was reduced and the resolution was increased . decreasing the fov and increasing the projections led to small changes in the calvarium dose in the promax 3d scanner ( 3% ) and the newtom vgi ( 1% ) , although there were no changes in the newtom 5 g . the reason for the smaller calvarium dose received in the newtom 5 g compared to the other two scanners might be the position of the patient , since the patient stands during imaging in the newtom vgi and promax 3d , but acquires a supine position in the newtom 5 g . the mandible ramus showed the highest increase in the case of the newtom vgi scanner ( about 2.2 fold ) when compared to the other two ( about 1.5 to 1.6 fold ) . similar increases was observed in the case of the mandible body : the newtom vgi scanner showed a 2.4 fold increase , while other two showed 1.8 to 1.9 fold increases . similar to the parotid , which showed a decrease when decreasing the fov , the vertebrae also received about 10% to 30% less radiation when the fov was reduced . in 2008 ( 14 ) , the doses received to the head and neck organs ( esophagus , midline thyroid , mandible body , submandibular , center c spine , midbrain , and orbital surface ) from the cbct ( cb mercuray ) scanner in different fovs of 6 , 9 and 12 inches were examined . the average dose absorbed by the thyroid gland , mandible body on the right and left sides , submandibular gland on the right and left , and the vertebrae were 40.8 , 70.6 , 30.6 , 60.7 , 30.7 , and 93.5 mgy , respectively . the average absorbed dose received by the thyroid gland from all three cbct devices investigated in this study when in the normal mode was greater , with doses of 0.335 , 0.376 , and 0.379 mgy , respectively , in the case of the promax 3d , new tom vgi , and new tom 5 g at fovs of 8 8 . ( 3 ) , the doses absorbed by the head and neck organs and generated by two different cbct scanners at different fovs and protocols were examined for the anterior region of the jaws . the average dose absorbed by the parotid gland from the vera view 3d scanner in the case of fovs of 4 8 and 4 4 were 2.49 and 2.22 mgy , respectively , while for the accuitomo scanner in the case of fovs of 6 6 and 4 4 , the absorbed doses were 2.24 and 1.26 mgy , respectively . ( 26 ) evaluated the absorbed dose of the head and neck organs in terms of four cbct scanners and two mdct scanners . their results pertaining to the promax 3d scanner at a fov of 8 5 were lower in the case of the submandibular and sublingual glands , calvarium , vertebrae , and mandible ramus and body ( 26 ) . ( 23 ) concluded , a slight change in exposure settings , size of fov , the location of the dosimeters and patient , and a slight shift in the fov of a few centimeters can notably alter the radiation received by the dosimeters ( 23 ) . using the same phantom this technique has proved reproducible , although some dosimeter locations might have some degree of variation , such as those placed close to the cranial and caudal ends of the x - ray beam , as well as those close to the skin , thyroid , and back of the neck . hence , patient position can also alter the dose of the head and neck organs such as the thyroid . since the absorbed dose is an average value , the only way to improve the accuracy of dosimetry is to use as many tlds as possible . in order to ensure the precision of the measurements in the present study , numerous tlds were placed throughout the head and neck area to cover the head and neck organs still , these results should be cautiously compared with those of other studies , since the number of tlds and their positions differ in all studies , especially as many prior studies have used too few tlds ( 3 , 10 , 11 , 16 , 19 , 21 , 23 ) . such excessive variations in the doses received by each organ , as caused by different cone beam collimations and exposure factors , imply that the average effective doses should not be used for comparisons between different radiographic techniques . however , in this study , reducing the fov did not reduce the absorbed dose , although it did increase the effective dose . we also manually simulated this reverse resolution fov association of the newtom devices on the promax 3d device . as we wanted to make the groups uniform , the promax 3d scanner , which allowed manual resolution adjustment , was manually configured at its high resolution option for the smaller fov . these resolution increases might be the reason for the increases observed in the promax 3d group when the smaller fov was used . these prior studies were limited to comparing the same - name settings of different devices , without attempting to match the exposure parameters ( 3 , 16 ) . furthermore , as another constraint , a cbct study should also evaluate the quality of the image together with the extent of the absorbed doses . ( 22 ) , the important factors in cbct imaging are the size and position of the fov and the quality of the image . however , we re - performed most of the steps involved in this study from the scratch ( with each step consisting of all exposures plus all calibration steps ) in order to ensure the validity of the results . when reducing the fov for the sake of x - ray safety , the resolution should be taken into consideration . the risk of absorbed doses is higher in the submandibular gland , mandible trunk and ramus , and red bone marrow . in terms of the three evaluated devices , it seems that resolution might play a more important role than fov in determining the absorbed dose of organs inside the fov or close to it such as the thyroid gland . however , the doses absorbed by organs farther away from the fov seem to be more affected by the size of the fov than the resolution .

traditional medicine is still the first point of healthcare for many people in sub - saharan africa , where there has been a long and rich tradition of obtaining treatments from herbs and trees . in the case of malaria , africa 's traditional healers use hundreds of indigenous plants for remedies . until the 1950s , when synthetic chemistry began to dominate drug research and development ( r and d ) efforts , most drugs developed and registered in the pharmacopoeia were in fact based on natural products . plant alkaloids , quinine among them , were the first components of natural herbal remedies to be extracted and refined for more effective use in the early 19th century . some 150 years later , quinine is still used as front - line therapy for severe malaria , even if it is not the recommended drug for this use when artemisinin combination therapies ( acts ) are available . in this context , it seems to be quite surprising that no african lead has emerged so far . meanwhile , there are efforts to assess plant remedies against malaria for their application in health care systems . b. aegyptiaca ( l. ) ( balanitaceae ) is a woody tree growing in various ecological conditions ( from 100 mm to 1000 mm annual rainfall ) , but mainly distributed in semiarid and arid zones in tropical africa . this tree reaches 10 m ( 33 ft ) in height with a generally narrow form and thorny branches . the tree produces several forms of inflorescence bearing yellow - green bisexual flowers which exude nectar . in egypt , it is confined to water - receiving sites , that is , wadis and river banks . its fruit has been the basis for an active trade in many centuries because of its oil , which consists of linear and branched chain alkanes while the kernel contains terpenes and sterols ( diosgenin ) , which showed a reduction of liver cholesterol . the bark was formerly used as a fish poison , presumably because of its steroidal saponin content as the active component . aqueous extracts of the bark showed antibacterial , antifungal and antiviral activity , but most notable was the antifungal effect of b. aegyptiaca against candida albicans infections . balanitin-6 and balanitin-7 are diosgenyl saponins from b. aegyptiaca kernels and they demonstrated appreciable anticancer effects in human cancer cell lines in vitro with a higher antiproliferative potential than etoposide and oxiplatin . the anticancer activity mainly results from a depletion of atp leading in turn to a major disorganization of actin cytoskeleton . the structures of balanitins , that is , balanitin-1 , -2 , -3 , are composed of steroid glycosides proven to be potent molluscicides for the control of freshwater snails that are carriers of bilharzia . moreover , the efficacy of b. aegyptiaca fruit mesocarp was compared with praziquantel in infected mice with a sudanese strain of schistosoma mansoni . a single dose of 200 mg / kg body weight reduced the recovery of adult worms significantly . recently , root - derived callus from b. aegyptiaca with 500 ppm extracted saponins showed a larvicidal effect of 100% against aedes aegypti mosquito larvae . over recent years , resistance of malaria parasites to therapy has increased the need to develop alternative treatments . since b. aegyptiaca bark shows promising anti - infective properties , we investigated its potential antiplasmodial activity in seeds and related this activity to its constituents in vitro . although phytochemical analyses and medicinal evaluations have been recently performed for fixed oil from b. aegyptiaca fruits , in vivo biological assays are missing . here , we monitored parasitemia after application of an aqueous extract obtained from the seeds of the plant in vitro and in vivo in balb / c mice . seeds of b. aegyptiaca ( balanitaceae ) were obtained from harraz pharmacy in cairo , collected in the area of ras mohammed national park and botanically identified by professor grtz , biological institute , university of stuttgart , germany . b. aegyptiaca seeds ( 5 g ) were powdered and incubated at 37c in 100 ml distilled water . to facilitate dissolution of the steroid aglyca , squalane solution ( 1 ml ) ( sigma - aldrich , munich , germany ) made with 2 n hcl was added , and the mixture was refluxed with 200 ml methanol for 24 hours in a soxhlet apparatus . the extract was dried with calcium sulfate ( caso42h2o ) ( carl roth , karlsruhe , germany ) and filtered . the residue obtained was diluted in 50 ml methanol by sonication and analyzed by gas chromatography - mass spectrometry ( gc / ms ) . for the preparation of an aqueous extract , 5 g of powdered b. aegyptiaca seeds were incubated at 37c in 100 ml distilled , autoclaved water for 24 hours in a sealed , sterilized falcon tube and filtrated in a laminar air flow under aseptic conditions . gc / ms measurements were made using a 7890a gas chromatograph with a series 7683b autosampler and a series 5975c quadrupole mass spectrometer ( agilent technologies inc . , santa clara , ca , usa ) operated in electron impact ionization ( ei ) mode . the fused silica capillary column , 30 m long , 0.25 mm i. d. with hp-5ms stationary phase , film thickness 0.25 m was used . gc / ms data were processed with the chemstation software ( agilent technologies ) and the nist 05 mass spectra library ( agilent technologies ) . the temperature of the column was programmed from 80c ( 1 min hold ) at 10c min to 280c ( 50 min hold ) . helium 5.0 grade ( westfalen ag , mnster , germany ) was used as a carrier gas . constant flow of helium of 1.0 cm min was used during the entire analysis . the temperature of the split / splitless injector was 250c , and the split flow was 10 cm min . the ionization occurred with a kinetic energy of the impacting electrons of 70 ev ( electron volts ) . mass spectra and reconstructed chromatograms ( total ion current [ tic ] ) were obtained after eluting of solvent ( 7 min solvent delay ) by automatic scanning in the mass range m / z 30750 u. p. falciparum isolate nf54 , which was obtained from malaria research and reference center , manassas , va , usa , was maintained in continuous culture with gentamicin ( 40 g / ml ) in petri dishes ( 5 cm in diameter ) with a gaseous phase of 90% n2 , 5% o2 , and 5% co2 , according to a protocol from molony et al . parasites were cultured in human erythrocytes ( blood group a ) in rpmi 1640 medium ( sigma ) supplemented with 25 mm hepes , 20 mm sodium bicarbonate , and 10% heat - inactivated human a plasma at 10% ( v / v ) hematocrit . the parasitemia of the infected erythrocytes was determined in giemsa - stained smears by light microscopy . parasitemias and morphological forms detected in the cultures were scored visually with a 100-fold oil - immersion objective , counting at least 1,000 erythrocytes to determine the percentage of the infected erythrocytes . a drug - free control ( methanol / water 50 : 50% v , v ) was used in all experiments . aliquots ( 200 l ) were suspended in 2 ml rpmi - medium ( sigma - aldrich ) supplemented with 25 mm hepes , 20 mm sodium bicarbonate , and 10% heat - inactivated human a plasma at 10% ( v / v ) hematocrit , dispensed into 12-well microculture trays , and incubated at 37c in a gaseous atmosphere of 90% n2 , 5% o2 , and 5% co2 . thereafter , growth medium was changed once a day for 4 days , and 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol stock solutions were dissolved in water / methanol mixture ( 50/50% , v / v ) before being diluted in rpmi growth medium in the concentrations given for each experiment . dilution of the crude balanite extract obtained after soxhlet extraction was performed with rpmi - growth medium as a solvent to protect the rbc membrane from perturbation . parasitemia and stage distribution were estimated as triplicates daily from giemsa - stained smears by counting 1,000 erythrocytes . the 50% inhibitory concentrations ( ic50s ) , defined as the drug concentration corresponding to 50% inhibition of parasitic growth were determined by nonlinear regression analysis . data obtained from the inhibitor - dependent concentration growth curves were computed into plots with nonlinear regression analysis from y - axis ( inhibition % ) to x - axis ( inhibitor concentration m ) . the animal studies were performed according to the guidelines of the german animal welfare act ( license number : 50.203 . balb / c2 mice were maintained and bred at the animal facility of the university of bonn and infected intravenously with 1 10 parasitized erythrocytes from a homologous donor mouse , which had been infected with frozen polyclonal stocks of p. berghei anka ( pba ) strain . balb / c mice developed a th2 response and did not succumb to cerebral malaria , but died after 1030 days because of high parasitemia and anemia . 50 l of the aqueous crude plant extract ( 1,25 g/100 ml ) were applied to the balb / c mice intraperitoneally ( i.p . ) on days 3 , 2 , 1 before infection , at the day of infection , and on days 1 , 2 , 3 , 4 , 5 , 6 , after infection . control mice were treated with 200 l pbs - buffer , and the same scheme of administration was applied . 2,6-di - tert - butyl - phenol and 2,4-di - tert - butyl - phenol were applied to the balb / c2 mice intraperitoneally ( i. p. ) on days 4 and 2 before infection , and on days 2 , 4 , and 6 before infection . chloroquine was applied i. p. on days 18 post infection in a concentration of 25 mg / kg for each mouse . giemsa - stained smears were performed on days 1,2 , 3,4 , 5,7 , 6,8 , 10 , and 12 , and 15 . 5 mice were treated in each group . the nucleic acid sequence from p. falciparum m18 aspartyl aminopeptidase was chemically synthesized by genscript ( nj , usa ) with optimization of codon usage as described by teuscher et al . a subclone was subsequently constructed with baculodirect c - terminal linear dna ( invitrogen , germany ) and transfected into sf9 ( spodoptera frugiperda ) cells for protein expression and purification according to teuscher et al . the activity assay was performed according to a high - throughput - screening ( htps ) described by teuscher et al . prior to the start of the assay , 2.5 l of assay buffer ( 50 mm tris hcl ph 7.5 , 4 mm cocl2 , 0.1% bsa ) containing 5 g / ml recombinant pfm 18 aap were dispensed into a 12-well microtiter plate . next , 37 l of the crude balanite extract and the depleted balanite extract where 6-phenyl-2(h)-1,2,4-triazin-5-one oxime was absent were added as a control reaction to the appropriate wells . depletion of the extract was performed by liquid chromatography - mass spectrometry ( lc / ms ) , and fractions were rechecked by gc / ms as described within for the presence and absence of the 6-phenyl-2(h)-1,2,4-triazin-5-one oxime . the assay was started by dispensing 2.5 l of 100 m h - glu - nhmec as a fluorogenic peptide ( genscript , nj , usa ) in buffer ( 50 mm tris hcl , ph 8.8 ) into all wells . fluorescence was read immediately ( t0 ) on the viewlux ( perkin - elmer , ma , usa ) and again after 90 minutes ( t90 ) of incubation at 25c . statistical analyses were performed using graphpad prism software to evaluate the in vivo inhibitor experiments performed with balb / c mice . nonparametrically distributed data were analyzed using the mann - whitney test for differences between the aqueous balanite extract and the chloroquine treated group . crude extracts from extracted balanites aegyptiaca seeds show antiplasmodial activity in the chloroquine susceptible p. falciparum strain nf54 in vitro . in vitro inhibition of growth of the chloroquine susceptible p. falciparum strain nf54 was tested with a crude extract from b. aegyptiaca ( the amount equivalent to 1 : 2000 of a seed ) obtained by soxhlet extraction which led to an eradication of the parasite within 3 days ( figure 1(a ) ) and a determined ic50 of 68.26 g/l 3,5 ( figure 1(b ) ) . by contrast , the untreated nf54 strain showed normal growth . the crude balanite extract and a dilution of the extract , in which the ratio of seeds to the extract was 1 : 200 , lysed red blood cells ( rbc ; data not shown ) . therefore , the amount equivalent to 1 : 2000 of a seed was applied with rpmi - medium as a solvent to avoid any perturbation of the erythrocyte membrane . figure 2 shows the chromatogram of the total ion current ( tic ) obtained by gc / ms analysis of the crude balanite extract to identify and characterize the active compounds of the crude extract after soxhlet extraction . figures 3(a)3(c ) represent the mass spectra of the identified peaks using pure standard substances and the peaks of the nist 05 mass spectra library of compounds for comparison and to elucidate the chemical structures of the detected substances 2,6-di - tert - butyl - phenol ( 18.43 min ) , 2,4-di - tert - butyl - phenol ( 19.36 min ) , and 6-phenyl-2(h)-1,2,4-triazin-5-one oxime ( 39.44 min ) , respectively . ion [ m ] at m / z 206 , the fragment ion [ m ch3 ] at m / z 191 and the [ c4h9 ] ion at m / z 57 are characteristic for both di - tert - butyl - phenol isomers ( figures 3(a)3(b ) ) . the crude balanite extract inhibited recombinant m18 aspartyl aminopeptidase from plasmodium falciparum ( pfm18aap ) with a determined ki of 2.34 m . the assay was performed with a commercially available fluorogenic peptide substrate h - glu - nhmec . cleavage of the substrate by recombinant pfmaap enzyme liberates the nhmec from the peptide , leading to increased fluorescence . different concentrations of the crude plant extract ( figure 4 , blue curve ) were applied to investigate the dose response of recombinant pfmaap . , we determined the inhibitory effect of recombinant pfm18aap after fractionation of the soxhlet extract pooling fractions in which 6-phenyl-2(h)-1,2,4-triazin-5-one oxime was absent ( figure 4 , pink curve ) . the pink curve in figure 4 shows that depletion of 6-phenyl-2(h)-1,2,4-triazin-5-one oxime from the extract leads to a 50% lower inhibition at concentrations ranging from 2.7 ng dry weight per ml to 73.5 ng dry weight per ml compared to the extract where the compound was present . derivatives of 6-phenyl-2(h)-1,2,4-triazin-5-one have been recently identified as important inhibitor lead structures for recombinant plasmodial m18 aminopeptidase ( pfm18aap ) in a biochemical high - throughput screening assay ( htps ) with fluorogenic peptide substrate ( the scripps research institute molecular screening center , http://mlpcn.florida.scripps.edu ) . the derivatives of 6-phenyl-2(h)-1,2,4-triazin-5-one that is , 3-(benzylamino)-4-ethyl-6-phenyl-1,2,4-triazin-5-one oxime were applied in a concentration of 7.35 m and inhibited the recombinant enzyme to 31% . this inhibitory effect has been attributed to formation of metal chelates with the cocatalytic zinc metal ions of the parasitic enzyme . taken together , these results suggest that the detected 6-phenyl-2(h)-1,2,4-triazin-5-one oxime might contribute to the antiplasmodial activity . the occurrence of the two phenolic compounds prompted us to perform in vitro inhibitor and dose - response experiments with p. falciparum strain nf54 at concentrations from 10 mm to 100 mm . for this experiment the commercially available pure phenolic compounds ( fluka , germany ) were applied . inhibition of growth started at a concentration of 10 mm independently of the compound . the most prominent growth arrest was observed at a concentration of 100 mm with either 2,4-di - tert - butyl - phenol or 2,6-di - tert - butyl - phenol ( figures 5(a ) and 5(c ) , resp . ) . the determined ic50 values were 50.29 m 3 for 2,6-di - tert - butyl - phenol ( figure 5(b ) ) and 47.82 m 2,5 for 2,4-di - tert - butyl - phenol ( figure 5(d ) ) , respectively . a complete eradication of the parasite was not observed , suggesting that these compounds may contribute to the biological activity of the crude plant extract . 2,4-di - tert - butyl - phenol was more potent than the 2,6-substituted compound but did not lead to complete eradication of the parasite . in the next set of experiments , the combination of both compounds showed an ic50 value of 42.5 m 3 ( figure 5(f ) ) . taken together , these results suggest that the combination of both drugs appear to have an additive effect . an aqueous extract from b. aegyptiaca seeds and the two phenolic compounds 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol tested in vivo in balb / c mice do not reduce parasitemia . hitherto , no in vivo biological activity assay has been performed to investigate antiplasmodial activity of the balanite extract . therefore , we monitored in vivo protection in balb / c infected mice after infection with 10 parasitized erythrocytes of p. berghei anka ( pba ) strain after administration of the balanite extract and the two phenolic compounds , that is , 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol . the crude aqueous plant extract ( 50 ml of 1,25 g dry extract/100 ml solvent ) was intraperitoneally applied on days 3 , 2 , and 1 before infection , at the day of infection , and on days 1,2 , 3,4 , 5,6 , 7 , and 8 after infection . the two phenolic compounds ( each dose was 6 mg / kg body weight ) were intraperitoneally applied on days 4 and 2 before infection and on days 2 , 4 , and 6 after infection . at day 8 , parasitemia was significantly increased in mice treated with the extract whereas , at day 12 p.i . , a reduction could be observed compared to controls ( figure 6 ) . extract - treated animals , a reduction of weight could be observed whereas chloroquine treatment prevented the weight loss ( data not shown ) . in a parallel experiment , we monitored the survival rate of balb / c infected mice with each of the phenolic compounds in a dose of 6 mg / kg body weight ( data not shown ) . however , both phenolics did not reduce parasitemia in comparison to the control and , therefore , did not prolong the survival of p. berghei infected mice . b. aegyptiaca ( balanitaceae ) is a widely distributed plant in africa and has been reported to show a lot of different biological activities , which were attributed to its saponin constituents mainly deriving from diosgenin as the sole aglycone . recent investigations proved a medicinal evaluation for the fixed oil of b. aegyptiaca fruits in vitro . in the past , moderate in vitro antiplasmodial activity has been determined in an extract from b. aegyptiaca stem bark with an ic50 value of 55 g / ml . no antiplasmodial activity was found in the mesocarp of the fruits from this species . since it was reported that a biosynthetic pathway to diosgenin exists in germinating seeds of the plant , these results challenged us to investigate an extract from balanite seeds obtained by soxhlet extraction . an aqueous dilution from the original extract in which the ratio of seeds was equivalent to a ratio of 1 : 2000 seeds was tested for in vitro inhibition of the chloroquine - susceptible p. falciparum strain nf54 , resulting in an ic50 value of 68.26 g/l 3,5 . although no saponin glycosides deriving from the aglycone diosgenin were identified by gc / ms ( kaiser unpublished ) , the crude , undiluted extract seems to contain constituents which lyse rbcs . in vitro activity is associated ( figure 4 blue curve ) with the occurrence of the oxime of 6-phenyl-2(h)-1,2,4-triazine-5-one , since the absence of the compound in the balanite extract leads to a 50% lower inhibition of recombinant plasmodial m18 aspartyl aminopeptidase ( pfm18aap ) ( figure 4 , pink curve ) compared to the crude extract where the oxime of 6-phenyl-2(h)-1,2,4-triazine-5-one was present ( figure 4 blue curve ) . however , the concentration of the compound in the extract was too low for a successful isolation and a proof of principle assay inhibiting growth of plasmodium in vitro . to our knowledge , there are no previous studies with the compound in the literature so far . taken together , these data are supported by the determined ki of 2.35 g/l of the crude balanite extract and the extract which was depleted of the 6-phenyl-2(h)-1,2,4-triazine-5-one oxime with a determined ki of 4.8 g/l . . the aspartyl aminopeptidase exhibits exopeptidase activity exclusively against the n - terminal acidic amino acids glutamate and aspartate from the host hemoglobin - derived peptides . the enzyme is mainly located in the cytosol but can also be trafficked to the parasitophorous vacuole . antisense - mediated knockdown of pfm18aap results in a lethal phenotype since the p. falciparum protein is expressed in all erythrocytic stages . the two phenolic compounds 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol were identified by gc / ms analysis ( figures 2 - 3 ) and contribute modestly to the in vitro biological activity ( figure 5 ) . the determined ic50 values were 50.29 m 3 for 2,6-di - tert - butyl - phenol and 47,82 m 2,5 for 2,4-di - tert - butyl - phenol , respectively ( figures 5(b ) and 5(d ) ) . however , the determined ic50 value of 42.5 m 3 , when both compounds were combined , appears to have a small additive effect but did not lead to the eradication of p. falciparum in vitro . these compounds have been recently associated with the antioxidant effect of a methanolic extract from the centipede scolopendra subspinipes mutilans ( scolopendridae ) which is used in traditional chinese and korean medicine for the treatment of diseases such as spasm , childhood convulsions , diphtheria and tetanus . moreover , 2,4-di - tert - butyl - phenol exhibited antioxidant activity against ldl - oxidation in a biological in vitro activity assay . consistent with the observation that 2,4-di - tert - butyl - phenol showed antioxidant activity , the drug exhibited also radical scavenging activity . recently , the antioxidant capacity was also proven for the oil present in balanites aegyptiaca fruits in vitro . the two phenolic compounds 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol have been detected in a variety of plant extracts . they occur as aromatic constituents in volatile oil fractions of five different species of chinese eaglewood such as aquilaria sinensis ( thymelaeaceae ) , in flowers of gynura cusimbua ( asteraceae ) , in flowers of clusia species ( guttiferae ) , and in leaves of pereskia bleo ( cactaceae ) . hitherto , the biogenesis of both phenolic compounds in the plant has not been elucidated . a possible precursor molecule which is a structural analogue of these tertiary phenolics might be a butylated hydroxyanisole deriving from the mevalonate pathway . secondly , in case of the extract obtained from pereskia bleo , ali et al . discussed the occurrence of both constituents as an uptake of the compounds from plastic waste being in the soil . we have no data which support the recent findings that the constituents are either of plant origin or due to uptake of both constituents . the content of total phenols in the methanol extract is 3.5% which is comparable to that of pereskia bleo and might protect the fatty oil in the seeds from oxidation . although the fixed oil in b. aegyptiaca fruits exhibited anticancer activity in lung , liver , and brain human carcinoma cell lines , no cytotoxicity could be detected for the seed extract ( data not shown ) . hitherto , only two in vivo experiments in mice have been performed with either the pure constituents of the plant or the crude balanite extract [ 6 , 24 ] . the steroidal saponins balanitin-6 and balanitin-7 increased the survival rate of mice bearing murine l1210 leukemia grafts to the same extent reported for vincristine . a second in vivo experiment was performed with a crude balanite extract to demonstrate moderate antihepatotoxic activity in comparison with the standard hepatoprotective silymarin . the lyophilized extract of balanites aegyptiaca was administered orally for 5 consecutive days in a dose of 1 g / kg . on the last day of treatment , a hepatotoxic oral dose of paracetamol ( 0.6 g / kg ) was given and the activity of diagnostic liver enzymes were determined . b. aegyptiaca had only a relatively modest hepatoprotective activity . to analyze parasitemia , balb / c infected mice were treated with a crude , aqueous balanite extract ( 50 ml of 1,25 g dry extract/100 ml solvent ) injected intraperitoneally on a daily basis , beginning on day 3,2 , 1 prior to infection , at the day of infection , and continuing until 8 days p.i . although a decrease in parasitemia could be observed on day 12 ( figure 6 ) , treatment did not clear the parasites over this time and therefore ; did not prolong survival . the plasmodial m18 aspartyl aminopeptidase inhibitor is present in the extract , however , its concentration might be too low to eradicate the parasite . the herbicide metamitron is a 4-amino-3-methyl-6-phenyl-1,2,4,triazin-5-one and , therefore , closely related from its structure to the 6-phenyl-2(h)-1,2,4-triazin-5-one oxime . the ld50 for metamitron for a female rat is approximately 1.4 , and for a male rat it is 2.9 mg / kg body weight . given that 6-phenyl-2(h)-1,2,4-triazin-5-one oxime has the same effect , it would have enhanced the neurological symptoms in the infected balb / c mice independently from the parasitic infection . since there is a lack of knowledge with respect to the half - life , absorption , distribution , and elimination of the three identified compounds in a mammalian organism , we , therefore , conclude that the crude plant extract in its current form and concentration does not reduce parasitemia in vivo .

balanites aegyptiaca ( balanitaceae ) is a widely grown desert plant with multiuse potential . in the present paper , a crude extract from b. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity . the determined ic50 value for the chloroquine - susceptible plasmodium falciparum nf54 strain was 68.26 g/l 3.5 . analysis of the extract by gas chromatography - mass spectrometry detected 6-phenyl-2(h)-1,2,4-triazin-5-one oxime , an inhibitor of the parasitic m18 aspartyl aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity . the crude plant extract had a ki of 2.35 g/l and showed a dose - dependent response . after depletion of the compound , a significantly lower inhibition was determined with a ki of 4.8 g/l . moreover , two phenolic compounds , that is , 2,6-di - tert - butyl - phenol and 2,4-di - tert - butyl - phenol , with determined ic50 values of 50.29 m 3 and 47.82 m 2.5 , respectively , were detected . these compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties . in an in vivo experiment , treatment of balb / c mice with the aqueous balanite extract did not lead to eradication of the parasites , although a reduced parasitemia at day 12 p.i . was observed .

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

plant alkaloids , quinine among them , were the first components of natural herbal remedies to be extracted and refined for more effective use in the early 19th century . b. aegyptiaca ( l. ) ( balanitaceae ) is a woody tree growing in various ecological conditions ( from 100 mm to 1000 mm annual rainfall ) , but mainly distributed in semiarid and arid zones in tropical africa . in egypt , it is confined to water - receiving sites , that is , wadis and river banks . balanitin-6 and balanitin-7 are diosgenyl saponins from b. aegyptiaca kernels and they demonstrated appreciable anticancer effects in human cancer cell lines in vitro with a higher antiproliferative potential than etoposide and oxiplatin . the structures of balanitins , that is , balanitin-1 , -2 , -3 , are composed of steroid glycosides proven to be potent molluscicides for the control of freshwater snails that are carriers of bilharzia . moreover , the efficacy of b. aegyptiaca fruit mesocarp was compared with praziquantel in infected mice with a sudanese strain of schistosoma mansoni . recently , root - derived callus from b. aegyptiaca with 500 ppm extracted saponins showed a larvicidal effect of 100% against aedes aegypti mosquito larvae . since b. aegyptiaca bark shows promising anti - infective properties , we investigated its potential antiplasmodial activity in seeds and related this activity to its constituents in vitro . although phytochemical analyses and medicinal evaluations have been recently performed for fixed oil from b. aegyptiaca fruits , in vivo biological assays are missing . here , we monitored parasitemia after application of an aqueous extract obtained from the seeds of the plant in vitro and in vivo in balb / c mice . seeds of b. aegyptiaca ( balanitaceae ) were obtained from harraz pharmacy in cairo , collected in the area of ras mohammed national park and botanically identified by professor grtz , biological institute , university of stuttgart , germany . the residue obtained was diluted in 50 ml methanol by sonication and analyzed by gas chromatography - mass spectrometry ( gc / ms ) . for the preparation of an aqueous extract , 5 g of powdered b. aegyptiaca seeds were incubated at 37c in 100 ml distilled , autoclaved water for 24 hours in a sealed , sterilized falcon tube and filtrated in a laminar air flow under aseptic conditions . mass spectra and reconstructed chromatograms ( total ion current [ tic ] ) were obtained after eluting of solvent ( 7 min solvent delay ) by automatic scanning in the mass range m / z 30750 u. p. falciparum isolate nf54 , which was obtained from malaria research and reference center , manassas , va , usa , was maintained in continuous culture with gentamicin ( 40 g / ml ) in petri dishes ( 5 cm in diameter ) with a gaseous phase of 90% n2 , 5% o2 , and 5% co2 , according to a protocol from molony et al . parasitemias and morphological forms detected in the cultures were scored visually with a 100-fold oil - immersion objective , counting at least 1,000 erythrocytes to determine the percentage of the infected erythrocytes . thereafter , growth medium was changed once a day for 4 days , and 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol stock solutions were dissolved in water / methanol mixture ( 50/50% , v / v ) before being diluted in rpmi growth medium in the concentrations given for each experiment . dilution of the crude balanite extract obtained after soxhlet extraction was performed with rpmi - growth medium as a solvent to protect the rbc membrane from perturbation . the animal studies were performed according to the guidelines of the german animal welfare act ( license number : 50.203 . balb / c mice developed a th2 response and did not succumb to cerebral malaria , but died after 1030 days because of high parasitemia and anemia . 50 l of the aqueous crude plant extract ( 1,25 g/100 ml ) were applied to the balb / c mice intraperitoneally ( i.p . ) 2,6-di - tert - butyl - phenol and 2,4-di - tert - butyl - phenol were applied to the balb / c2 mice intraperitoneally ( i. p. ) on days 4 and 2 before infection , and on days 2 , 4 , and 6 before infection . prior to the start of the assay , 2.5 l of assay buffer ( 50 mm tris hcl ph 7.5 , 4 mm cocl2 , 0.1% bsa ) containing 5 g / ml recombinant pfm 18 aap were dispensed into a 12-well microtiter plate . next , 37 l of the crude balanite extract and the depleted balanite extract where 6-phenyl-2(h)-1,2,4-triazin-5-one oxime was absent were added as a control reaction to the appropriate wells . depletion of the extract was performed by liquid chromatography - mass spectrometry ( lc / ms ) , and fractions were rechecked by gc / ms as described within for the presence and absence of the 6-phenyl-2(h)-1,2,4-triazin-5-one oxime . statistical analyses were performed using graphpad prism software to evaluate the in vivo inhibitor experiments performed with balb / c mice . nonparametrically distributed data were analyzed using the mann - whitney test for differences between the aqueous balanite extract and the chloroquine treated group . crude extracts from extracted balanites aegyptiaca seeds show antiplasmodial activity in the chloroquine susceptible p. falciparum strain nf54 in vitro . in vitro inhibition of growth of the chloroquine susceptible p. falciparum strain nf54 was tested with a crude extract from b. aegyptiaca ( the amount equivalent to 1 : 2000 of a seed ) obtained by soxhlet extraction which led to an eradication of the parasite within 3 days ( figure 1(a ) ) and a determined ic50 of 68.26 g/l 3,5 ( figure 1(b ) ) . the crude balanite extract and a dilution of the extract , in which the ratio of seeds to the extract was 1 : 200 , lysed red blood cells ( rbc ; data not shown ) . therefore , the amount equivalent to 1 : 2000 of a seed was applied with rpmi - medium as a solvent to avoid any perturbation of the erythrocyte membrane . figure 2 shows the chromatogram of the total ion current ( tic ) obtained by gc / ms analysis of the crude balanite extract to identify and characterize the active compounds of the crude extract after soxhlet extraction . figures 3(a)3(c ) represent the mass spectra of the identified peaks using pure standard substances and the peaks of the nist 05 mass spectra library of compounds for comparison and to elucidate the chemical structures of the detected substances 2,6-di - tert - butyl - phenol ( 18.43 min ) , 2,4-di - tert - butyl - phenol ( 19.36 min ) , and 6-phenyl-2(h)-1,2,4-triazin-5-one oxime ( 39.44 min ) , respectively . ion [ m ] at m / z 206 , the fragment ion [ m ch3 ] at m / z 191 and the [ c4h9 ] ion at m / z 57 are characteristic for both di - tert - butyl - phenol isomers ( figures 3(a)3(b ) ) . the crude balanite extract inhibited recombinant m18 aspartyl aminopeptidase from plasmodium falciparum ( pfm18aap ) with a determined ki of 2.34 m . different concentrations of the crude plant extract ( figure 4 , blue curve ) were applied to investigate the dose response of recombinant pfmaap . , we determined the inhibitory effect of recombinant pfm18aap after fractionation of the soxhlet extract pooling fractions in which 6-phenyl-2(h)-1,2,4-triazin-5-one oxime was absent ( figure 4 , pink curve ) . the pink curve in figure 4 shows that depletion of 6-phenyl-2(h)-1,2,4-triazin-5-one oxime from the extract leads to a 50% lower inhibition at concentrations ranging from 2.7 ng dry weight per ml to 73.5 ng dry weight per ml compared to the extract where the compound was present . this inhibitory effect has been attributed to formation of metal chelates with the cocatalytic zinc metal ions of the parasitic enzyme . taken together , these results suggest that the detected 6-phenyl-2(h)-1,2,4-triazin-5-one oxime might contribute to the antiplasmodial activity . the occurrence of the two phenolic compounds prompted us to perform in vitro inhibitor and dose - response experiments with p. falciparum strain nf54 at concentrations from 10 mm to 100 mm . inhibition of growth started at a concentration of 10 mm independently of the compound . the most prominent growth arrest was observed at a concentration of 100 mm with either 2,4-di - tert - butyl - phenol or 2,6-di - tert - butyl - phenol ( figures 5(a ) and 5(c ) , resp . ) the determined ic50 values were 50.29 m 3 for 2,6-di - tert - butyl - phenol ( figure 5(b ) ) and 47.82 m 2,5 for 2,4-di - tert - butyl - phenol ( figure 5(d ) ) , respectively . a complete eradication of the parasite was not observed , suggesting that these compounds may contribute to the biological activity of the crude plant extract . 2,4-di - tert - butyl - phenol was more potent than the 2,6-substituted compound but did not lead to complete eradication of the parasite . in the next set of experiments , the combination of both compounds showed an ic50 value of 42.5 m 3 ( figure 5(f ) ) . an aqueous extract from b. aegyptiaca seeds and the two phenolic compounds 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol tested in vivo in balb / c mice do not reduce parasitemia . hitherto , no in vivo biological activity assay has been performed to investigate antiplasmodial activity of the balanite extract . therefore , we monitored in vivo protection in balb / c infected mice after infection with 10 parasitized erythrocytes of p. berghei anka ( pba ) strain after administration of the balanite extract and the two phenolic compounds , that is , 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol . the two phenolic compounds ( each dose was 6 mg / kg body weight ) were intraperitoneally applied on days 4 and 2 before infection and on days 2 , 4 , and 6 after infection . at day 8 , parasitemia was significantly increased in mice treated with the extract whereas , at day 12 p.i . in a parallel experiment , we monitored the survival rate of balb / c infected mice with each of the phenolic compounds in a dose of 6 mg / kg body weight ( data not shown ) . however , both phenolics did not reduce parasitemia in comparison to the control and , therefore , did not prolong the survival of p. berghei infected mice . b. aegyptiaca ( balanitaceae ) is a widely distributed plant in africa and has been reported to show a lot of different biological activities , which were attributed to its saponin constituents mainly deriving from diosgenin as the sole aglycone . recent investigations proved a medicinal evaluation for the fixed oil of b. aegyptiaca fruits in vitro . in the past , moderate in vitro antiplasmodial activity has been determined in an extract from b. aegyptiaca stem bark with an ic50 value of 55 g / ml . no antiplasmodial activity was found in the mesocarp of the fruits from this species . an aqueous dilution from the original extract in which the ratio of seeds was equivalent to a ratio of 1 : 2000 seeds was tested for in vitro inhibition of the chloroquine - susceptible p. falciparum strain nf54 , resulting in an ic50 value of 68.26 g/l 3,5 . in vitro activity is associated ( figure 4 blue curve ) with the occurrence of the oxime of 6-phenyl-2(h)-1,2,4-triazine-5-one , since the absence of the compound in the balanite extract leads to a 50% lower inhibition of recombinant plasmodial m18 aspartyl aminopeptidase ( pfm18aap ) ( figure 4 , pink curve ) compared to the crude extract where the oxime of 6-phenyl-2(h)-1,2,4-triazine-5-one was present ( figure 4 blue curve ) . however , the concentration of the compound in the extract was too low for a successful isolation and a proof of principle assay inhibiting growth of plasmodium in vitro . to our knowledge , there are no previous studies with the compound in the literature so far . taken together , these data are supported by the determined ki of 2.35 g/l of the crude balanite extract and the extract which was depleted of the 6-phenyl-2(h)-1,2,4-triazine-5-one oxime with a determined ki of 4.8 g/l . the enzyme is mainly located in the cytosol but can also be trafficked to the parasitophorous vacuole . the two phenolic compounds 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol were identified by gc / ms analysis ( figures 2 - 3 ) and contribute modestly to the in vitro biological activity ( figure 5 ) . the determined ic50 values were 50.29 m 3 for 2,6-di - tert - butyl - phenol and 47,82 m 2,5 for 2,4-di - tert - butyl - phenol , respectively ( figures 5(b ) and 5(d ) ) . however , the determined ic50 value of 42.5 m 3 , when both compounds were combined , appears to have a small additive effect but did not lead to the eradication of p. falciparum in vitro . these compounds have been recently associated with the antioxidant effect of a methanolic extract from the centipede scolopendra subspinipes mutilans ( scolopendridae ) which is used in traditional chinese and korean medicine for the treatment of diseases such as spasm , childhood convulsions , diphtheria and tetanus . moreover , 2,4-di - tert - butyl - phenol exhibited antioxidant activity against ldl - oxidation in a biological in vitro activity assay . consistent with the observation that 2,4-di - tert - butyl - phenol showed antioxidant activity , the drug exhibited also radical scavenging activity . recently , the antioxidant capacity was also proven for the oil present in balanites aegyptiaca fruits in vitro . the two phenolic compounds 2,4-di - tert - butyl - phenol and 2,6-di - tert - butyl - phenol have been detected in a variety of plant extracts . discussed the occurrence of both constituents as an uptake of the compounds from plastic waste being in the soil . although the fixed oil in b. aegyptiaca fruits exhibited anticancer activity in lung , liver , and brain human carcinoma cell lines , no cytotoxicity could be detected for the seed extract ( data not shown ) . hitherto , only two in vivo experiments in mice have been performed with either the pure constituents of the plant or the crude balanite extract [ 6 , 24 ] . a second in vivo experiment was performed with a crude balanite extract to demonstrate moderate antihepatotoxic activity in comparison with the standard hepatoprotective silymarin . the lyophilized extract of balanites aegyptiaca was administered orally for 5 consecutive days in a dose of 1 g / kg . to analyze parasitemia , balb / c infected mice were treated with a crude , aqueous balanite extract ( 50 ml of 1,25 g dry extract/100 ml solvent ) injected intraperitoneally on a daily basis , beginning on day 3,2 , 1 prior to infection , at the day of infection , and continuing until 8 days p.i . although a decrease in parasitemia could be observed on day 12 ( figure 6 ) , treatment did not clear the parasites over this time and therefore ; did not prolong survival . the plasmodial m18 aspartyl aminopeptidase inhibitor is present in the extract , however , its concentration might be too low to eradicate the parasite . the herbicide metamitron is a 4-amino-3-methyl-6-phenyl-1,2,4,triazin-5-one and , therefore , closely related from its structure to the 6-phenyl-2(h)-1,2,4-triazin-5-one oxime . given that 6-phenyl-2(h)-1,2,4-triazin-5-one oxime has the same effect , it would have enhanced the neurological symptoms in the infected balb / c mice independently from the parasitic infection . since there is a lack of knowledge with respect to the half - life , absorption , distribution , and elimination of the three identified compounds in a mammalian organism , we , therefore , conclude that the crude plant extract in its current form and concentration does not reduce parasitemia in vivo .

in 1938 bankart reported excellent results of re - attachment of the capsulolabral complex in patients with anterior shoulder instability , a method first described by perthes . this technique is based on the premis that detachment of the anteroinferior capsulolabral complex is the cause for recurrent dislocations and therefore refixation is the therapy of choice . since then open " bankart repair " with minor modifications has been performed with good results and low recurrence rates and therefore was considered as the " golden standard " . rowe reported in 1978 excellent and good functional results in 97% and a recurrence rate of 3.5% with this technique after an average follow - up duration of 6 years . different studies have proven this technique as a sucessful treatment for traumatic anterior shoulder instability with recurrence rates below 10% . however , long - term follow - up studies and investigations including subluxation as a failure demonstrated a higher recurrence rate than expected . problems reported to be associated with open bankart repair include restriction in external rotation and subscapularis muscle insufficiency . therefore less invasive arthroscopic techniques have been developed and have improved from disappointingly high recurrence rates with staple capsulorrhaphy , transglenoid sutures and bioabsorbable tacks to promising results with suture anchors equal to open procedures . arthroscopic stabilization is considered advantageous in terms of decreased morbidity with reduced pain and shorter hospitalization ( if necessary at all ) , faster rehabilitation , no violation of the subscapularis tendon and no loss in range of motion . nevertheless , higher recurrence rates compared to the open procedure have been reported . the intention of this investigation was to compare open and arthroscopic bankart repair concerning functional outcome and stability at mid - term follow - up . in this retrospective cross - sectional study patients were reviewed , who underwent surgical treatment for shoulder instability at the authors ' orthopedic department . in 1995 a modified bankart procedure without osteotomy of the coracoid process became the standard operation for traumatic anterior shoulder instability at the authors ' department . a total of 212 patients were treated between 1995 and june 2004 for traumatic anterior shoulder dislocation ( tubs ) according to the criteria of matsen . most patients ( 206 , 97% ) had recurrent dislocations , 169 ( 80% ) were male and 43 ( 20% ) female . in 109 patients ( 51% ) age at first dislocation was in median 22 years ( range 5 to 67 years ) , the time from first dislocation to surgery was in median 28 month ( ranging from 1 month to 8 years ) . 21 patients had an osseus bankart lesion , in four patients the bankart fragment could be fixed with screws , nine patients suffering from loss of the anterior glenoid of more than 20% required a reconstruction with iliac crest bone grafting and were excluded from this study . eight patients with only small osseus fragments were treated with refixation of the capsulolabral complex with suture anchors and were included in this study . in a total of 199 patients a bankart procedure with suture anchors was performed ( median age at surgery 26 years , 20% females ) , either arthroscopically in presence of an detached , but not elongated capsulolabral complex ( 40 , 20% ) or open ( 159 , 80% ) . in 56 of these 159 patients ( 28% ) with a wide capsule an additional inferior capsular shift as described by neer was performed . patients who underwent open surgery showed a median age of 27 years ( 22% females ) versus 25 years among the arthroscopically treated patients ( 13% females ) . a total of 36% ( open ) and 40% ( arthroscopic ) of them reported more than five dislocations before surgery . three patients treated with open surgery and four patients treated arthroscopically had an additional slap lesion . there were 12 partial lesion of the supraspinatus tendon in the open group , the rotator cuff was intakt in all arthroscopic treated patients the demographic data for the patient samples are summarized in table 1 . distribution characteristics for demographic data of patients after open and arthroscopic bankart procedure ( absolute and relative frequencies for gender and dislocations before surgery , medians and quartile ranges for age and time from trauma to surgery ) all patients were operated on under general anesthesia and in beach - chair position . after diagnostic arthroscopy through the standard dorsal portal the decision was made whether to continue with arthroscopic or open surgery . in cases of detached , but intact capsulolabral complex and no visually elongated capsule an arthroscopic fixation using suture anchors was performed . additional antero - superior and anterior portals were used . in the other cases an open procedure using a deltoideo - pectoral approach with an l - shaped incision of the upper two third of the subscapularis tendon was performed . the capsular incision was done laterally , in cases of elongated capsule a lateral based t - shaped incision was used and an inferior shift was done . in all cases the anteroinferior capsulolabral complex was separated from the glenoid and anatomical repair was performed with two to three suture anchors ( fas - tak , fa.arthrex ; mini - revo , fa.linvatec ; mitek gii , fa.depuy-mitek ) . instructions were given to the patient by a physiotherapist during hospital stay and in written form for the further therapy . the arm was immobilized in a gilchrist cast for 6 weeks and only passive motion twice a day ( anteversion up to 90 , no abduction , no external rotation ) was allowed . during the 7th to the 12th week after surgery active movement was allowed and full range of motion was achieved assisted by physiotherapy . ( 12 to 67 months ) 174 patients were contacted and agreed to follow - up ; a total of 143 of them underwent a clinical examination and a written interview with a questionnaire , further 31 patients only answered the questionaire . among these recall patients 135 the questionnaire included information about recurrent instability ( dislocation , subluxation or feeling of instability ) as well as questions to loss of function and subjective rating . the clinical examination included assessment of range of motion , stability ( apprehension and relocation test 36 ) , loss of function and strength in order to obtain the rowe score 33 and a modified rowe score 34 adapted for subluxations . the primary analysis was performed via kaplan / meier estimation of the dislocation - free time after initial surgery ; the description of the survival estimates was based on the mean dislocation - free times after the respective surgical treatment and the corresponding 95% confidence intervals . the kaplan / meier estimates for the time to dislocation after open versus arthroscopic treatment were then compared by means of a univariate logrank test and by means of a multivariate cox regression analysis based on likelihood ratio tests . the latter adjusted the dislocation patterns of the samples for putative prognostically relevant cofactors such as age and gender . results of both the univariate and multivariate analysis were summarized in terms of p - values . due to the exploratory character of the multivariate analysis p - values derived from the latter were not formally adjusted for multiplicity ; a p - value < 0.05 therefore indicates a locally significant difference between samples . in general the data were analysed according to the respective endpoints ' scale level : the description of continuous endpoints was based on medians and quartiles , the description of categorical endpoints of appropriate absolute and relative frequencies . the significance comparison of sub samples was based on pairwise two sample wilcoxon and fisher tests , accordingly . all numeric and graphic analyses were performed with spss software ( release 12.0 for windows ; spss inc , chicago , il ) . most patients ( 206 , 97% ) had recurrent dislocations , 169 ( 80% ) were male and 43 ( 20% ) female . in 109 patients ( 51% ) age at first dislocation was in median 22 years ( range 5 to 67 years ) , the time from first dislocation to surgery was in median 28 month ( ranging from 1 month to 8 years ) . 21 patients had an osseus bankart lesion , in four patients the bankart fragment could be fixed with screws , nine patients suffering from loss of the anterior glenoid of more than 20% required a reconstruction with iliac crest bone grafting and were excluded from this study . eight patients with only small osseus fragments were treated with refixation of the capsulolabral complex with suture anchors and were included in this study . in a total of 199 patients a bankart procedure with suture anchors was performed ( median age at surgery 26 years , 20% females ) , either arthroscopically in presence of an detached , but not elongated capsulolabral complex ( 40 , 20% ) or open ( 159 , 80% ) . in 56 of these 159 patients ( 28% ) with a wide capsule an additional inferior capsular shift as described by neer was performed . patients who underwent open surgery showed a median age of 27 years ( 22% females ) versus 25 years among the arthroscopically treated patients ( 13% females ) . a total of 36% ( open ) and 40% ( arthroscopic ) of them reported more than five dislocations before surgery . three patients treated with open surgery and four patients treated arthroscopically had an additional slap lesion . there were 12 partial lesion of the supraspinatus tendon in the open group , the rotator cuff was intakt in all arthroscopic treated patients the demographic data for the patient samples are summarized in table 1 . distribution characteristics for demographic data of patients after open and arthroscopic bankart procedure ( absolute and relative frequencies for gender and dislocations before surgery , medians and quartile ranges for age and time from trauma to surgery ) all patients were operated on under general anesthesia and in beach - chair position . after diagnostic arthroscopy through the standard dorsal portal the decision was made whether to continue with arthroscopic or open surgery . in cases of detached , but intact capsulolabral complex and no visually elongated capsule an arthroscopic fixation using suture anchors was performed . additional antero - superior and anterior portals were used . in the other cases an open procedure using a deltoideo - pectoral approach with an l - shaped incision of the upper two third of the subscapularis tendon was performed . the capsular incision was done laterally , in cases of elongated capsule a lateral based t - shaped incision was used and an inferior shift was done . in all cases the anteroinferior capsulolabral complex was separated from the glenoid and anatomical repair was performed with two to three suture anchors ( fas - tak , fa.arthrex ; mini - revo , fa.linvatec ; mitek gii , fa.depuy-mitek ) . instructions were given to the patient by a physiotherapist during hospital stay and in written form for the further therapy . the arm was immobilized in a gilchrist cast for 6 weeks and only passive motion twice a day ( anteversion up to 90 , no abduction , no external rotation ) was allowed . during the 7th to the 12th week after surgery active movement was allowed and full range of motion was achieved assisted by physiotherapy . after a median time of 31 months ( 12 to 67 months ) 174 patients were contacted and agreed to follow - up ; a total of 143 of them underwent a clinical examination and a written interview with a questionnaire , further 31 patients only answered the questionaire . among these recall patients 135 the questionnaire included information about recurrent instability ( dislocation , subluxation or feeling of instability ) as well as questions to loss of function and subjective rating . the clinical examination included assessment of range of motion , stability ( apprehension and relocation test 36 ) , loss of function and strength in order to obtain the rowe score 33 and a modified rowe score 34 adapted for subluxations . the primary analysis was performed via kaplan / meier estimation of the dislocation - free time after initial surgery ; the description of the survival estimates was based on the mean dislocation - free times after the respective surgical treatment and the corresponding 95% confidence intervals . the kaplan / meier estimates for the time to dislocation after open versus arthroscopic treatment were then compared by means of a univariate logrank test and by means of a multivariate cox regression analysis based on likelihood ratio tests . the latter adjusted the dislocation patterns of the samples for putative prognostically relevant cofactors such as age and gender . results of both the univariate and multivariate analysis were summarized in terms of p - values . due to the exploratory character of the multivariate analysis p - values derived from the latter were not formally adjusted for multiplicity ; a p - value < 0.05 therefore indicates a locally significant difference between samples . in general the data were analysed according to the respective endpoints ' scale level : the description of continuous endpoints was based on medians and quartiles , the description of categorical endpoints of appropriate absolute and relative frequencies . the significance comparison of sub samples was based on pairwise two sample wilcoxon and fisher tests , accordingly . all numeric and graphic analyses were performed with spss software ( release 12.0 for windows ; spss inc , chicago , il ) . re - dislocations occurred in 11 ( 8% ) of 135 patients after open and in 6 ( 15% ) of 39 patients after arthroscopic bankart procedure . after open surgery 4 of these 11 re - dislocations occurred after a new adequate trauma and 1 of the 6 re - dislocations after arthroscopic surgery . this corresponds to an adjusted re - dislocation rate without new adequate trauma of 5% ( 7 of 135 patients ) after open versus 13% ( 5 of 39 patients ) after arthroscopic treatment ( table 2 ) . absolute and relative rates for recurrence of instability in patients after open and arthroscopic bankart procedure furthermore 4 patients after open and 3 patients after arthroscopic surgery reported subluxations . if both " dislocation for any cause " and " subluxation " are considered as recurrence of instability , the open treatment failed in 15 patients ( 11% ) versus 9 patients after arthroscopic treatment ( 23% ) . after arthroscopic surgery there was an increased rate of failure ( re - dislocation and subluxation ) observed among patients , who had reported more than 5 dislocations before initial surgery ( 6 of 16 patients [ 38% ] ) versus patients with fewer previous dislocations ( 3 of 23 patients [ 13% ] ) . the mean time interval between surgery and dislocation for any cause was 64 month after open and 41 month after arthroscopic procedure ( 95% confidence intervals 62 - 67 versus 37 - 45 months , respectively ) . the underlying dislocation - free time distributions differed statistically significantly ( p = 0.0268 ) . kaplan / meier survival estimate for the dislocation - free time [ months ] after initial surgery of patients after open and arthroscopic bankart procedure ( crosses indicate censoring of patients ) . the cox regression analysis of the above re - dislocation rates confirmed the surgical procedure as the dominating and statistically significant determinant ( likelihood ratio p = 0.039 ) : neither age at first dislocation ( p = 0.441 ) or at recall ( p = 0.219 ) , the patient 's gender ( p = 0.984 ) , the duration between first luxation and initial surgery ( p = 0.297 ) or the self - reported number of dislocations before surgery ( p = 0.088 ) showed a statistically significant association with the time to re - dislocation after surgery . a total of 125 patients ( 93% ) after open surgery versus 30 patients ( 77% ) after arthroscopic treatment were satisfied with the operation ( fisher p = 0.016 ) . only 2 patients ( 2% ) after open surgery and 2 patients ( 5% ) after arthroscopic treatment would not agree to undergo the same type of surgery again . among the 143 patients who underwent clinical examination ( 108 after open and 35 after arthroscopic treatment ) a total of 6 patients ( 6% ) after open and 5 patients ( 14% ) after arthroscopic treatment showed a positive apprehension and relocation test during clinical examination ( fisher p = 0.144 ) . concerning range of motion compared to the contralateral side no significant difference between open and arthroscopic bankart procedure were observed for abduction . an external rotation lag of 20 or more was observed more often ( 16% ) after open than after arthroscopic surgery ( 3% , fisher p = 0.073 ) . using the lift - off test 12 to determine the subscapularis function the rowe score demonstrated " good " or " excellent " functional results in 94 of 108 ( 87% ) patients after open and in 28 of 35 ( 80% ) patients after arthroscopic treatment ( fisher p = 0.409 ) . the modified rowe 34 score demonstrated " good " or " excellent " functional results in 91 of 108 ( 84% ) patients after open and in 26 of 35 ( 74% ) patients after arthroscopic treatment ( fisher p = 0.210 ; table 3 ) . taking only the patients without re - dislocations there are similar results in both groups . functional outcome of patients after open and arthroscopic bankart procedure assessed and classified by means of the rowe and the modified rowe score a total of 125 patients ( 93% ) after open surgery versus 30 patients ( 77% ) after arthroscopic treatment were satisfied with the operation ( fisher p = 0.016 ) . only 2 patients ( 2% ) after open surgery and 2 patients ( 5% ) after arthroscopic treatment would not agree to undergo the same type of surgery again . among the 143 patients who underwent clinical examination ( 108 after open and 35 after arthroscopic treatment ) a total of 6 patients ( 6% ) after open and 5 patients ( 14% ) after arthroscopic treatment showed a positive apprehension and relocation test during clinical examination ( fisher p = 0.144 ) . concerning range of motion compared to the contralateral side no significant difference between open and arthroscopic bankart procedure were observed for abduction . an external rotation lag of 20 or more was observed more often ( 16% ) after open than after arthroscopic surgery ( 3% , fisher p = 0.073 ) . using the lift - off test 12 to determine the subscapularis function the rowe score demonstrated " good " or " excellent " functional results in 94 of 108 ( 87% ) patients after open and in 28 of 35 ( 80% ) patients after arthroscopic treatment ( fisher p = 0.409 ) . the modified rowe 34 score demonstrated " good " or " excellent " functional results in 91 of 108 ( 84% ) patients after open and in 26 of 35 ( 74% ) patients after arthroscopic treatment ( fisher p = 0.210 ; table 3 ) . taking only the patients without re - dislocations there are similar results in both groups . functional outcome of patients after open and arthroscopic bankart procedure assessed and classified by means of the rowe and the modified rowe score this retrospective investigation demonstrated an numerically increased recurrence rate after arthroscopic compared to open bankart repair as already reported in previous studies ( table 4 ) and a recent meta - analysis 25 , but the observed difference did not reach statistically significance . however , the recurrences of instability after artroscopically treated patients were observed earlier than after open treatment ( figure 1 ) . studies comparing open and arthroscopic bankart procedure these findings may be due to methodological limitation of the study with its retrospective cross - sectional implementation . the treatment samples differed , for example , in the number of dislocations before surgery , in the duration from first dislocation to surgery and in the degree of bankart lesion and capsular elongation . another reason for this difference might be the different surgical treatment of the capsule . although decision for arthroscopic treatment was done after arthroscopic assesment and only patients with considered not elongated capsule were treated arthroscopically the higher recurrence rate in the arthroscopic group may be caused by patient selection . 40% of arthroscopic repairs were performed in patients with more than 5 dislocations before surgery and showed a higher failure rate as has been reported before . we did no capsulorraphy during arthroscopic procedure but an additional capsular shift was performed in 28% of the open bankart repairs . although operated on by a single surgeon ( mb ) experienced in arthroscopic surgery the number of arthroscopic procedures is quite low and the individual learning curve is included which can lead to a greater recurrence rate . however , there was no increased failure rate in the earlier compared to the later treated patients . despite its limitations , this investigation indicates , that the clinical outcome after arthroscopic bankart procedure with no capsular shift seems to be inferior to the results after open surgery and corresponds to previous studies [ 6,10,17,20,21,37 - 39 ] ( table 4 ) . in these studies arthroscopic repair has been performed with different techniques ( transglenoid sutures , tacks ) and mostly no capsular tightening has been done . recent studies with suture anchor fixation demonstrate , that equally good results can be achieved for selected patients and in specialized institutions after arthroscopic bankart repair . main disadvantage of open bankart procedure is the violation of of the subscapularis tendon and therefore the possibility of subscapularis muscle insufficiency . in our patients with open procedures we did not observe any case of clinical subscapularis muscle insufficiency and therefore we consider this not to be a problem as long as thorough dissection and reattachment of the tendon is done . we observed an external rotation lag of 20 or more in 16% after open and only 3% after artroscopic treatment . this external rotation lag was tolerated by most patients as the better rating after open procedure suggests . nonetheless this can be a problem for some patients who require full range of motion . various scoring systems are established but most studies used the rowe score for assessment of functional results after operative shoulder stabilization . we found similar good results as reported by these studies in both groups regarding the rowe score as well as the modified rowe score . in this retrospective investigation the open bankart procedure demonstrated good functional results , a gradual loss of external rotation which was tolerated by most patients and no clinical signs of subcapularis muscle insufficiency . the arthroscopic treatment without capsular shift resulted in a better range of motion , but showed a tendency towards more frequently and earlier recurrence of instability . the authors recommend sensitive patient selection for arthroscopic bankart repair especially in patients with more than five dislocations . the data of this paper has been presented at the 8efort congress , 11 - 15 may 2007 in firenze , italy , at the 24 . aga congress , 27 - 29 september 2007 in kln , germany and at the congress of the german orthopedic society , 24 - 27 october 2007 , berlin . parts of this presentation are contained in the doctoral thesis of ms juliane lbke ( dresden university of technology , medical faculty ) . the authors have no commercial or political interests in the methodological or medical aspects presented in this paper . there are no financial or other relationships that might lead to a conflict of interest .

purposeboth open and arthroscopic bankart repair are established procedures in the treatment of anterior shoulder instability . while the open procedure is still considered as the " golden standard " functional outcome is supposed to be better in the arthroscopic procedure . the aim of this retrospective study was to compare the functional outcome between open and arthroscopic bankart repair.materials and methodsin 199 patients a bankart procedure with suture anchors was performed , either arthroscopically in presence of an detached , but not elongated capsulolabral complex ( 40 ) or open ( 159 ) . after a median time of 31 months ( 12 to 67 months ) 174 patients were contacted and agreed to follow - up , 135 after open and 39 after arthroscopic bankart procedure.resultsre-dislocations occurred in 8% after open and 15% after arthroscopic bankart procedure . after open surgery 4 of the 11 re - dislocations occurred after a new adequate trauma and 1 of the 6 re - dislocations after arthroscopic surgery . re - dislocations after arthroscopic procedure occured earlier than after open bankart repair . an external rotation lag of 20 or more was observed more often ( 16% ) after open than after arthroscopic surgery ( 3% ) . the rowe score demonstrated " good " or " excellent " functional results in 87% after open and in 80% patients after arthroscopic treatment.conclusionin this retrospective investigation the open bankart procedure demonstrated good functional results . the arthroscopic treatment without capsular shift resulted in a better range of motion , but showed a tendency towards more frequently and earlier recurrence of instability . sensitive patient selection for arthroscopic bankart repair is recommended especially in patients with more than five dislocations .

Introduction Materials and methods Patient samples Surgical procedure Postoperative rehabilitation Recall Statistical analysis Results Functional and subjective outcome Discussion Conclusion Conflict of interests

in 1938 bankart reported excellent results of re - attachment of the capsulolabral complex in patients with anterior shoulder instability , a method first described by perthes . this technique is based on the premis that detachment of the anteroinferior capsulolabral complex is the cause for recurrent dislocations and therefore refixation is the therapy of choice . since then open " bankart repair " with minor modifications has been performed with good results and low recurrence rates and therefore was considered as the " golden standard " . rowe reported in 1978 excellent and good functional results in 97% and a recurrence rate of 3.5% with this technique after an average follow - up duration of 6 years . different studies have proven this technique as a sucessful treatment for traumatic anterior shoulder instability with recurrence rates below 10% . however , long - term follow - up studies and investigations including subluxation as a failure demonstrated a higher recurrence rate than expected . problems reported to be associated with open bankart repair include restriction in external rotation and subscapularis muscle insufficiency . therefore less invasive arthroscopic techniques have been developed and have improved from disappointingly high recurrence rates with staple capsulorrhaphy , transglenoid sutures and bioabsorbable tacks to promising results with suture anchors equal to open procedures . arthroscopic stabilization is considered advantageous in terms of decreased morbidity with reduced pain and shorter hospitalization ( if necessary at all ) , faster rehabilitation , no violation of the subscapularis tendon and no loss in range of motion . nevertheless , higher recurrence rates compared to the open procedure have been reported . the intention of this investigation was to compare open and arthroscopic bankart repair concerning functional outcome and stability at mid - term follow - up . in this retrospective cross - sectional study patients were reviewed , who underwent surgical treatment for shoulder instability at the authors ' orthopedic department . in 1995 a modified bankart procedure without osteotomy of the coracoid process became the standard operation for traumatic anterior shoulder instability at the authors ' department . a total of 212 patients were treated between 1995 and june 2004 for traumatic anterior shoulder dislocation ( tubs ) according to the criteria of matsen . 21 patients had an osseus bankart lesion , in four patients the bankart fragment could be fixed with screws , nine patients suffering from loss of the anterior glenoid of more than 20% required a reconstruction with iliac crest bone grafting and were excluded from this study . eight patients with only small osseus fragments were treated with refixation of the capsulolabral complex with suture anchors and were included in this study . in a total of 199 patients a bankart procedure with suture anchors was performed ( median age at surgery 26 years , 20% females ) , either arthroscopically in presence of an detached , but not elongated capsulolabral complex ( 40 , 20% ) or open ( 159 , 80% ) . in 56 of these 159 patients ( 28% ) with a wide capsule an additional inferior capsular shift as described by neer was performed . patients who underwent open surgery showed a median age of 27 years ( 22% females ) versus 25 years among the arthroscopically treated patients ( 13% females ) . a total of 36% ( open ) and 40% ( arthroscopic ) of them reported more than five dislocations before surgery . there were 12 partial lesion of the supraspinatus tendon in the open group , the rotator cuff was intakt in all arthroscopic treated patients the demographic data for the patient samples are summarized in table 1 . distribution characteristics for demographic data of patients after open and arthroscopic bankart procedure ( absolute and relative frequencies for gender and dislocations before surgery , medians and quartile ranges for age and time from trauma to surgery ) all patients were operated on under general anesthesia and in beach - chair position . after diagnostic arthroscopy through the standard dorsal portal the decision was made whether to continue with arthroscopic or open surgery . in cases of detached , but intact capsulolabral complex and no visually elongated capsule an arthroscopic fixation using suture anchors was performed . in the other cases an open procedure using a deltoideo - pectoral approach with an l - shaped incision of the upper two third of the subscapularis tendon was performed . in all cases the anteroinferior capsulolabral complex was separated from the glenoid and anatomical repair was performed with two to three suture anchors ( fas - tak , fa.arthrex ; mini - revo , fa.linvatec ; mitek gii , fa.depuy-mitek ) . instructions were given to the patient by a physiotherapist during hospital stay and in written form for the further therapy . the arm was immobilized in a gilchrist cast for 6 weeks and only passive motion twice a day ( anteversion up to 90 , no abduction , no external rotation ) was allowed . during the 7th to the 12th week after surgery active movement was allowed and full range of motion was achieved assisted by physiotherapy . ( 12 to 67 months ) 174 patients were contacted and agreed to follow - up ; a total of 143 of them underwent a clinical examination and a written interview with a questionnaire , further 31 patients only answered the questionaire . the clinical examination included assessment of range of motion , stability ( apprehension and relocation test 36 ) , loss of function and strength in order to obtain the rowe score 33 and a modified rowe score 34 adapted for subluxations . the primary analysis was performed via kaplan / meier estimation of the dislocation - free time after initial surgery ; the description of the survival estimates was based on the mean dislocation - free times after the respective surgical treatment and the corresponding 95% confidence intervals . the kaplan / meier estimates for the time to dislocation after open versus arthroscopic treatment were then compared by means of a univariate logrank test and by means of a multivariate cox regression analysis based on likelihood ratio tests . 21 patients had an osseus bankart lesion , in four patients the bankart fragment could be fixed with screws , nine patients suffering from loss of the anterior glenoid of more than 20% required a reconstruction with iliac crest bone grafting and were excluded from this study . eight patients with only small osseus fragments were treated with refixation of the capsulolabral complex with suture anchors and were included in this study . in a total of 199 patients a bankart procedure with suture anchors was performed ( median age at surgery 26 years , 20% females ) , either arthroscopically in presence of an detached , but not elongated capsulolabral complex ( 40 , 20% ) or open ( 159 , 80% ) . in 56 of these 159 patients ( 28% ) with a wide capsule an additional inferior capsular shift as described by neer was performed . patients who underwent open surgery showed a median age of 27 years ( 22% females ) versus 25 years among the arthroscopically treated patients ( 13% females ) . a total of 36% ( open ) and 40% ( arthroscopic ) of them reported more than five dislocations before surgery . there were 12 partial lesion of the supraspinatus tendon in the open group , the rotator cuff was intakt in all arthroscopic treated patients the demographic data for the patient samples are summarized in table 1 . distribution characteristics for demographic data of patients after open and arthroscopic bankart procedure ( absolute and relative frequencies for gender and dislocations before surgery , medians and quartile ranges for age and time from trauma to surgery ) all patients were operated on under general anesthesia and in beach - chair position . after diagnostic arthroscopy through the standard dorsal portal the decision was made whether to continue with arthroscopic or open surgery . in cases of detached , but intact capsulolabral complex and no visually elongated capsule an arthroscopic fixation using suture anchors was performed . in the other cases an open procedure using a deltoideo - pectoral approach with an l - shaped incision of the upper two third of the subscapularis tendon was performed . in all cases the anteroinferior capsulolabral complex was separated from the glenoid and anatomical repair was performed with two to three suture anchors ( fas - tak , fa.arthrex ; mini - revo , fa.linvatec ; mitek gii , fa.depuy-mitek ) . instructions were given to the patient by a physiotherapist during hospital stay and in written form for the further therapy . the arm was immobilized in a gilchrist cast for 6 weeks and only passive motion twice a day ( anteversion up to 90 , no abduction , no external rotation ) was allowed . during the 7th to the 12th week after surgery active movement was allowed and full range of motion was achieved assisted by physiotherapy . after a median time of 31 months ( 12 to 67 months ) 174 patients were contacted and agreed to follow - up ; a total of 143 of them underwent a clinical examination and a written interview with a questionnaire , further 31 patients only answered the questionaire . the clinical examination included assessment of range of motion , stability ( apprehension and relocation test 36 ) , loss of function and strength in order to obtain the rowe score 33 and a modified rowe score 34 adapted for subluxations . the primary analysis was performed via kaplan / meier estimation of the dislocation - free time after initial surgery ; the description of the survival estimates was based on the mean dislocation - free times after the respective surgical treatment and the corresponding 95% confidence intervals . the kaplan / meier estimates for the time to dislocation after open versus arthroscopic treatment were then compared by means of a univariate logrank test and by means of a multivariate cox regression analysis based on likelihood ratio tests . re - dislocations occurred in 11 ( 8% ) of 135 patients after open and in 6 ( 15% ) of 39 patients after arthroscopic bankart procedure . after open surgery 4 of these 11 re - dislocations occurred after a new adequate trauma and 1 of the 6 re - dislocations after arthroscopic surgery . this corresponds to an adjusted re - dislocation rate without new adequate trauma of 5% ( 7 of 135 patients ) after open versus 13% ( 5 of 39 patients ) after arthroscopic treatment ( table 2 ) . absolute and relative rates for recurrence of instability in patients after open and arthroscopic bankart procedure furthermore 4 patients after open and 3 patients after arthroscopic surgery reported subluxations . if both " dislocation for any cause " and " subluxation " are considered as recurrence of instability , the open treatment failed in 15 patients ( 11% ) versus 9 patients after arthroscopic treatment ( 23% ) . after arthroscopic surgery there was an increased rate of failure ( re - dislocation and subluxation ) observed among patients , who had reported more than 5 dislocations before initial surgery ( 6 of 16 patients [ 38% ] ) versus patients with fewer previous dislocations ( 3 of 23 patients [ 13% ] ) . the mean time interval between surgery and dislocation for any cause was 64 month after open and 41 month after arthroscopic procedure ( 95% confidence intervals 62 - 67 versus 37 - 45 months , respectively ) . kaplan / meier survival estimate for the dislocation - free time [ months ] after initial surgery of patients after open and arthroscopic bankart procedure ( crosses indicate censoring of patients ) . the cox regression analysis of the above re - dislocation rates confirmed the surgical procedure as the dominating and statistically significant determinant ( likelihood ratio p = 0.039 ) : neither age at first dislocation ( p = 0.441 ) or at recall ( p = 0.219 ) , the patient 's gender ( p = 0.984 ) , the duration between first luxation and initial surgery ( p = 0.297 ) or the self - reported number of dislocations before surgery ( p = 0.088 ) showed a statistically significant association with the time to re - dislocation after surgery . a total of 125 patients ( 93% ) after open surgery versus 30 patients ( 77% ) after arthroscopic treatment were satisfied with the operation ( fisher p = 0.016 ) . only 2 patients ( 2% ) after open surgery and 2 patients ( 5% ) after arthroscopic treatment would not agree to undergo the same type of surgery again . among the 143 patients who underwent clinical examination ( 108 after open and 35 after arthroscopic treatment ) a total of 6 patients ( 6% ) after open and 5 patients ( 14% ) after arthroscopic treatment showed a positive apprehension and relocation test during clinical examination ( fisher p = 0.144 ) . concerning range of motion compared to the contralateral side no significant difference between open and arthroscopic bankart procedure were observed for abduction . an external rotation lag of 20 or more was observed more often ( 16% ) after open than after arthroscopic surgery ( 3% , fisher p = 0.073 ) . using the lift - off test 12 to determine the subscapularis function the rowe score demonstrated " good " or " excellent " functional results in 94 of 108 ( 87% ) patients after open and in 28 of 35 ( 80% ) patients after arthroscopic treatment ( fisher p = 0.409 ) . the modified rowe 34 score demonstrated " good " or " excellent " functional results in 91 of 108 ( 84% ) patients after open and in 26 of 35 ( 74% ) patients after arthroscopic treatment ( fisher p = 0.210 ; table 3 ) . taking only the patients without re - dislocations there are similar results in both groups . functional outcome of patients after open and arthroscopic bankart procedure assessed and classified by means of the rowe and the modified rowe score a total of 125 patients ( 93% ) after open surgery versus 30 patients ( 77% ) after arthroscopic treatment were satisfied with the operation ( fisher p = 0.016 ) . only 2 patients ( 2% ) after open surgery and 2 patients ( 5% ) after arthroscopic treatment would not agree to undergo the same type of surgery again . among the 143 patients who underwent clinical examination ( 108 after open and 35 after arthroscopic treatment ) a total of 6 patients ( 6% ) after open and 5 patients ( 14% ) after arthroscopic treatment showed a positive apprehension and relocation test during clinical examination ( fisher p = 0.144 ) . concerning range of motion compared to the contralateral side no significant difference between open and arthroscopic bankart procedure were observed for abduction . an external rotation lag of 20 or more was observed more often ( 16% ) after open than after arthroscopic surgery ( 3% , fisher p = 0.073 ) . using the lift - off test 12 to determine the subscapularis function the rowe score demonstrated " good " or " excellent " functional results in 94 of 108 ( 87% ) patients after open and in 28 of 35 ( 80% ) patients after arthroscopic treatment ( fisher p = 0.409 ) . the modified rowe 34 score demonstrated " good " or " excellent " functional results in 91 of 108 ( 84% ) patients after open and in 26 of 35 ( 74% ) patients after arthroscopic treatment ( fisher p = 0.210 ; table 3 ) . taking only the patients without re - dislocations there are similar results in both groups . functional outcome of patients after open and arthroscopic bankart procedure assessed and classified by means of the rowe and the modified rowe score this retrospective investigation demonstrated an numerically increased recurrence rate after arthroscopic compared to open bankart repair as already reported in previous studies ( table 4 ) and a recent meta - analysis 25 , but the observed difference did not reach statistically significance . however , the recurrences of instability after artroscopically treated patients were observed earlier than after open treatment ( figure 1 ) . studies comparing open and arthroscopic bankart procedure these findings may be due to methodological limitation of the study with its retrospective cross - sectional implementation . the treatment samples differed , for example , in the number of dislocations before surgery , in the duration from first dislocation to surgery and in the degree of bankart lesion and capsular elongation . another reason for this difference might be the different surgical treatment of the capsule . although decision for arthroscopic treatment was done after arthroscopic assesment and only patients with considered not elongated capsule were treated arthroscopically the higher recurrence rate in the arthroscopic group may be caused by patient selection . 40% of arthroscopic repairs were performed in patients with more than 5 dislocations before surgery and showed a higher failure rate as has been reported before . we did no capsulorraphy during arthroscopic procedure but an additional capsular shift was performed in 28% of the open bankart repairs . despite its limitations , this investigation indicates , that the clinical outcome after arthroscopic bankart procedure with no capsular shift seems to be inferior to the results after open surgery and corresponds to previous studies [ 6,10,17,20,21,37 - 39 ] ( table 4 ) . recent studies with suture anchor fixation demonstrate , that equally good results can be achieved for selected patients and in specialized institutions after arthroscopic bankart repair . main disadvantage of open bankart procedure is the violation of of the subscapularis tendon and therefore the possibility of subscapularis muscle insufficiency . in our patients with open procedures we did not observe any case of clinical subscapularis muscle insufficiency and therefore we consider this not to be a problem as long as thorough dissection and reattachment of the tendon is done . we observed an external rotation lag of 20 or more in 16% after open and only 3% after artroscopic treatment . this external rotation lag was tolerated by most patients as the better rating after open procedure suggests . nonetheless this can be a problem for some patients who require full range of motion . various scoring systems are established but most studies used the rowe score for assessment of functional results after operative shoulder stabilization . we found similar good results as reported by these studies in both groups regarding the rowe score as well as the modified rowe score . in this retrospective investigation the open bankart procedure demonstrated good functional results , a gradual loss of external rotation which was tolerated by most patients and no clinical signs of subcapularis muscle insufficiency . the arthroscopic treatment without capsular shift resulted in a better range of motion , but showed a tendency towards more frequently and earlier recurrence of instability . the authors recommend sensitive patient selection for arthroscopic bankart repair especially in patients with more than five dislocations . aga congress , 27 - 29 september 2007 in kln , germany and at the congress of the german orthopedic society , 24 - 27 october 2007 , berlin . parts of this presentation are contained in the doctoral thesis of ms juliane lbke ( dresden university of technology , medical faculty ) .

traumatic brain injury ( tbi ) is heterogeneous disorder caused by the action of external mechanical forces on the brain . it is one of the leading causes of mortality in young adults with mortality rates in the usa and europe ranging between 15 and 20 per 100,000 per annum ( tagliaferri et al . this is due to increasing use of motorised vehicles , while in developed nations there is an increasing incidence of tbi in the older population due to falls ( roozenbeek et al . moreover , it is estimated that approximately 5.3 and 7.7 million people are living with disability due to tbi in the usa and europe respectively ( roozenbeek et al . the primary injurious event is followed in the hours and days after injury by secondary pathological processes that can exacerbate damage , and are thus targets for therapy . the management of tbi centres on the maintenance of adequate cerebral blood flow ( cbf ) and the delivery of sufficient glucose and oxygen to preserve tissue not irreparably damaged by the initial physical insult . thus , cerebral perfusion is maintained , hypoxia avoided , and with modern neurocritical care management , secondary ischaemia is not thought to play a major role . however , despite progress in management of tbi patients , many still suffer long - term or lifelong disabilities , thus placing heavy demands on carers and healthcare resources , and there is scope for better treatments ( kolias et al . disruption to cell membranes and a failure to maintain normal ionic homeostasis results in cytotoxic oedema , which further challenges membrane integrity ( unterberg et al . excitotoxic release of glutamate and other excitatory neurotransmitters exacerbate the disturbance of trans - membrane ionic gradients ( katayama et al . the generation of free radicals , highly reactive oxygen and nitrogen species , normally kept in check by cellular antioxidant systems , leads to the oxidative modification of proteins , lipids and dna ( hall et al . 1993 ; marklund et al . 2001 ) . this results in increased permeability of the inner mitochondrial membrane , release of cytochrome c leading to caspase - dependent apoptosis , and activation of poly - adp ribose polymerase ( parp ) , a nuclear dna repair enzyme that consumes nad , a coenzyme essential for glycolysis ( sullivan et al . although initially potentially reversible , these metabolic and mitochondrial perturbations , result in energy failure and activation of both apoptotic and necrotic pathways ( lewn et al . these and other , as yet uncharacterised changes may be important in tbi patients brains . the pathophysiology of energy failure after tbi is not yet fully understood but has important implications for how tbi patients are managed . improving our understanding of these metabolic disturbances will help lead to strategies that reduce secondary injury and improve outcomes . the metabolic perturbations that result from tbi have largely been studied using animal models of tbi ( andersen and marmarou 1992 ; xiong et al . 2013 ) . in general , for the purposes of metabolic studies , metabolites are measured and/or tracers are used to follow biochemical processes at set time points after experimental injury . many of the techniques used require the brain to be extracted and so are not applicable in clinical studies . animal models of tbi produce a relatively homogenous injury in comparison to clinical tbi and the majority of studies do not consider co - morbidities or other systemic factors that frequently complicate clinical tbi . patients with severe tbi commonly have injuries to other organs , such as chest and abdominal injuries , with consequent risk of systemic hypoxia and/or hypotension , which is deleterious to outcome ( chesnut et al . 1993 ; stocchetti et al . 1996 ) . the impact of sedative drugs and other pharmaceutical agents , which also likely influence cerebral metabolism , are also frequently overlooked . hence , we have to be careful in applying what we learn about glucose metabolism after tbi in the laboratory to what actually happens in clinical practice . for an in - depth review of experimental tbi models , see ( xiong et al . there are various methods for investigating metabolism after tbi in patients ( fig . 1 ) . measuring arterial , jugular and/or cerebrospinal fluid ( csf ) concentrations of metabolites provides a measure of how much the brain as a whole imports or exports . microdialysis , which samples extracellular fluid , is a focal technique that allows the immediate microenvironment of the brain to be sampled in vivo . microdialysis can also be used to deliver substances and in this way has been used to deliver glucose and other metabolites labelled with carbon-13 ( c ) . this is combined with ex vivo nuclear magnetic resonance ( nmr ) spectroscopy of the microdialysis - collected extracellular fluid yielding information on different intracellular pathways . autoradiography experiments in animals , and analogous positron emission tomography ( pet ) studies in humans , use glucose labelled with a radioactive tracer . . in vivo magnetic resonance spectroscopy ( mrs ) has also been used to measure the combined extra- and intracellular regional concentrations of lactate and high - energy phosphate compounds . our ability to study cerebral metabolism using these techniques importantly , there is also the potential for these techniques to help in the management of patients with tbi and improve clinical outcomes although their role in the clinical setting is still being defined.fig . 1methods for measuring glucose metabolism in the human brain : various techniques lend themselves to measuring glucose metabolism in the human brain . ( 1 ) microdialysis , which allows metabolites in the extracellular fluid to be measured , is an invasive technique requiring insertion of a microdialysis catheter ( possessing a semipermeable membrane , with nominal molecular weight cut - off typically 20 kda or 100 kda ) into brain parenchyma . a pump is used to infuse perfusion fluid at a low flow rate ( e.g. 0.3 l / min ) into the catheter and returned microdialysate is collected into small vials for subsequent analysis . ( 2 ) imaging techniques include positron emission tomography ( pet ) , which uses detectors to measure ionising radiation emitted from the glucose analogue fdg administered intravenously . ( 3 ) h and p magnetic resonance spectroscopy ( mrs ) can be used to measure lactate and high - energy phosphate compounds respectively , in different regions of the brain . ( 4 ) a global measure of uptake or release from the brain can be achieved by using a jugular bulb catheter , which allows the venous outflow of the brain to be sampled . measured concentrations in blood sampled from the venous catheter can be compared with arterial concentrations of metabolites , typically measured in blood taken from an arterial line . this enables the net uptake or release of metabolites by the brain to be calculated and , if cerebral blood flow is known , the cerebral metabolic rate to be calculated methods for measuring glucose metabolism in the human brain : various techniques lend themselves to measuring glucose metabolism in the human brain . ( 1 ) microdialysis , which allows metabolites in the extracellular fluid to be measured , is an invasive technique requiring insertion of a microdialysis catheter ( possessing a semipermeable membrane , with nominal molecular weight cut - off typically 20 kda or 100 kda ) into brain parenchyma . a pump is used to infuse perfusion fluid at a low flow rate ( e.g. 0.3 l / min ) into the catheter and returned microdialysate is collected into small vials for subsequent analysis . ( 2 ) imaging techniques include positron emission tomography ( pet ) , which uses detectors to measure ionising radiation emitted from the glucose analogue fdg administered intravenously . ( 3 ) h and p magnetic resonance spectroscopy ( mrs ) can be used to measure lactate and high - energy phosphate compounds respectively , in different regions of the brain . ( 4 ) a global measure of uptake or release from the brain can be achieved by using a jugular bulb catheter , which allows the venous outflow of the brain to be sampled . measured concentrations in blood sampled from the venous catheter can be compared with arterial concentrations of metabolites , typically measured in blood taken from an arterial line . this enables the net uptake or release of metabolites by the brain to be calculated and , if cerebral blood flow is known , the cerebral metabolic rate to be calculated the aim of this review is to summarise what we know about the metabolic perturbations that characterise tbi with a focus on clinical studies . specifically , we will focus on changes to glucose metabolism after moderate to severe tbi . the most noticeable feature is that the injured brain can exhibit a relative rise in non - oxygen - consuming glucose metabolism . with the increasing clinical availability of techniques that provide insight into glucose metabolism , such as microdialysis and mrs it is important to review the available evidence with a view to improving our ability to interpret this data.fig . 2glycolysis and the tricarboxylic acid cycle : glucose is the preferred substrate for the brain , although the brain can take up lactate , other monocarboxylic acids and ketone bodies under certain circumstances , for example , during the perinatal period . glycolysis ( also termed embden - meyerhof pathway ) is the series of reactions that results in the breakdown of glucose , generating pyruvate , adenosine triphosphate ( atp ) and nicotinamide adenine dinucleotide ( nadh ) . it is a truly fundamental pathway , found throughout nature and proceeds without the need for oxygen . the ten key enzymatic steps , in which 2 atp molecules are consumed early on but then paid back later with the generation of 4 atp molecules per glucose molecule , so the net production of atp is 2 molecules per molecule of glucose , takes place in the cytoplasm . there are three key regulatory points catalysed by the enzymes hexokinase , phosphofructokinase , and pyruvate kinase . pyruvate is converted to acetyl coa , which enters the tricarboxylic acid ( tca ) cycle , within mitochondria . the tca cycle results in the transfer of electrons ( from nadh and succinate ) , to electron transport chains ( etc . ) located in the inner mitochondrial membrane , which ultimately deposit on oxygen molecules . thus the tca cycle generates carbon dioxide ( also generated by the pyruvate dehydrogenase step prior to tca cycle ) and the etc generates water . the etcs pump protons across the inner mitochondrial membrane , maintaining a gradient of protons across the membrane . protons then flow down their concentration gradient , through atp synthetases ( atpase ) , resulting in the generation of atp , the cells widely used energy currency . glutamate spins off the tca cycle from -ketoglutarate ( kg ) , an intermediate of the tca cycle . there is a constant cycle of glutamate released during neurotransmission , retrieved from synaptic junctions by astrocytes and returned to neurons as glutamine glycolysis and the tricarboxylic acid cycle : glucose is the preferred substrate for the brain , although the brain can take up lactate , other monocarboxylic acids and ketone bodies under certain circumstances , for example , during the perinatal period . glycolysis ( also termed embden - meyerhof pathway ) is the series of reactions that results in the breakdown of glucose , generating pyruvate , adenosine triphosphate ( atp ) and nicotinamide adenine dinucleotide ( nadh ) . it is a truly fundamental pathway , found throughout nature and proceeds without the need for oxygen . the ten key enzymatic steps , in which 2 atp molecules are consumed early on but then paid back later with the generation of 4 atp molecules per glucose molecule , so the net production of atp is 2 molecules per molecule of glucose , takes place in the cytoplasm . there are three key regulatory points catalysed by the enzymes hexokinase , phosphofructokinase , and pyruvate kinase . pyruvate is converted to acetyl coa , which enters the tricarboxylic acid ( tca ) cycle , within mitochondria . the tca cycle results in the transfer of electrons ( from nadh and succinate ) , to electron transport chains ( etc . ) located in the inner mitochondrial membrane , which ultimately deposit on oxygen molecules . thus the tca cycle generates carbon dioxide ( also generated by the pyruvate dehydrogenase step prior to tca cycle ) and the etc generates water . the etcs pump protons across the inner mitochondrial membrane , maintaining a gradient of protons across the membrane . protons then flow down their concentration gradient , through atp synthetases ( atpase ) , resulting in the generation of atp , the cells widely used energy currency . glutamate spins off the tca cycle from -ketoglutarate ( kg ) , an intermediate of the tca cycle . there is a constant cycle of glutamate released during neurotransmission , retrieved from synaptic junctions by astrocytes and returned to neurons as glutamine arterio - venous ( av ) studies provided the first means for studying human cerebral energy metabolism . this was first achieved when kety and schmidt ( 1948 ) applied the fick principle to calculate cbf and cerebral oxygen consumption . the amount of substance that a tissue removes from or releases into the circulation is equal to the product of the blood flow to the tissue and the arterio - venous ( av ) concentration difference . using this principle , the steady - state rate of utilisation or production , known as the cerebral metabolic rate ( cmr ) , av studies investigating the cmr of glucose ( cmrglc ) after tbi demonstrate a depression in cmrglc indicating reduced glucose uptake and utilisation by the injured brain ( cruz 1995 ; leegsma - vogt et al . glenn et al . ( 2003 ) compared 49 tbi patients with 31 awake volunteers and used av difference measurements and a xenon-133 clearance technique to calculate cbf . a mean cmrglc of 4.46 1.16 mg/100 g / min was found in normal subjects whereas this was significantly lower in tbi patients who had a mean cmrglc of 3.43 2.32 mg/100 g / min during post - injury days 0 to 5 . alongside this global depression in glucose metabolism , they calculated the metabolic ratio ( mr ) by calculating cmro2/cmrglc ( in molar units ) and found that this was significantly lower in tbi patients compared to normal subjects . the mr in normal subjects was 5.83 1.41 , which demonstrates the efficiency of the brains aerobic metabolism of glucose . six moles of oxygen are needed for the oxidation of one mole of glucose hence a mr value close to 6 indicates almost complete aerobic metabolism of glucose . a mr value greater than 6 implies aerobic metabolism of other substrates , and less than 6 the anaerobic metabolism of glucose . other substrates potentially metabolised by the injured brain that would in theory account for an mr greater than 6 include lactate ( see below ) and ketone bodies ( prins and giza 2006 ) . further non - glucose substrates for brain could include fatty acids , although a recent review has argued against fatty acids being a good brain energy source ( schnfeld and reiser 2013 ) . in the tbi patients the mr was 4.11 2.11 , i.e. more moles of glucose metabolised per mole of oxygen , which indicates that there is proportionally greater anaerobic ( non - oxygen - consuming ) metabolism of glucose . in several patients a mr of less than 3.44 was observed and this was interpreted as indicating relative hyperglycolysis ; proportionally much greater anaerobic glucose metabolism than aerobic , although only a small proportion of samples ( 5 % ) demonstrated absolute hyperglycolysis with an elevated cmrglc . in a similar metabolic ratio calculation based on av difference of oxygen and glucose measurements in 69 tbi patients , which they termed the oxygen - glucose index ( ogi ) , holbein et al . ( 2009 ) found values mostly above 6 in 69 tbi patients suggesting maintained aerobic metabolism of glucose . however , higher arterial glucose levels were associated with greater anaerobic ( non - oxygen - consuming ) glucose metabolism . as well as perturbations to the relationship between cerebral glucose and oxygen metabolism , the metabolism of lactate is also affected by tbi . av measurements in healthy controls reveal a small net release of lactate from the brain into the circulation ( glenn et al . 2003 ; larsen et al . 2008 ) . however , several authors have revealed periods during which av measurements indicate uptake of lactate by the injured brain after tbi ( glenn et al . ( 2013 ) examined av gradients in 19 patients with diffuse tbi and found periods of lactate uptake in 17 of these patients . the likelihood is that , after tbi , lactate from the circulation is taken up by the injured brain and used as a metabolic substrate . supporting this concept , intravenous administration of c - lactate to a rodent tbi model demonstrated accumulation of radiolabel at the injury site ( chen et al . 2000 ) . moreover , in humans , lactate labelled with c and delivered to the brains of tbi patients by microdialysis is metabolised via the tca cycle to glutamine ( gallagher et al . av studies provide a measure of the uptake or release of substance by the whole brain at a single time point ; they lack the spatial and temporal resolution to explore how changes to glucose metabolism relate to other pathophysiological changes . moreover , given the heterogeneity of tbi this technique will not detect metabolic differences between injured and non - injured areas of brain . another important consideration when using this method is that jugular venous samples are not representative of the entire brain ; only one vein is sampled excluding contralateral and extra - jugular venous drainage ( ferris and engel 1946 ) . these limitations and the difficulty of obtaining reliable venous oxygen saturation measurements means that they are not used routinely in clinical practice ( pattinson et al . 2005 ) . microdialysis allows continuous in vivo sampling of molecules from the extracellular fluid from the human brain and permits the longitudinal analysis of certain energy - related small molecules . clinical microdialysis catheters are often inserted alongside brain tissue oxygen sensors such that changes in concentrations of small molecules related to energy metabolism can be monitored in conjunction with variations in tissue oxygen levels . metabolic features of microdialysates statistically associated with adverse outcomes after tbi include high concentrations of lactate and high lactate / pyruvate ratio , while for glucose the situation appears more complex with both high and low concentrations having adverse associations . patients with poorer outcomes after tbi have been observed to have lower average glucose concentrations for the monitoring period compared to those with better outcomes although the relationship is complex and high glucose concentrations are also associated with worse outcomes ( zauner et al . ( 2003 ) identified three patterns of daily mean glucose concentrations in 30 patients with severe tbi . then later declined as opposed to initially low at the start of their monitoring ( fig . they also found the lowest overall mean glucose in the patients that had the worse outcomes , although , no independent effect of low glucose was observed using a multivariate model with other known predictors . ( 2011 ) found that glucose gradually declines until the fourth day after the onset of monitoring and then increases . the largest investigation of tbi patients to date found that the total averaged monitoring glucose concentration was a positive predictor of mortality in a multivariate model such that higher concentrations of glucose were associated with mortality ( fig . hence , the likelihood is that there is an optimum range for cerebral glucose with both low and high glucose having been associated with worse clinical outcomes.fig . normal followed by declining mean microdialysate glucose concentrations after post - injury day 5 in those patients who went on to have a good outcome ( glasgow outcome scale extended ( gose ) 5 to 8) . figure originally published in the journal of cerebral blood flow & metabolism ( vespa et al . lower panel : pooled microdialysis glucose concentrations averaged by day of monitoring and split by outcome categories demonstrating declining median concentrations over the course of a week of post - injury monitoring . 2011a ) pattern of declining microdialysate glucose after tbi : upper panel : graph showing initially normal followed by declining mean microdialysate glucose concentrations after post - injury day 5 in those patients who went on to have a good outcome ( glasgow outcome scale extended ( gose ) 5 to 8) . figure originally published in the journal of cerebral blood flow & metabolism ( vespa et al . lower panel : pooled microdialysis glucose concentrations averaged by day of monitoring and split by outcome categories demonstrating declining median concentrations over the course of a week of post - injury monitoring . 2011a ) low brain glucose after tbi might be attributable to ischaemia , i.e. a deficiency in supply of oxygen , glucose and other blood - borne nutrients . nowadays , with modern protocol - driven therapy , the injured brain may however be at risk from micro - vascular ischaemia , failure of vascular autoregulation , elevated intracranial pressure , and systemic problems that result in hypoxia and/or hypotension ( chesnut et al . because of the confounding effects of a reduced conscious level and sedative drugs after tbi ischaemic thresholds can not simply be determined by cbf measurements . instead an increase in the oxygen gradient across the brain is used to indicate a relative supply failure . ( 2004 ) used oxygen-15 ( o ) pet to measure oxygen extraction fraction ( oef ) and to define ischaemic voxels . they based their oef ischaemia threshold on a venous oxygen threshold of 3.5 ml/100 ml and estimated the volume of ischaemic brain in 15 tbi patients within 24 h of tbi . they found ischaemic volumes of between 1 and 16 % of the total brain volume , particularly in voxels adjacent to contusions or haematomas . ( 2005 ) , using similar methodology and the same oef threshold in 19 patients found that the incidence of ischaemic voxels averaged only 0.14 0.28 % , although patients underwent pet imaging at later time points ; on average 60 30 h from injury . in the same study , using a different threshold based on an oef greater than 0.75 ( a value based on previous o pet studies performed in patients with ischaemic stroke ) they found the mean incidence of ischaemic voxels was 0.11 0.18 % with a maximum of 1 % of voxels in a single patient . furthermore , no significant correlation was found between microdialysis - measured brain glucose and pet measured cbf or oef . interestingly , in areas of brain deemed to be at risk of non - survival , defined as o pet - measured oxygen metabolism below a critical threshold , normobaric hyperoxia increases oxygen metabolism but does not have a consistent effect on microdialysis measures of metabolism ( nortje et al . although the incidence of frank ischaemia after tbi appears to be low , marked changes to cbf , induced by hyperventilation , or marked reductions in cerebral perfusion pressure ( cpp ) can affect brain glucose . hutchinson et al . ( 2002 ) combined triple oxygen pet with microdialysis and found no significant correlation between cbf and brain microdialysate glucose in 17 patients with tbi . however , during periods of hyperventilation , which significantly increased the oef , a significant reduction of brain glucose was observed . ( 2004 ) found that in a subset of 57 patients with tbi who experienced a marked reduction in cpp , due to high intracranial pressure ( icp ) or systemic hypotension , there was a fall in brain glucose . a reduction in brain glucose after tbi is partly a consequence of greater cellular uptake and utilisation , reflected by elevation of downstream metabolites . pyruvate and lactate are both derived from glucose and so their extracellular concentrations partly reflect glycolytic activity . in turn , the relative proportions of lactate and pyruvate , the lactate / pyruvate ratio , reflects the degree that glycolytically produced pyruvate is subsequently metabolised oxidatively within the mitochondria via the tca cycle or anaerobically in the cytosol to lactate . a greater lactate / pyruvate ratio indicates a greater proportion of glycolytic metabolism in relation to mitochondrial oxidative metabolism . ( 2011a ) in the largest study to date found the most consistent trends of all the microdialysis parameters measured after tbi were for brain lactate and the lactate / pyruvate ratio both of which were lower on a daily basis in patients with favourable outcome as compared with those patients with a poor outcome . moreover , the lactate / pyruvate ratio was found to be a significant positive predictor and pyruvate a significant negative predictor of mortality . the association of a higher lactate and higher lactate / pyruvate ratio with a poor outcome is a consistent finding across the tbi - microdialysis literature and is found in the most methodologically robust of studies that use patient - averaged data and multivariate statistical techniques ( timofeev et al . the association between high microdialysate lactate and poor outcome might seem , taken at face value , at odds with increasing evidence for lactate s role as an energy substrate . however , this apparent contradiction may be explained by uncoupling of neuronal and glial metabolism . animal studies have provided evidence for brain utilisation of intravenously administered 3-c lactate via the tca cycle ( tyson et al . 2003 ) . in human brain , direct evidence for utilisation of lactate via the tca cycle , in tbi patients , has been obtained in a cerebral microdialysis study in which 3-c lactate was infused directly into brain via the microdialysis catheter ( gallagher et al . a recent intravenous lactate supplementation study in tbi patients revealed evidence for a beneficial effect judged by surrogate endpoints ( bouzat et al . lactate has conventionally been regarded as a waste product of glucose metabolism , though a more recent idea is that neurons take up and metabolise lactate that has been generated by astrocytes . this has become known as the astrocyte - neuron lactate shuttle hypothesis ( pellerin and magistretti 1994 ) . 2011a ) , might be because astrocyte - derived glycolytic lactate is efficiently taken up by neurons and utilised via the tca cycle ( gallagher et al . conversely , where neurons are too damaged to utilise the lactate produced from glucose by astrocytes , i.e. uncoupling of neuronal and glial metabolism , high extracellular levels of lactate would accumulate , explaining one potential mechanism behind the association between high extracellular lactate and poor outcome ( carpenter et al . 2014 ) . in support of brain glucose concentrations being influenced by uptake and cellular utilization , a combined f - fluoro - deoxyglucose - pet ( fdg - pet ) and cerebral microdialysis study in tbi patients found that the cmrglc measurements in a 2 cm region of interest around the microdialysis catheter tip showed significant positive correlations of cmrglc with lactate and pyruvate concentrations , no relationship between cmrglc and l / p ratio , and a weak inverse trend for cmrglc with glucose concentrations in the microdialysates ( fig . thus low concentration of brain extracellular glucose seems a consequence of increased substrate demand rather than inadequate substrate delivery . a caveat is that at low brain glucose concentrations there is a risk of underestimating lumped constant and hence of overestimating cmrglc in such situations . the main conclusion of the study is that in tbi brain an increase in glucose metabolism leads to increases in both lactate and pyruvate , as opposed to a shift towards anaerobic metabolism . for further discussion of pet in the context of cerebral metabolism see autoradiography and positron emission tomography 4fdg - pet measurement of cmrglc and its relationship to brain microdialysate composition : a c fdg - pet cmrglc map demonstrating relatively high fdg uptake at sites of injury , in contrast to less injured areas of the brain . a computed tomography ( ct ) scan showing gold tip of microdialysis catheter ( indicated by arrow ) . b co - registered fdg - pet cmrglc map showing high fdg uptake at sites of injury . c overlay of ct and co - registered cmrglc map , showing microdialysis catheter tip location ( arrow ) . ( d f graphs illustrating relationships by linear regression ( for 22 rois in 17 tbi patients ) between fdg - pet derived cmrglc and the microdialysis parameters measured during the scan ( d ) lactate , ( e ) pyruvate , ( f ) lactate / pyruvate ( l / p ) ratio , and ( g ) glucose . for the linear regressions in ( d g ) , corresponding values of p ( anova ) data - points from catheters at craniotomy sites ( 4 patients ) are differentiated by grey triangles . data - points from a second fdg - pet scan ( one patient ) are differentiated by grey diamonds . all other data - points are depicted as black circles ( catheters inserted via cranial access device ) . linear regressions presented on the graphs are for the entire ( combined black plus grey symbols ) dataset consisting of all 22 rois . figure originally published in acta neurochirurgica ( hutchinson et al . 2009 ) fdg - pet measurement of cmrglc and its relationship to brain microdialysate composition : a c fdg - pet cmrglc map demonstrating relatively high fdg uptake at sites of injury , in contrast to less injured areas of the brain . a computed tomography ( ct ) scan showing gold tip of microdialysis catheter ( indicated by arrow ) . b co - registered fdg - pet cmrglc map showing high fdg uptake at sites of injury . c overlay of ct and co - registered cmrglc map , showing microdialysis catheter tip location ( arrow ) . ( d f graphs illustrating relationships by linear regression ( for 22 rois in 17 tbi patients ) between fdg - pet derived cmrglc and the microdialysis parameters measured during the scan ( d ) lactate , ( e ) pyruvate , ( f ) lactate / pyruvate ( l / p ) ratio , and ( g ) glucose . for the linear regressions in ( d g ) , corresponding values of p ( anova ) data - points from catheters at craniotomy sites ( 4 patients ) are differentiated by grey triangles . data - points from a second fdg - pet scan ( one patient ) are differentiated by grey diamonds . all other data - points are depicted as black circles ( catheters inserted via cranial access device ) . linear regressions presented on the graphs are for the entire ( combined black plus grey symbols ) dataset consisting of all 22 rois . figure originally published in acta neurochirurgica ( hutchinson et al . 2009 ) brain glucose reflects systemic glucose levels , glycaemic control and the use of insulin in tbi patients . several studies have explored the relationship between systemic glucose and brain microdialysis parameters in tbi patients and found that higher arterial glucose concentrations are associated with higher brain glucose concentrations although this relationship may be lost when the brain is injured ( vespa et al . ( 2010 ) observed that arterial glucose concentrations greater than 6 mmol / l were associated with both higher brain microdialysate glucose and glutamate concentration . the relationship between systemic glucose and cerebral metabolism has also been explored using av measures of metabolism . holbein et al . ( 2009 ) observed increased glucose uptake , decreased lactate export and reduced oxygen consumption by the brain at higher arterial glucose concentrations . the retrospective nature of these studies limits our ability to be clear about how changes in systemic glucose concentrations affect cerebral metabolism or to clearly define an optimal systemic glucose concentration for tbi patients . in a prospective study , 2012 ) randomised 13 tbi patients using a within - subject crossover design to tight ( 80110 mg / dl ) versus loose ( 120150 mg / dl ) glycaemic control and assessed metabolism using both microdialysis and fdg - pet . they observed that microdialysate glucose , pyruvate and lactate were significantly lower , that more time was spent with a high lactate / pyruvate ratio ( > 28 ) , and that fdg - pet indicated increased glucose metabolism ( although in a heterogeneous manner depending on the patient and injury pattern ) when tight glycaemic control was used . hence , loose glycaemic control was interpreted as being preferable for cerebral metabolism . in summary , microdialysis parameters can be difficult to interpret due to the various factors that influence the extracellular concentrations of metabolites . trends of values and ratios , as opposed to single values , are more clinically useful in predicting clinical outcomes and for guiding treatments . moreover , microdialysis is a focal technique limited to sampling a small area of brain . data from microdialysis should be interpreted with this in mind ; peri - contusional brain expresses a different metabolic profile to macroscopically normal brain . however , the relative ease of placement , safety and ability to follow trends in brain chemistry over extended periods of time has resulted in the adoption of microdialysis as a clinical monitoring tool in many neurocritical care units . autoradiography and pet imaging studies that use labelled 2-deoxy - d - glucose ( 2dg ) as a tracer allow the in vivo delivery , uptake and initial metabolism of glucose to be assessed both globally and regionally in the brain . ( 1977 ) provided some of the first experimental techniques for interrogating regional changes in cerebral metabolism in vivo . they used 2-deoxy - d-[c]glucose as a tracer ( [ c]dg ) , which is taken up from the circulation and phosphorylated by hexokinase in the first stage of glycolysis . [ c]dg , following phosphorylation , does not continue in the next and subsequent steps of glycolysis , essentially remaining trapped . ( 1977 ) injected awake and anaesthetised rats with the tracer and used quantitative autoradiography of brain sections to calculate regional rates of glucose metabolism . pet is a tomographic technique that involves measuring the accumulation of radiolabel in tissue following injection of labelled compound into the circulation . for the purposes of exploring regional glucose metabolism , the patient is injected with deoxy - glucose , labelled with the positron emitter f ( fdg ) ( phelps 1991 ) . the emitted positrons are measured using a ring - shaped array of detectors ( scintillator crystals and photomultipliers ) and then algorithms used to reconstruct the location and local concentration of radioisotopes . rate constants relating to the movement of tracer between the circulation , extracellular space and the intracellular compartment are used with the plasma glucose concentration and a lumped constant to give a measure of the metabolic rate of glucose . the lumped constant ( lc ) of sokoloff et al . ( 1977 ) accounts for the differences in transport and phosphorylation rates between d - glucose and 2dg . 2dg is more readily transported into the cell whereas glucose is more readily phosphorylated by hexokinase . however , there is no universal value for lc and values may differ between centres or even within - centre at different times . while many studies have assumed that the lc is uniform over the whole brain and in different states of disease or conscious level , this is not necessarily the case . 2009 ) demonstrated regional differences in the uptake of c - glucose relative to 2dg in rats after experimental tbi , suggesting regional differences in lc , and wu et al . ( 2004 ) showed that the global lc value was significantly lower in tbi patients compared with normal controls . furthermore , various mathematical models have been used to calculate cmrglc from the pet data . these include the huang autoradiographic model and dynamic methods such as patlak graphical analysis and full kinetic modelling with 3 or 4 rate constants ( termed 3 k or 4 k methods ) ( huang et al . the different models apply different rate constants , which are either pre - defined ( standard , as in the huang method ) or estimated from the fdg - pet data ( as in the dynamic methods ) . another approach is statistical parametric mapping to compare fdg uptake in different areas of the brain without calculating absolute cmrglc values ( zhang et al . hence , it is not necessarily straightforward to compare fdg - pet quantitative results between studies performed in different centres , or within the same centre at different times . autoradiography and pet studies in animals have demonstrated a stereotyped acute period of high glucose metabolism followed by longer lasting metabolic depression . this acute period of hypermetabolism is observed in animal models for up to 6 h , and affects both hemispheres but more so on the ipsilateral hemisphere ( sunami et al . following this acute period of hypermetabolism , a depression in glucose metabolic activity ensues that may last several weeks . this appears to be widespread throughout various regions of the brain and has been found to be associated with neurological outcome in clinical studies ( kato et al . most clinical reports demonstrate a depression of glucose metabolism following tbi when compared to control subjects with only discrete areas of supra - normal glucose metabolism , particularly in relation to mass lesions ( fig . 4 ) . ( 2000 ) observed that 20 of 26 ( 77 % ) patients with a range of tbi severities within seven days post - injury demonstrated a cmrglc in cortical grey matter of the whole brain at least two standard deviations lower than a value for cmrglc derived from a historical control group ( 7.3 1.2 mg/100 g / min ) . there was no clear correlation between the severity of injury and cmrglc values although patients that were on dual sedative drugs ( morphine and a benzodiazepine ) exhibited lower cmrglc values than those on a single sedative . some peri - contusional cortical grey matter areas demonstrated markedly raised cmrglc values compared to the control value whereas others demonstrated depressed cmrglc . a later study from the same group , which used aged - matched controls and lc values calculated from a cohort of subjects who underwent additional arterio - venous 2dg gradient measurements , used to estimate a global lc value , found significantly lower cmrglc in the whole brain , striatum and thalamus in tbi patients compared to control subjects ( hattori et al . whole brain cmrglc in control subjects in this study was 3.8 0.7 mg/100 g / min , which was considerably lower than the value previously calculated from a historical cohort study . although glucose metabolism in tbi patients is found to be relatively depressed compared to awake and non - sedated control subjects , pet studies suggest that glucose metabolism in tbi is increased relative to the rate of oxygen utilisation . in a subset of six patients undergoing fdg - pet bergsneider et al . ( 1997 ) used a combination of arterio - venous oxygen measurements and intravenous xenon-133 cbf measurements to calculate both oxygen and glucose metabolism . in all six of these patients , who underwent fdg - pet imaging between days 2 and 8 post - injury , glucose metabolism wu et al . ( 2013 ) combined triple - oxygen ( o ) pet , used to measure regional oxygen utilisation , with fdg - pet to explore the relationship between glucose and oxygen metabolism in contusional and peri - contusional regions in eight tbi patients . cmrglc in contusional and peri - contusional regions was depressed relative to cmrglc measured in the surrounding normal - appearing white matter of the same patient . moreover , oxygen metabolism was even more depressed suggesting a shift in glucose metabolism from oxygen consuming to anaerobic metabolism . the changes in glucose metabolic activity observed with deoxy - glucose studies relate to both the rate of glucose transport into cells and the rate of phosphorylation by hexokinase . whether the changes in glucose metabolic activity that follow tbi are caused by changes in glucose transport , changes in hexokinase activity or are due to a combination of both changes in transport and hexokinase activity is not readily appreciated by autoradiography studies . manipulating the mathematical models used in order to try and separate out these different aspects suggests that glucose transport activity is impaired in peri - contusional areas only whereas hexokinase activity is impaired more globally ( hattori et al . 2004 ) . whether blood brain barrier ( bbb ) permeability impacts significantly on glucose metabolism in tbi patients is as yet unknown . a leaky bbb might conceivably allow more circulating glucose to enter the brain , while damaged cells might have less efficient specific transporters and/or exhibit a more glycolytic phenotype . in a microdialysis comparison of sites in tbi patients brains , glucose had a tendency to be lower in peri - lesional sites than in less - injured sites , while lactate / pyruvate ratio , lactate concentration and pyruvate concentration were all significantly higher in peri - lesional sites than in less - injured brain ( timofeev et al . although this was a large study ( 97 patients ) , a limitation of the statistical analysis was that catheters were not paired within - patient . a much smaller fdg - pet and microdialysis combined study included a subset of four patients with paired catheters ( hutchinson et al . the peri - lesional sites had higher pyruvate and higher regional cmrglc ( together with a tendency towards higher lactate ) than in the corresponding less - injured sites , while glucose and lactate / pyruvate ratio were not significantly different between sites , albeit in this very small subset of patients . more patients need to be studied for site - dependent differences , which may shed light on effects of bbb within the injured brain . the infrastructure required to generate pet ligands as well as the ionising nature of the radiation limits their widespread use as a clinical tool . furthermore , fdg - pet provides information on the uptake of glucose and on the first part of glycolysis but does not evaluate glucose metabolism beyond this . applying a tracer to glucose that can then be identified in downstream metabolites derived from glucose allows the metabolic fate of glucose to be evaluated . classically , this was achieved with radioisotope labelling using carbon-14 ( c ) . due to the ionising nature of c moreover , most investigators using c - glucose have identified the labelled metabolites in extracted and purified brains . carbon-13 ( c ) is a naturally occurring stable isotope with magnetic properties that can be detected by nuclear magnetic resonance ( nmr ) spectroscopy and by gas chromatography mass spectrometry ( gc - ms ) . the ability of nmr to identify not only the metabolite but also the precise intra - molecular position(s ) of c means that the relative activity of metabolic pathways can be determined from the particular labelling patterns observed in metabolites . see fig . 5 for an example of labelling patterns observed after administration of 1-c glucose and 3-c lactate.fig . 5carbon-13 labelling patterns after administration of 1-c glucose : when the first carbon of glucose ( c1 ) is c , the resulting pyruvate maintains the c as the third carbon ( c3 ) . glycolysis converts one molecule of glucose into two molecules of pyruvate and so only one of the two 3-carbon chain length pyruvate molecules will contain c. pyruvate can be converted to lactate , by the action of lactate dehydrogenase . alternatively , through the action of pyruvate dehydrogenase on pyruvate , acetyl - coa is produced containing just a 2-carbon backbone with one of the carbons ( c1 , the carboxylate carbon of pyruvate ) lost as carbon dioxide . the 2 carbons of acetyl - coa are then condensed with oxaloacetate and continue in the tca cycle , ultimately emerging as amino acid spin - offs or as carbon dioxide . the main products identifiable with nmr ( in animal brain tissue extracts ex vivo ) or mrs ( in human or animal brain in vivo ) include glutamine , glutamate , gaba and aspartate . the c1 of a glucose molecule will become the c4 of glutamate and glutamine on the first turn of the tca cycle . subsequent turns of the tca cycle will result in the c label shifting to the c3 or c2 of glutamate and glutamine . hence , it is possible to determine , through the relative enrichment of the c4 , c3 , or c2 carbons , the proportion of glutamate or glutamine produced on the first turn of the tca and subsequent cycles . in this way , c - nmr provides a convenient way to study the transformation of the carbon skeleton that takes place during the metabolism of glucose . the relative proportions of c incorporation at the different carbon positions of the relevant metabolites have been used in numerous studies to compare pathway activities . this schematic diagram is based on results obtained with singly labelled substrates , by gallagher et al . ( 2013 ) abbreviations : pdh pyruvate dehydrogenase , pc , pyruvate carboxylase , me malic enzyme carbon-13 labelling patterns after administration of 1-c glucose : when the first carbon of glucose ( c1 ) is c , the resulting pyruvate maintains the c as the third carbon ( c3 ) . glycolysis converts one molecule of glucose into two molecules of pyruvate and so only one of the two 3-carbon chain length pyruvate molecules will contain c. pyruvate can be converted to lactate , by the action of lactate dehydrogenase . alternatively , through the action of pyruvate dehydrogenase on pyruvate , acetyl - coa is produced containing just a 2-carbon backbone with one of the carbons ( c1 , the carboxylate carbon of pyruvate ) lost as carbon dioxide . the 2 carbons of acetyl - coa are then condensed with oxaloacetate and continue in the tca cycle , ultimately emerging as amino acid spin - offs or as carbon dioxide . the main products identifiable with nmr ( in animal brain tissue extracts ex vivo ) or mrs ( in human or animal brain in vivo ) include glutamine , glutamate , gaba and aspartate . the c1 of a glucose molecule will become the c4 of glutamate and glutamine on the first turn of the tca cycle . subsequent turns of the tca cycle will result in the c label shifting to the c3 or c2 of glutamate and glutamine . hence , it is possible to determine , through the relative enrichment of the c4 , c3 , or c2 carbons , the proportion of glutamate or glutamine produced on the first turn of the tca and subsequent cycles . in this way , c - nmr provides a convenient way to study the transformation of the carbon skeleton that takes place during the metabolism of glucose . the relative proportions of c incorporation at the different carbon positions of the relevant metabolites have been used in numerous studies to compare pathway activities . this schematic diagram is based on results obtained with singly labelled substrates , by gallagher et al . ( 2013 ) abbreviations : pdh pyruvate dehydrogenase , pc , pyruvate carboxylase , me malic enzyme c - labelled substrates have been delivered to the brain intravenously , or directly into the brain extracellular fluid using microdialysis . for an in - depth review see carpenter et al . hyperglycolysis observed in studies using av measurements or fdg - pet relate to actual increased glycolysis or are due to the metabolism of glucose by other alternative pathways , and have also investigated the metabolism of other substrates by the brain such as lactate and acetate ( gallagher et al . c - labelled glucose administered intravenously to cci and fpi rat injury models of tbi , analysed by c nmr of brain tissue extracts , suggest that glucose is metabolised differently after tbi with up regulation of the pentose phosphate pathway ( ppp ) ( kaibara et al . it results in the production of ribose sugars , needed for dna synthesis and repair , and the nadph generated by the ppp also acts to maintain glutathione in a reduced state , which serves a vital antioxidant role ( herrero - mendez et al . ( 2007 ) used intravenously delivered 1,2-c2 glucose and used gc - ms to measure the c - labelled products in arterial and jugular venous blood , in six tbi patients and six healthy controls . their arterio - venous difference analysis of c - labelled lactate , differently labelled depending on whether it was derived by either glycolysis or ppp supported the preclinical finding of ppp up - regulation after tbi . this includes spectra showing c signals for glutamine c4 after delivery of c - lactate to the brains of tbi patients indicating tca cycle operation followed by conversion of glutamate to glutamine , which demonstrates that the injured brain is able to utilise lactate as a metabolic fuel ( gallagher et al . 6illustrative c - nmr spectra achieved by ex vivo nmr of microdialysate after delivery of 3-c lactate and 1,2-c2 glucose to tbi patients : upper panel : a and b examples of c nmr spectra of brain microdialysates from a tbi patient , who received perfusion with 3-c lactate ( 4 mm ) by microdialysis catheters via a craniotomy ( cto ) ; red stars indicate c signals for glutamine c4 , c3 and c2 indicating metabolism via tca cycle . d c nmr spectrum of brain microdialysate from an unlabelled patient . for further details , lower panel : examples of c nmr spectra of brain microdialysates from patients who received 1,2-c2 glucose ( 4 mm ) perfused via the microdialysis catheter . uninjured brain is normal - appearing brain in a patient operated on for a benign tumour elsewhere in the brain . the part of the spectrum illustrated in each case is for the c3 carbon of lactate . also present in this part of the spectrum is one of the signals due to the internal standard dss ( 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt ) . the remainder of the spectrum ( including the main dss signal at 0 ppm ) is not shown . the c3 doublet indicated by red stars represents lactate doubly labelled with c , produced by glycolysis ; the c3 signal for c is split into 2 peaks by coupling to c also present at the neighbouring c2 position within the same molecule . the c3 singlet indicated by green stars represents lactate singly labelled with c , produced via the ppp . 2014 ) illustrative c - nmr spectra achieved by ex vivo nmr of microdialysate after delivery of 3-c lactate and 1,2-c2 glucose to tbi patients : upper panel : a and b examples of c nmr spectra of brain microdialysates from a tbi patient , who received perfusion with 3-c lactate ( 4 mm ) by microdialysis catheters via a craniotomy ( cto ) ; red stars indicate c signals for glutamine c4 , c3 and c2 indicating metabolism via tca cycle . c c nmr spectrum of the 3-c lactate substrate solution prior to perfusing . d c nmr spectrum of brain microdialysate from an unlabelled patient . for further details , lower panel : examples of c nmr spectra of brain microdialysates from patients who received 1,2-c2 glucose ( 4 mm ) perfused via the microdialysis catheter . uninjured brain is normal - appearing brain in a patient operated on for a benign tumour elsewhere in the brain . the part of the spectrum illustrated in each case is for the c3 carbon of lactate . also present in this part of the spectrum is one of the signals due to the internal standard dss ( 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt ) . the remainder of the spectrum ( including the main dss signal at 0 ppm ) is not shown . the c3 doublet indicated by red stars represents lactate doubly labelled with c , produced by glycolysis ; the c3 signal for c is split into 2 peaks by coupling to c also present at the neighbouring c2 position within the same molecule . the c3 singlet indicated by green stars represents lactate singly labelled with c , produced via the ppp . 2014 ) the relatively novel combination of c - labelled substrate delivered by microdialysis with ex vivo nmr has some important advantages over other methods of studying metabolism in the clinical setting . firstly , the technique has proved to be safe and easy to use in ventilated sedated tbi patients and does not require the transfer of the patient to a scanner ( gallagher et al . it is relatively inexpensive ; only small amounts of c substrates ( typically less than 1 g ) are required to manufacture sufficient microdialysis perfusion fluid to study tens of patients . furthermore , this technique allows the downstream metabolism of glucose to be evaluated beyond that previously afforded by arterio - venous studies and pet . the intravenous delivery of exogenous c - labelled substrates can be combined with in vivo magnetic resonance spectroscopy ( mrs ) to investigate cerebral metabolism in humans , although this technique has not yet been applied to tbi patients . in vivo c - mrs , performed on a conventional mri scanner with specialised coils , has been used to follow the rate of change of fractional enrichment of a metabolic product , performed in real time , after intravenous delivery of the substrate . in this way , infusing 1-c glucose and measuring the rate of fractional enrichment for glutamate c4 has been used in metabolic modelling data analysis to calculate tca cycle kinetics in healthy volunteers ( mason et al . the extension to tbi patients of such in vivo c mrs methodology will enable calculation of rates of metabolism beyond that previously afforded by fdg - pet , which only provides kinetic data on glucose uptake and phosphorylation , and will thus permit more in - depth evaluation of metabolism after tbi . the tca cycle is inherently mitochondrial , and the above c - labelling strategies with measurements by ex vivo high resolution nmr and in vivo mrs are thus relevant to mitochondrial activity in the human brain . in particular , microdialysis studies with c - labelled lactate and acetate have already demonstrated tca cycle utilisation of these substrates in tbi patients brains ( gallagher et al . 2009 ) . an important implication of these results is that mitochondria are evidently still active in injured brain , even in sites close to focal lesions . invoked , the mitochondria are certainly functioning , although there is obviously much scope for debate about how healthy or otherwise they are . also relevant in this context are results from a combined fdg - pet and microdialysis study in 17 tbi patients ( hutchinson et al . , there was significant positive linear relationship between cmrglc and levels of lactate ( r = 0.778 , p < 0.0001 ) and pyruvate ( r = 0.799 , p the results imply that therapeutic strategies aimed at supporting or rescuing mitochondria may be helpful in tbi patients . possibly relevant are experimental tbi studies in which cyclosporin a , which inhibits opening of the mitochondrial permeability transition pore ( mptp ) , restored mitochondrial membrane potential , reduced tissue damage and improved neurological outcomes ( scheff and sullivan 1999 ; alessandri et al . 2002 ) . very recently , another experimental tbi study reported neuroprotective effects with improved behavioural outcomes for n - acetylcysteine amide ( naca ) , apparently due to maintenance of mitochondrial glutathione and mitochondrial bioenergetics , and to reduction in oxidative damage ( pandya et al . these or other mitochondrial support and rescue strategies may prove useful in future development of novel therapies for tbi . we have already mentioned ( see above ) the relevance of c labelling to address the tca cycle in clinical studies of tbi patients . in addition , p in vivo mrs ( discussed in proton & phosphorus magnetic resonance spectroscopy section below ) has the potential to be useful in future clinical trials of drugs directed at improving mitochondrial function by assessing the response of cellular atp levels to treatment . magnetic resonance spectroscopy ( mrs ) is an in vivo technique carried out on suitably equipped mri scanners . mrs was developed from the principles of nmr and can identify metabolites relevant to disordered energy metabolism in voxels and regions of interest ( rois ) defined on the mri scan . the most commonly used nucleus is the proton ( h ) ; however other nuclei that possess nuclei spin values of 1/2 , and so in essence behave like miniature bar magnets , can also be used in mrs . h and phosphorus ( p ) mrs have been used to interrogate energy metabolism after tbi . an important advantage of h and p mrs techniques is their ability to yield biochemical information non - invasively without the need for tracers . different localisation techniques are used to produce spectra that represent either a broad region of the brain ( e.g. a hemispherical roi relative to the plane of a surface coil ) or individual voxels ( e.g. if a head - coil is used ) . the sensitivity of h mrs is sufficient to allow the identification of n - acetylaspartate ( naa , typically the largest signal ) , choline - containing phospholipids , creatine and phosphocreatine , lactate , myo - inositol , glutamate , gaba , and lipids . the appearance of signals in h mrs is influenced by choice of echo time ( te ) . in particular , lactate signals are can appear positive , disappear or appear inverted depending on the duration of te . typically , in h mrs levels of lactate and other metabolites are expressed as ratios to creatine ; absolute and accurate quantification of metabolite signals is more difficult ( marino et al . the most relevant h mrs metabolite for demonstrating perturbed energy metabolism after tbi is lactate . increases in lactate have been demonstrated after tbi particularly in children . in normal controls , a lactate peak is not readily detectable . in a study of 51 infants and children with tbi , 91 % of infants and 80 % of children with a poor outcome demonstrated a lactate mrs peak in the grey matter of the occipital lobe ( ashwal et al . a later study also showed that the presence of a lactate peak , this time with the mrs voxel located in the corpus callosum and frontal white matter , was predictive of a poor outcome in children ( aaen et al . the ability to detect lactate with mrs after tbi in children has not been consistently replicated in studies of adult patients . several studies that demonstrate other markers of disordered biochemistry such as a reduced naa / creatine ratio and an increased choline / creatine ratio did not find a detectable lactate signal in their patients ( cecil et al . this may be due to the particular echo times used or the timing post - injury of the mrs scan . in support of this , mrs performed within 72 h of tbi was able to demonstrate a lactate peak in five of ten patients and this correlated with gcs at presentation and clinical outcome ( marino et al . adenosine triphosphate ( atp ) an important end - point of cellular energy metabolism , and phosphocreatine ( pcr ) , a readily mobilised store of high - energy phosphate , are compounds that can be measured in vivo using p mrs . when mitochondrial atp synthesis is running normally , the pcr store is well stocked ; when atp synthesis is struggling to meet demand the pcr store runs down . p mrs has been used to determine the molar ratio of pcr to inorganic phosphate ( pcr / pi ) and the pcr / atp ratio as indicators of oxidative phosphorylation status and mitochondrial function ( eleff et al . both pcr / pi and pcr / atp ratios are notionally easier to measure than absolute concentrations and have been measured in both chronic and acute tbi patients ( cadoux - hudson et al . garnett et al . ( 2001 ) found increased pcr / pi ratio and reduced pi / atp in predominantly normal appearing white matter of seven patients scanned between 2 and 21 days after severe tbi suggesting adequate mitochondrial energy metabolism . however , animal studies using p mrs after fpi or weight - drop model of diffuse injury reveal a decline in pcr / pi suggestive of a failure of mitochondrial function ( vink et al . 1987 ; heath and vink 1995 ) . more studies are needed to determine whether the differences between clinical and experimental findings are due to differences in ph , free intracellular magnesium concentrations , or cellularity , all of which might alter the measurement of phosphate compounds using p mrs . understanding cerebral energy metabolism is complicated by a series of specialisations that are unique to the brain . the brain exists in its own unique chemical environment , shielded by the blood brain barrier ; it has high demands for energy and is vulnerable to reduced substrate provision . unlike other organs , there are limited glycogen stores and the energy generating pathways are intrinsically linked to its main function of neurotransmission . increasingly recognised are the functional differences in how the two main cell types , neurons and astrocytes , use glucose . pet studies , both alone and in combination with microdialysis , suggest that ischaemia , when it occurs , is only detectable to any great extent in the first few hours after tbi or when cbf is challenged by hyperventilation or marked falls in cpp . more relevant is a change in the cellular utilisation of glucose with an increase in the proportion of glucose that is anaerobically metabolised . more studies are needed to determine whether this simply serves an increased requirement for a rapid supply of energy , maintaining the availability of high - energy phosphate compounds , or whether greater amounts of glucose are used for non - energy generating , reparative and biosynthetic roles such as the ppp . along with a change in the metabolic role of glucose , lactate appears to become important as a metabolic substrate , imported from the circulation and oxidatively metabolised via the tca cycle . the methods discussed in this review are not yet employed widely in the clinical setting . choice of technique is inevitably dictated by what is available locally . in the present review we have gathered together findings from several technologies , and have discussed in each of the above sections what the particular technique is good for , and any limitations and caveats . debate still exists on how best to integrate results from multiple techniques , including how to fit together snapshot techniques ( such as scans ) with data collected over an extended timescale of days ( e.g. microdialysis ) , how to integrate global , regional and focal measures , and intracellular vs. extracellular information . amalgamation of these diverse components may emerge from advances in data handling methods , including multivariate methods and machine learning technologies , when applied to the large databases that are growing in specialist centres and as a result of multicentre collaborations.table 1summary of methodology for measuring glucose metabolism in human braintechniqueextent of measurementtimeframe ( and frequency)invasive?examples in human brainarterio - venous differenceglobalmulti - day ( sampling twice daily)yes ( insertion of arterial line and jugular venous catheter)net changes ( import or export ) by brain for glucose and lactate ( jalloh et al . 2013)microdialysisfocalmulti - day ( hourly vial changes)yes ( insertion of catheter into brain)brain extracellular concentrations of small molecules ( e.g. glucose , lactate , pyruvate , glutamate and glycerol ) ( timofeev et al . 2011a).petglobal and regionalusually single scan session ( < 1 h ) , sometimes repeated after a few daysyes ( i.v . injection of radioactivity with short half - life)regional cerebral metabolic rate of glucose ( cmrglc ) or oxygen ( cmro2 ) ( hutchinson et al . h mrsregional and voxelusually single scan session ( < 1 h ) , sometimes repeated after a few daysnosmall molecules in brain tissue ( e.g. naa , creatine , choline , myo - inositol , glutamate and glutamine , gaba , lactate ) ( marino et al . 2007 ) p mrsregional and voxelusually single scan session ( < 1 h ) , sometimes repeated after a few daysnophosphorus - containing small molecules in brain tissue ( e.g. atp , phosphocreatine , inorganic phosphate ) and brain intracellular ph ( hamilton et al . 2006 ) . 2 h)moderately ( i.v . bolus and infusion of stable - isotope c - labelled substrate e.g. glucose ) c - labelling in metabolites ( glutamate and glutamine ) for calculating tca cycle rate , other c - labelled species also detectable ( e.g. gaba , aspartate , naa ) ( rothman et al . c - labelled microdialysisfocaltypically 24 h ( 24 1 h vials pooled)yes ( insertion of catheter into brain , perfused with solution of stable isotope c - labelled substrate e.g. glucose , lactate , or acetate ) c- labelling patterns in metabolites ( e.g. glutamine or lactate ) diagnostic for biochemical pathways such as tca cycle , glycolysis , ppp ( gallagher et al . abbreviations : atp adenosine triphosphate , cmr glc cerebral metabolic rate of glucose , cmro 2 cerebral metabolic rate of oxygen , gaba gamma - aminobutyric acid ; i.v . intravenous , mrs magnetic resnance spectroscopy , naa n - acetylaspartate , pet positron emission tomography , ppp pentose phosphate pathway cmro2 measurement by pet additionally involves inhalation of radioactive ( short half - life ) gases summary of methodology for measuring glucose metabolism in human brain abbreviations : atp adenosine triphosphate , cmr glc cerebral metabolic rate of glucose , cmro 2 cerebral metabolic rate of oxygen , gaba gamma - aminobutyric acid ; i.v . intravenous , mrs magnetic resnance spectroscopy , naa n - acetylaspartate , pet positron emission tomography , ppp pentose phosphate pathway cmro2 measurement by pet additionally involves inhalation of radioactive ( short half - life ) gases the precise biological questions to be addressed need to be appropriate to the techniques available . mrs and pet are suited to observing regional changes in metabolism and can demonstrate changes associated with structural lesions such as contusions . currently , pet is limited by the availability of pet ligands , while mrs is limited by mr technology , improvements in which will lead to greater spatial resolution and the ability to interrogate all areas of the brain including deep structures such as the brain stem . this offers the potential to better characterise the injured brain , assisting with outcome prediction , and helping to direct clinical and rehabilitation management decisions . however , transferring patients from neurocritical care wards for pet or mrs scans is not a risk free undertaking , limiting the serial use of these investigations , so the time - variant aspect of such data is relatively unexplored and these scanning techniques are effectively snapshots for the duration of the scan . moreover , only a few centres worldwide have scanners adjacent to neurocritical care wards and the necessary expertise for fully ventilated patients to routinely receive pet and mri scans . moreover , the relative ease of obtaining data in the early stages following injury means they are particularly well placed to direct interventions and guide treatment . in particular , brain chemistry monitored by microdialysis has been demonstrated in large numbers of patients to relate statistically to clinical outcome and there is increasing evidence of how it is influenced by different therapeutic interventions . hence , microdialysis is well placed to monitor glucose delivery to the brain , assist in the management of systemic glucose levels , and optimise cpp . future clinical studies are needed to establish the role of microdialysis - directed interventions alongside icp / cpp - guided management . the current aims of tbi management focus on icp / cpp - guided management to maintain adequate cerebral perfusion . the development of methods that sense metabolic changes means that we can tailor our interventions to maintain sufficient energy metabolism and ultimately limit the natural end - point of metabolic failure , cell death . the ability to monitor the changing metabolism of glucose after tbi raises interesting questions about the potential to manipulate these different pathways and reduce secondary injury . multimodality monitoring , including microdialysis measures of brain chemistry , simultaneously with local pbto2 , together with icp and derived pressure parameters such as cpp and prx , is not only useful in standard neurocritical care to assist in patient management but is also a research approach that is being utilised to explore metabolic changes in the injured brain . multimodality continuous monitoring is complemented by specialised scanning techniques ( including pet , mri and mrs ) , which provide a snapshot of the brain . the ability to understand and modulate glucose metabolism in the brain is likely to be a crucial factor in achieving the best clinical outcomes for tbi patients .

the pathophysiology of traumatic brain ( tbi ) injury involves changes to glucose uptake into the brain and its subsequent metabolism . we review the methods used to study cerebral glucose metabolism with a focus on those used in clinical tbi studies . arterio - venous measurements provide a global measure of glucose uptake into the brain . microdialysis allows the in vivo sampling of brain extracellular fluid and is well suited to the longitudinal assessment of metabolism after tbi in the clinical setting . a recent novel development is the use of microdialysis to deliver glucose and other energy substrates labelled with carbon-13 , which allows the metabolism of glucose and other substrates to be tracked . positron emission tomography and magnetic resonance spectroscopy allow regional differences in metabolism to be assessed . we summarise the data published from these techniques and review their potential uses in the clinical setting .

Introduction Arterio-venous gradient studies Microdialysis Autoradiography and positron emission tomography Carbon-13 labelling studies Proton & phosphorus magnetic resonance spectroscopy Summary and conclusions

traumatic brain injury ( tbi ) is heterogeneous disorder caused by the action of external mechanical forces on the brain . this is due to increasing use of motorised vehicles , while in developed nations there is an increasing incidence of tbi in the older population due to falls ( roozenbeek et al . the pathophysiology of energy failure after tbi is not yet fully understood but has important implications for how tbi patients are managed . many of the techniques used require the brain to be extracted and so are not applicable in clinical studies . hence , we have to be careful in applying what we learn about glucose metabolism after tbi in the laboratory to what actually happens in clinical practice . there are various methods for investigating metabolism after tbi in patients ( fig . microdialysis , which samples extracellular fluid , is a focal technique that allows the immediate microenvironment of the brain to be sampled in vivo . microdialysis can also be used to deliver substances and in this way has been used to deliver glucose and other metabolites labelled with carbon-13 ( c ) . autoradiography experiments in animals , and analogous positron emission tomography ( pet ) studies in humans , use glucose labelled with a radioactive tracer . in vivo magnetic resonance spectroscopy ( mrs ) has also been used to measure the combined extra- and intracellular regional concentrations of lactate and high - energy phosphate compounds . our ability to study cerebral metabolism using these techniques importantly , there is also the potential for these techniques to help in the management of patients with tbi and improve clinical outcomes although their role in the clinical setting is still being defined.fig . 1methods for measuring glucose metabolism in the human brain : various techniques lend themselves to measuring glucose metabolism in the human brain . ( 1 ) microdialysis , which allows metabolites in the extracellular fluid to be measured , is an invasive technique requiring insertion of a microdialysis catheter ( possessing a semipermeable membrane , with nominal molecular weight cut - off typically 20 kda or 100 kda ) into brain parenchyma . ( 2 ) imaging techniques include positron emission tomography ( pet ) , which uses detectors to measure ionising radiation emitted from the glucose analogue fdg administered intravenously . ( 3 ) h and p magnetic resonance spectroscopy ( mrs ) can be used to measure lactate and high - energy phosphate compounds respectively , in different regions of the brain . ( 4 ) a global measure of uptake or release from the brain can be achieved by using a jugular bulb catheter , which allows the venous outflow of the brain to be sampled . this enables the net uptake or release of metabolites by the brain to be calculated and , if cerebral blood flow is known , the cerebral metabolic rate to be calculated methods for measuring glucose metabolism in the human brain : various techniques lend themselves to measuring glucose metabolism in the human brain . ( 1 ) microdialysis , which allows metabolites in the extracellular fluid to be measured , is an invasive technique requiring insertion of a microdialysis catheter ( possessing a semipermeable membrane , with nominal molecular weight cut - off typically 20 kda or 100 kda ) into brain parenchyma . ( 2 ) imaging techniques include positron emission tomography ( pet ) , which uses detectors to measure ionising radiation emitted from the glucose analogue fdg administered intravenously . ( 3 ) h and p magnetic resonance spectroscopy ( mrs ) can be used to measure lactate and high - energy phosphate compounds respectively , in different regions of the brain . ( 4 ) a global measure of uptake or release from the brain can be achieved by using a jugular bulb catheter , which allows the venous outflow of the brain to be sampled . this enables the net uptake or release of metabolites by the brain to be calculated and , if cerebral blood flow is known , the cerebral metabolic rate to be calculated the aim of this review is to summarise what we know about the metabolic perturbations that characterise tbi with a focus on clinical studies . specifically , we will focus on changes to glucose metabolism after moderate to severe tbi . with the increasing clinical availability of techniques that provide insight into glucose metabolism , such as microdialysis and mrs it is important to review the available evidence with a view to improving our ability to interpret this data.fig . glycolysis ( also termed embden - meyerhof pathway ) is the series of reactions that results in the breakdown of glucose , generating pyruvate , adenosine triphosphate ( atp ) and nicotinamide adenine dinucleotide ( nadh ) . the ten key enzymatic steps , in which 2 atp molecules are consumed early on but then paid back later with the generation of 4 atp molecules per glucose molecule , so the net production of atp is 2 molecules per molecule of glucose , takes place in the cytoplasm . located in the inner mitochondrial membrane , which ultimately deposit on oxygen molecules . there is a constant cycle of glutamate released during neurotransmission , retrieved from synaptic junctions by astrocytes and returned to neurons as glutamine glycolysis and the tricarboxylic acid cycle : glucose is the preferred substrate for the brain , although the brain can take up lactate , other monocarboxylic acids and ketone bodies under certain circumstances , for example , during the perinatal period . glycolysis ( also termed embden - meyerhof pathway ) is the series of reactions that results in the breakdown of glucose , generating pyruvate , adenosine triphosphate ( atp ) and nicotinamide adenine dinucleotide ( nadh ) . the amount of substance that a tissue removes from or releases into the circulation is equal to the product of the blood flow to the tissue and the arterio - venous ( av ) concentration difference . using this principle , the steady - state rate of utilisation or production , known as the cerebral metabolic rate ( cmr ) , av studies investigating the cmr of glucose ( cmrglc ) after tbi demonstrate a depression in cmrglc indicating reduced glucose uptake and utilisation by the injured brain ( cruz 1995 ; leegsma - vogt et al . the mr in normal subjects was 5.83 1.41 , which demonstrates the efficiency of the brains aerobic metabolism of glucose . a mr value greater than 6 implies aerobic metabolism of other substrates , and less than 6 the anaerobic metabolism of glucose . more moles of glucose metabolised per mole of oxygen , which indicates that there is proportionally greater anaerobic ( non - oxygen - consuming ) metabolism of glucose . as well as perturbations to the relationship between cerebral glucose and oxygen metabolism , the metabolism of lactate is also affected by tbi . av measurements in healthy controls reveal a small net release of lactate from the brain into the circulation ( glenn et al . av studies provide a measure of the uptake or release of substance by the whole brain at a single time point ; they lack the spatial and temporal resolution to explore how changes to glucose metabolism relate to other pathophysiological changes . microdialysis allows continuous in vivo sampling of molecules from the extracellular fluid from the human brain and permits the longitudinal analysis of certain energy - related small molecules . instead an increase in the oxygen gradient across the brain is used to indicate a relative supply failure . interestingly , in areas of brain deemed to be at risk of non - survival , defined as o pet - measured oxygen metabolism below a critical threshold , normobaric hyperoxia increases oxygen metabolism but does not have a consistent effect on microdialysis measures of metabolism ( nortje et al . although the incidence of frank ischaemia after tbi appears to be low , marked changes to cbf , induced by hyperventilation , or marked reductions in cerebral perfusion pressure ( cpp ) can affect brain glucose . however , during periods of hyperventilation , which significantly increased the oef , a significant reduction of brain glucose was observed . ( 2011a ) in the largest study to date found the most consistent trends of all the microdialysis parameters measured after tbi were for brain lactate and the lactate / pyruvate ratio both of which were lower on a daily basis in patients with favourable outcome as compared with those patients with a poor outcome . the association of a higher lactate and higher lactate / pyruvate ratio with a poor outcome is a consistent finding across the tbi - microdialysis literature and is found in the most methodologically robust of studies that use patient - averaged data and multivariate statistical techniques ( timofeev et al . for further discussion of pet in the context of cerebral metabolism see autoradiography and positron emission tomography 4fdg - pet measurement of cmrglc and its relationship to brain microdialysate composition : a c fdg - pet cmrglc map demonstrating relatively high fdg uptake at sites of injury , in contrast to less injured areas of the brain . 2009 ) fdg - pet measurement of cmrglc and its relationship to brain microdialysate composition : a c fdg - pet cmrglc map demonstrating relatively high fdg uptake at sites of injury , in contrast to less injured areas of the brain . several studies have explored the relationship between systemic glucose and brain microdialysis parameters in tbi patients and found that higher arterial glucose concentrations are associated with higher brain glucose concentrations although this relationship may be lost when the brain is injured ( vespa et al . the relationship between systemic glucose and cerebral metabolism has also been explored using av measures of metabolism . they observed that microdialysate glucose , pyruvate and lactate were significantly lower , that more time was spent with a high lactate / pyruvate ratio ( > 28 ) , and that fdg - pet indicated increased glucose metabolism ( although in a heterogeneous manner depending on the patient and injury pattern ) when tight glycaemic control was used . however , the relative ease of placement , safety and ability to follow trends in brain chemistry over extended periods of time has resulted in the adoption of microdialysis as a clinical monitoring tool in many neurocritical care units . autoradiography and pet imaging studies that use labelled 2-deoxy - d - glucose ( 2dg ) as a tracer allow the in vivo delivery , uptake and initial metabolism of glucose to be assessed both globally and regionally in the brain . ( 1977 ) injected awake and anaesthetised rats with the tracer and used quantitative autoradiography of brain sections to calculate regional rates of glucose metabolism . for the purposes of exploring regional glucose metabolism , the patient is injected with deoxy - glucose , labelled with the positron emitter f ( fdg ) ( phelps 1991 ) . rate constants relating to the movement of tracer between the circulation , extracellular space and the intracellular compartment are used with the plasma glucose concentration and a lumped constant to give a measure of the metabolic rate of glucose . 2009 ) demonstrated regional differences in the uptake of c - glucose relative to 2dg in rats after experimental tbi , suggesting regional differences in lc , and wu et al . this appears to be widespread throughout various regions of the brain and has been found to be associated with neurological outcome in clinical studies ( kato et al . most clinical reports demonstrate a depression of glucose metabolism following tbi when compared to control subjects with only discrete areas of supra - normal glucose metabolism , particularly in relation to mass lesions ( fig . a later study from the same group , which used aged - matched controls and lc values calculated from a cohort of subjects who underwent additional arterio - venous 2dg gradient measurements , used to estimate a global lc value , found significantly lower cmrglc in the whole brain , striatum and thalamus in tbi patients compared to control subjects ( hattori et al . although glucose metabolism in tbi patients is found to be relatively depressed compared to awake and non - sedated control subjects , pet studies suggest that glucose metabolism in tbi is increased relative to the rate of oxygen utilisation . ( 1997 ) used a combination of arterio - venous oxygen measurements and intravenous xenon-133 cbf measurements to calculate both oxygen and glucose metabolism . the peri - lesional sites had higher pyruvate and higher regional cmrglc ( together with a tendency towards higher lactate ) than in the corresponding less - injured sites , while glucose and lactate / pyruvate ratio were not significantly different between sites , albeit in this very small subset of patients . furthermore , fdg - pet provides information on the uptake of glucose and on the first part of glycolysis but does not evaluate glucose metabolism beyond this . applying a tracer to glucose that can then be identified in downstream metabolites derived from glucose allows the metabolic fate of glucose to be evaluated . subsequent turns of the tca cycle will result in the c label shifting to the c3 or c2 of glutamate and glutamine . in this way , c - nmr provides a convenient way to study the transformation of the carbon skeleton that takes place during the metabolism of glucose . in this way , c - nmr provides a convenient way to study the transformation of the carbon skeleton that takes place during the metabolism of glucose . ( 2013 ) abbreviations : pdh pyruvate dehydrogenase , pc , pyruvate carboxylase , me malic enzyme c - labelled substrates have been delivered to the brain intravenously , or directly into the brain extracellular fluid using microdialysis . hyperglycolysis observed in studies using av measurements or fdg - pet relate to actual increased glycolysis or are due to the metabolism of glucose by other alternative pathways , and have also investigated the metabolism of other substrates by the brain such as lactate and acetate ( gallagher et al . it results in the production of ribose sugars , needed for dna synthesis and repair , and the nadph generated by the ppp also acts to maintain glutathione in a reduced state , which serves a vital antioxidant role ( herrero - mendez et al . their arterio - venous difference analysis of c - labelled lactate , differently labelled depending on whether it was derived by either glycolysis or ppp supported the preclinical finding of ppp up - regulation after tbi . uninjured brain is normal - appearing brain in a patient operated on for a benign tumour elsewhere in the brain . 2014 ) the relatively novel combination of c - labelled substrate delivered by microdialysis with ex vivo nmr has some important advantages over other methods of studying metabolism in the clinical setting . furthermore , this technique allows the downstream metabolism of glucose to be evaluated beyond that previously afforded by arterio - venous studies and pet . the intravenous delivery of exogenous c - labelled substrates can be combined with in vivo magnetic resonance spectroscopy ( mrs ) to investigate cerebral metabolism in humans , although this technique has not yet been applied to tbi patients . in vivo c - mrs , performed on a conventional mri scanner with specialised coils , has been used to follow the rate of change of fractional enrichment of a metabolic product , performed in real time , after intravenous delivery of the substrate . the extension to tbi patients of such in vivo c mrs methodology will enable calculation of rates of metabolism beyond that previously afforded by fdg - pet , which only provides kinetic data on glucose uptake and phosphorylation , and will thus permit more in - depth evaluation of metabolism after tbi . possibly relevant are experimental tbi studies in which cyclosporin a , which inhibits opening of the mitochondrial permeability transition pore ( mptp ) , restored mitochondrial membrane potential , reduced tissue damage and improved neurological outcomes ( scheff and sullivan 1999 ; alessandri et al . in addition , p in vivo mrs ( discussed in proton & phosphorus magnetic resonance spectroscopy section below ) has the potential to be useful in future clinical trials of drugs directed at improving mitochondrial function by assessing the response of cellular atp levels to treatment . magnetic resonance spectroscopy ( mrs ) is an in vivo technique carried out on suitably equipped mri scanners . h and phosphorus ( p ) mrs have been used to interrogate energy metabolism after tbi . different localisation techniques are used to produce spectra that represent either a broad region of the brain ( e.g. the most relevant h mrs metabolite for demonstrating perturbed energy metabolism after tbi is lactate . in a study of 51 infants and children with tbi , 91 % of infants and 80 % of children with a poor outcome demonstrated a lactate mrs peak in the grey matter of the occipital lobe ( ashwal et al . the ability to detect lactate with mrs after tbi in children has not been consistently replicated in studies of adult patients . understanding cerebral energy metabolism is complicated by a series of specialisations that are unique to the brain . the brain exists in its own unique chemical environment , shielded by the blood brain barrier ; it has high demands for energy and is vulnerable to reduced substrate provision . along with a change in the metabolic role of glucose , lactate appears to become important as a metabolic substrate , imported from the circulation and oxidatively metabolised via the tca cycle . the methods discussed in this review are not yet employed widely in the clinical setting . amalgamation of these diverse components may emerge from advances in data handling methods , including multivariate methods and machine learning technologies , when applied to the large databases that are growing in specialist centres and as a result of multicentre collaborations.table 1summary of methodology for measuring glucose metabolism in human braintechniqueextent of measurementtimeframe ( and frequency)invasive?examples in human brainarterio - venous differenceglobalmulti - day ( sampling twice daily)yes ( insertion of arterial line and jugular venous catheter)net changes ( import or export ) by brain for glucose and lactate ( jalloh et al . intravenous , mrs magnetic resnance spectroscopy , naa n - acetylaspartate , pet positron emission tomography , ppp pentose phosphate pathway cmro2 measurement by pet additionally involves inhalation of radioactive ( short half - life ) gases summary of methodology for measuring glucose metabolism in human brain abbreviations : atp adenosine triphosphate , cmr glc cerebral metabolic rate of glucose , cmro 2 cerebral metabolic rate of oxygen , gaba gamma - aminobutyric acid ; i.v . intravenous , mrs magnetic resnance spectroscopy , naa n - acetylaspartate , pet positron emission tomography , ppp pentose phosphate pathway cmro2 measurement by pet additionally involves inhalation of radioactive ( short half - life ) gases the precise biological questions to be addressed need to be appropriate to the techniques available . mrs and pet are suited to observing regional changes in metabolism and can demonstrate changes associated with structural lesions such as contusions . hence , microdialysis is well placed to monitor glucose delivery to the brain , assist in the management of systemic glucose levels , and optimise cpp . the ability to monitor the changing metabolism of glucose after tbi raises interesting questions about the potential to manipulate these different pathways and reduce secondary injury . multimodality continuous monitoring is complemented by specialised scanning techniques ( including pet , mri and mrs ) , which provide a snapshot of the brain . the ability to understand and modulate glucose metabolism in the brain is likely to be a crucial factor in achieving the best clinical outcomes for tbi patients .

lots of research is performed on epitaxial thin films and heterostructures of complex oxides because of the wide range of functional properties that can be obtained by tuning the composition and structure of the materials . due to the development of several growth techniques , nowadays it is possible to make a large range of films with compositions and crystalline qualities that can not be reached in bulk.together with the fact that the properties of these materials are highly anisotropic , this makes that in epitaxial films phenomena and functionalities are observed that are not obtained in bulk . besides , epitaxial strain and the creation of heterostructures can be used to obtain new or enhanced properties . in order to grow epitaxial films and heterostructures with the desired properties , substrates with well - defined surfaces are required . local differences in surface chemistry or morphology cause inhomogeneous nucleation and growth , which gives rise to undesired defects and grain boundaries in the film . furthermore , the interface between film and substrate plays an important role in determining the properties because of the limited thickness of the film . this means that substrates are required that are smooth and homogeneous on the atomic level . this criterion is hard to reach when substrates are used that naturally do not have well - defined surfaces , e.g. , other complex oxides . from this perspective , perovskite oxides are one of the most studied substrate materials . perovskite oxides can be represented by the general formula abo3 , in which a and b stand for metal ions . almost all metals can be incorporated in the a or b site , which makes it possible to fabricate a wide range of different substrates . the versatility of the substrate material allows one to tune the properties of the film grown on top of it by tuning the applied epitaxial strain and the structure at the interface . however , growth on these substrates is not straightforward due to the ambiguous nature of the perovskite surface , which is especially visible in ( 001 ) oriented substrates . in the ( 001 ) direction , when a ( 001 ) oriented substrate is made by cleaving from a larger crystal , both oxides are present at the surface . this phenomenon is shown in figure 1 . since the crystal is never perfectly cleaved along the ( 001 ) plane , a surface forms consisting of terraces with unit cell height differences . however , height differences of half a unit cell exist as well , which indicates the presence of both types of surface terminations . it is important to have single terminated perovskite substrates in order to grow a continuous film with homogeneous properties , as has been shown especially for the growth of perovskite oxide films . the termination can cause a large difference in growth kinetics , leading to growth of non - continuous films . furthermore , the stacking order should be similar across the complete film - substrate interface , since ao - bo interfaces can have totally different properties than bo - ao interfaces . the first successful method to obtain a single terminated perovskite oxide surface kawasaki et al.introduced a wet etching method , which was later ameliorated by koster et al . the method consists of increasing the sensitivity of the sro towards acidic etching by hydroxylating this oxide in water , followed by a short etch in buffered hydrogen fluoride ( bhf ) . subsequent annealing to increase the crystallinity yields an atomically smooth surface were only tio2 is present . later , a method to obtain single terminated rare earth scandates was developed by using the higher solubility of rare earth oxides compared to scandates in basic solution . this method was especially described for the orthorhombic ( 110 ) oriented dysco3 , and it was shown that it is possible to obtain completely scandate terminated surfaces . the methods to obtain these single terminated srtio3 and dysco3 substrates are described in this protocol . though the value of single crystalline perovskite substrates is clear , alternatively , arbitrary substrates without suitable crystal structures can be used for epitaxial film growth as well . substrates that are unsuitable for epitaxial film growth by themselves can be made into suitable templates by covering them with a layer of nanosheets . nanosheets are essentially two - dimensional single crystals , with a thickness of a few nanometers and a lateral size in the micrometer range , and thus possess the ability to direct epitaxial growth of thin films . by depositing a layer of nanosheets on an arbitrary substrate , a seed layer is created for oriented growth of any film material with matching lattice parameters . this approach has been reported successful for the oriented growth of for example zno , tio2 , srtio3 , lanio3 , pb(zr , ti)o3 and srruo3.by using nanosheets , the relatively high prices and size limitations of regular single crystalline substrates can be avoided and nanosheets can be deposited on virtually any substrate material . nanosheets are generally obtained by delamination of a layered parent compound into its discrete layers , with their specific thickness determined by the crystal structure of the parent compound . delamination can be achieved in aqueous environment by exchanging the interlayer metal ions in the parent compound with bulky organic ions , which causes the structure to swell and ultimately delaminate into unilamellar nanosheets . this results in a colloidal dispersion of charged nanosheets that are surrounded by counter - charged organic ions . a schematic representation of the delamination process is shown in figure 2 . in the present protocol , ca2nb3o10 nanosheets were used as a model system and these can be obtained from the perovskite parent compound hca2nb3o10 . ca2nb3o10 nanosheets have in - plane lattice parameters almost equal to those of srtio3 and display an atomically smooth , single terminated surface . once an aqueous dispersion of nanosheets is obtained , they can be deposited on an arbitrary substrate by langmuir - blodgett ( lb ) deposition . this method enables nanosheet deposition in monolayers with a high controllability that generally can not be achieved by other conventional techniques like electrophoretic deposition or flocculation . the organic ions surrounding the nanosheets are surface - active molecules and tend to diffuse to the surface of the dispersion , creating a monolayer of floating nanosheets . a schematic representation of the deposition process is shown in figure 3 ; a surface coverage of over 95% is generally achievable and this occurs mainly without stacking of nanosheets or overlapping edges . ca2nb3o10 nanosheets were used as a model system , but the principle of using nanosheets as a seed layer for epitaxial film growth is more widely applicable . though oxide nanosheets receive more attention as seed layers in literature , the concept may be extended to non - oxide nanosheets such as bn , gaas , tis2 , zns and mgb2as well . furthermore , since nanosheets inherit the composition of their parent compound , various functionalities can be inserted by appropriate design of the parent structure . in addition to their use as seed layer for oriented film growth , a wide variety of nanosheets has proven to be a valuable toolbox in studying fundamental material properties and engineering new functional structures . this protocol shows the experimental procedures to obtain the different types of templates for epitaxial growth oxide thin films . the complete procedures to obtain well - defined single terminated srtio3 and dysco3 substrates are described , as well as the procedure to fabricate ca2nb3o10 nanosheet layers on arbitrary of substrates . cleaning srtio3 and dysco3 substrates immerse the substrates in a beaker filled with acetone ( purity 99.5% ) and place it in an ultrasonic bath ( ub ) for 10 min . repeat this step with ethanol ( purity 99.8% ) , without drying the substrate in between . use a nitrogen gun to dry the samples by blowing the ethanol drops from the surface . in this way , particles that are present in ethanol will not stay on the surface after drying.check the surface with an optical microscope . remove any leftover particles by rubbing the substrate gently on a lens tissue , which is soaked in ethanol . immerse the substrates in a beaker filled with acetone ( purity 99.5% ) and place it in an ultrasonic bath ( ub ) for 10 min . repeat this step with ethanol ( purity 99.8% ) , without drying the substrate in between . use a nitrogen gun to dry the samples by blowing the ethanol drops from the surface . in this way , particles that are present in ethanol will not stay on the surface after drying . remove any leftover particles by rubbing the substrate gently on a lens tissue , which is soaked in ethanol . dysco3 treatment annealing load the cleaned substrates in a quartz boat and anneal them in a clean tube oven at 1,000 c in flowing oxygen ( 150 ml / hr ) for 4 hr.check the surface with an optical microscope . if dirt is visible , use the procedure described in step 1.1.2 to clean the surface . load the cleaned substrates in a quartz boat and anneal them in a clean tube oven at 1,000 c in flowing oxygen ( 150 ml / hr ) for 4 hr . check the surface with an optical microscope . if dirt is visible , use the procedure described in step 1.1.2 to clean the surface . dysco3 treatment surface roughening by bhf immerse the clean substrates in a 100 ml beaker containing 40 ml deionized ( di ) water and place it in an ub for 30 min . use a teflon holder to carry the substrates.fill three hf resistant 100 ml beakers with 40 ml di water . fill one 100 ml beaker with 40 ml of ethanol . fill one hf resistant beaker with 40 ml 12.5% buffered hydrogen fluoride ( bhf , nh4f : hf = 87.5:12.5 , ph = 5.5 ) . proper precautions should be taken.transfer the teflon holder carrying the substrates from the beaker with di water to the beaker containing bhf . put the beaker in the ub for 30 sec.transfer the teflon holder to an hf resistant beaker containing di water and immerse for 20 sec , gently moving the holder up and down . leave the holder with samples in the beaker containing ethanol.dispose of all bhf containing liquid.dry the substrates using a nitrogen gun . check the surface with an optical microscope . if dirt is visible , repeat step 1.1.2 . immerse the clean substrates in a 100 ml beaker containing 40 ml deionized ( di ) water and place it in an ub for 30 min . use a teflon holder to carry the substrates . fill one hf resistant beaker with 40 ml 12.5% buffered hydrogen fluoride ( bhf , nh4f : hf = 87.5:12.5 , ph = 5.5 ) . transfer the teflon holder carrying the substrates from the beaker with di water to the beaker containing bhf . put the beaker in the ub for 30 sec . transfer the teflon holder to an hf resistant beaker containing di water and immerse for 20 sec , gently moving the holder up and down . check the surface with an optical microscope . if dirt is visible , repeat step 1.1.2 . dysco3 treatment selective etching by naoh fill a 100 ml beaker with 40 ml 12 m naoh ( aq ) . immerse the samples using a teflon holder and place the beaker in an ub for 30 min.transfer the samples to a 100 ml beaker containing 40 ml 1 m naoh ( aq ) . put it in the ub for 30 min.fill three beakers with di water and one beaker with ethanol . rinse the samples by subsequent immersing in the three beakers with water and finally in the beaker with ethanol . dry the samples using a nitrogen gun.check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . fill a 100 ml beaker with 40 ml 12 m naoh ( aq ) . immerse the samples using a teflon holder and place the beaker in an ub for 30 min . transfer the samples to a 100 ml beaker containing 40 ml 1 m naoh ( aq ) . put it in the ub for 30 min . rinse the samples by subsequent immersing in the three beakers with water and finally in the beaker with ethanol . check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step.anneal the samples at 950 c in flowing oxygen ( 150 ml / hr ) for 90 min . check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step . anneal the samples at 950 c in flowing oxygen ( 150 ml / hr ) for 90 min . check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . preparation of ca2nb3o10 nanosheets make a dispersion of hca2nb3o10 powder in di water with a concentration of 0.40 g/100 ml and add an equimolar amount of tetra - butyl ammonium hydroxide ( tbaoh ) . please refer to ebina et al.for the solid - state synthesis of kca2nb3o10 powder and its protonation to hca2nb3o10 . note : tbaoh is corrosive ; wear gloves at all times and handle with care.gently shake the bottle by hand and place it horizontally on a rocking shaker at 30 hz for 14 days . re - disperse the precipitation five to six times during these 14 days by slowly rolling the bottle.dilute the dispersion to 0.40 g / l and gently shake it again . let it stand for at least 24 hr before use in order to let large aggregates settle to the lower part of the dispersion . initial batches of 100 ml and diluted batches of 500 ml were made , both in polypropylene bottles . make a dispersion of hca2nb3o10 powder in di water with a concentration of 0.40 g/100 ml and add an equimolar amount of tetra - butyl ammonium hydroxide ( tbaoh ) . please refer to ebina et al.for the solid - state synthesis of kca2nb3o10 powder and its protonation to hca2nb3o10 . note : tbaoh is corrosive ; wear gloves at all times and handle with care . gently shake the bottle by hand and place it horizontally on a rocking shaker at 30 hz for 14 days . re - disperse the precipitation five to six times during these 14 days by slowly rolling the bottle . let it stand for at least 24 hr before use in order to let large aggregates settle to the lower part of the dispersion . initial batches of 100 ml and diluted batches of 500 ml were made , both in polypropylene bottles . deposition of ca2nb3o10 nanosheets note : various versions of equipment and software yield various operational settings clean the wilhelmy plate by rinsing with di water and cleaning with oxygen plasma at high energy for at least 3 min for each side . store the wilhelmy plate in di water immediately afterwards . note : if multiple depositions are done successively , the wilhelmy plate does not need to be treated with oxygen plasma every time.clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . make sure the setup is placed on an anti - vibration table to protect against vibrations , and in a box that can be closed during deposition to protect against flowing air and dust.take 50 ml from the upper part of a fresh nanosheet dispersion with a syringe and slowly put it in the trough . . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly.let the dispersion rest for 15 min.choose an arbitrary substrate compatible with aqueous solutions and clean it properly . attach the substrate to the holder of the lb setup and give it a final blow with nitrogen gas . note : keep in mind that atomically flat films have to be grown on atomically flat substrates . example data in this report were obtained with silicon substrates , which were cleaned with ethanol , a jet of supercritical co2 for 30 sec and oxygen plasma at high energy for 5 min.attach the substrate holder to the setup . take the wilhelmy plate , dip it in the trough and carefully attach it to the spring . remove droplets from the wire of the plate with a piece of paper.lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . make sure that the substrate holder does not touch the nanosheet dispersion.set the surface pressure in the software to zero and let the dispersion rest for 15 min . after 15 min the surface pressure typically reaches 1 to 2 mn / m . large deviations may indicate poor quality of the following deposition.set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. , 20 mn / m).monitor the development of surface pressure and surface area . wait until the increase of the pressure slows down significantly and the pressure approaches its maximum . the maximum pressure typically reaches 15.0 to 18.0 mn / m , but this is neither an absolute nor a constant value.enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . monitor the surface pressure.remove the wilhelmy plate when the deposition is finished , rinse it with di water and store it in di water again.remove the substrate after it has dried completely.for multilayer deposition , decompose the organic residues from the previous layer . this can be done for example by heating to 600 c in a microwave oven for 30 min or by ultraviolet irradiation for 30 min . repeat the protocol from step 2.2.2 , but do not clean the substrate other than with a blow of nitrogen gas . clean the wilhelmy plate by rinsing with di water and cleaning with oxygen plasma at high energy for at least 3 min for each side . store the wilhelmy plate in di water immediately afterwards . note : if multiple depositions are done successively , the wilhelmy plate does not need to be treated with oxygen plasma every time . clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . make sure the setup is placed on an anti - vibration table to protect against vibrations , and in a box that can be closed during deposition to protect against flowing air and dust . take 50 ml from the upper part of a fresh nanosheet dispersion with a syringe and slowly put it in the trough . make sure the edges of the trough and the barriers are free of droplets . note : the amount required for one deposition depends on the size of the trough . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly . attach the substrate to the holder of the lb setup and give it a final blow with nitrogen gas . note : keep in mind that atomically flat films have to be grown on atomically flat substrates . example data in this report were obtained with silicon substrates , which were cleaned with ethanol , a jet of supercritical co2 for 30 sec and oxygen plasma at high energy for 5 min . take the wilhelmy plate , dip it in the trough and carefully attach it to the spring . lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . set the surface pressure in the software to zero and let the dispersion rest for 15 min . after 15 min the surface pressure typically reaches 1 to 2 mn / m . large deviations may indicate poor quality of the following deposition . set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. , 20 mn / m ) . wait until the increase of the pressure slows down significantly and the pressure approaches its maximum . the maximum pressure typically reaches 15.0 to 18.0 mn / m , but this is neither an absolute nor a constant value . enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . remove the wilhelmy plate when the deposition is finished , rinse it with di water and store it in di water again . this can be done for example by heating to 600 c in a microwave oven for 30 min or by ultraviolet irradiation for 30 min . repeat the protocol from step 2.2.2 , but do not clean the substrate other than with a blow of nitrogen gas . cleaning srtio3 and dysco3 substrates immerse the substrates in a beaker filled with acetone ( purity 99.5% ) and place it in an ultrasonic bath ( ub ) for 10 min . repeat this step with ethanol ( purity 99.8% ) , without drying the substrate in between . use a nitrogen gun to dry the samples by blowing the ethanol drops from the surface . in this way , particles that are present in ethanol will not stay on the surface after drying.check the surface with an optical microscope . remove any leftover particles by rubbing the substrate gently on a lens tissue , which is soaked in ethanol . immerse the substrates in a beaker filled with acetone ( purity 99.5% ) and place it in an ultrasonic bath ( ub ) for 10 min . repeat this step with ethanol ( purity 99.8% ) , without drying the substrate in between . use a nitrogen gun to dry the samples by blowing the ethanol drops from the surface . in this way , particles that are present in ethanol will not stay on the surface after drying . remove any leftover particles by rubbing the substrate gently on a lens tissue , which is soaked in ethanol . dysco3 treatment annealing load the cleaned substrates in a quartz boat and anneal them in a clean tube oven at 1,000 c in flowing oxygen ( 150 ml / hr ) for 4 hr.check the surface with an optical microscope . if dirt is visible , use the procedure described in step 1.1.2 to clean the surface . load the cleaned substrates in a quartz boat and anneal them in a clean tube oven at 1,000 c in flowing oxygen ( 150 ml / hr ) for 4 hr . check the surface with an optical microscope . if dirt is visible , use the procedure described in step 1.1.2 to clean the surface . dysco3 treatment surface roughening by bhf immerse the clean substrates in a 100 ml beaker containing 40 ml deionized ( di ) water and place it in an ub for 30 min . use a teflon holder to carry the substrates.fill three hf resistant 100 ml beakers with 40 ml di water . fill one 100 ml beaker with 40 ml of ethanol . fill one hf resistant beaker with 40 ml 12.5% buffered hydrogen fluoride ( bhf , nh4f : hf = 87.5:12.5 , ph = 5.5 ) . proper precautions should be taken.transfer the teflon holder carrying the substrates from the beaker with di water to the beaker containing bhf . put the beaker in the ub for 30 sec.transfer the teflon holder to an hf resistant beaker containing di water and immerse for 20 sec , gently moving the holder up and down . leave the holder with samples in the beaker containing ethanol.dispose of all bhf containing liquid.dry the substrates using a nitrogen gun . check the surface with an optical microscope . if dirt is visible , repeat step 1.1.2 . immerse the clean substrates in a 100 ml beaker containing 40 ml deionized ( di ) water and place it in an ub for 30 min . use a teflon holder to carry the substrates . fill one hf resistant beaker with 40 ml 12.5% buffered hydrogen fluoride ( bhf , nh4f : hf = 87.5:12.5 , ph = 5.5 ) . transfer the teflon holder carrying the substrates from the beaker with di water to the beaker containing bhf . put the beaker in the ub for 30 sec . transfer the teflon holder to an hf resistant beaker containing di water and immerse for 20 sec , gently moving the holder up and down . check the surface with an optical microscope . if dirt is visible , repeat step 1.1.2 . dysco3 treatment selective etching by naoh fill a 100 ml beaker with 40 ml 12 m naoh ( aq ) . immerse the samples using a teflon holder and place the beaker in an ub for 30 min.transfer the samples to a 100 ml beaker containing 40 ml 1 m naoh ( aq ) . put it in the ub for 30 min.fill three beakers with di water and one beaker with ethanol . rinse the samples by subsequent immersing in the three beakers with water and finally in the beaker with ethanol . dry the samples using a nitrogen gun.check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . fill a 100 ml beaker with 40 ml 12 m naoh ( aq ) . immerse the samples using a teflon holder and place the beaker in an ub for 30 min . transfer the samples to a 100 ml beaker containing 40 ml 1 m naoh ( aq ) . put it in the ub for 30 min . rinse the samples by subsequent immersing in the three beakers with water and finally in the beaker with ethanol . check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step.anneal the samples at 950 c in flowing oxygen ( 150 ml / hr ) for 90 min . check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step . anneal the samples at 950 c in flowing oxygen ( 150 ml / hr ) for 90 min . check the surface with an optical microscope and clean if necessary , using the procedure described in step 1.1.2 . preparation of ca2nb3o10 nanosheets make a dispersion of hca2nb3o10 powder in di water with a concentration of 0.40 g/100 ml and add an equimolar amount of tetra - butyl ammonium hydroxide ( tbaoh ) . please refer to ebina et al.for the solid - state synthesis of kca2nb3o10 powder and its protonation to hca2nb3o10 . note : tbaoh is corrosive ; wear gloves at all times and handle with care.gently shake the bottle by hand and place it horizontally on a rocking shaker at 30 hz for 14 days . re - disperse the precipitation five to six times during these 14 days by slowly rolling the bottle.dilute the dispersion to 0.40 let it stand for at least 24 hr before use in order to let large aggregates settle to the lower part of the dispersion . initial batches of 100 ml and diluted batches of 500 ml were made , both in polypropylene bottles . make a dispersion of hca2nb3o10 powder in di water with a concentration of 0.40 g/100 ml and add an equimolar amount of tetra - butyl ammonium hydroxide ( tbaoh ) . please refer to ebina et al.for the solid - state synthesis of kca2nb3o10 powder and its protonation to hca2nb3o10 . note : tbaoh is corrosive ; wear gloves at all times and handle with care . gently shake the bottle by hand and place it horizontally on a rocking shaker at 30 hz for 14 days . re - disperse the precipitation five to six times during these 14 days by slowly rolling the bottle . let it stand for at least 24 hr before use in order to let large aggregates settle to the lower part of the dispersion . initial batches of 100 ml and diluted batches of 500 ml were made , both in polypropylene bottles . deposition of ca2nb3o10 nanosheets note : various versions of equipment and software yield various operational settings . clean the wilhelmy plate by rinsing with di water and cleaning with oxygen plasma at high energy for at least 3 min for each side . store the wilhelmy plate in di water immediately afterwards . note : if multiple depositions are done successively , the wilhelmy plate does not need to be treated with oxygen plasma every time.clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . make sure the setup is placed on an anti - vibration table to protect against vibrations , and in a box that can be closed during deposition to protect against flowing air and dust.take 50 ml from the upper part of a fresh nanosheet dispersion with a syringe and slowly put it in the trough . note : the amount required for one deposition depends on the size of the trough . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly.let the dispersion rest for 15 min.choose an arbitrary substrate compatible with aqueous solutions and clean it properly . attach the substrate to the holder of the lb setup and give it a final blow with nitrogen gas . note : keep in mind that atomically flat films have to be grown on atomically flat substrates . example data in this report were obtained with silicon substrates , which were cleaned with ethanol , a jet of supercritical co2 for 30 sec and oxygen plasma at high energy for 5 min.attach the substrate holder to the setup . take the wilhelmy plate , dip it in the trough and carefully attach it to the spring . remove droplets from the wire of the plate with a piece of paper.lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . make sure that the substrate holder does not touch the nanosheet dispersion.set the surface pressure in the software to zero and let the dispersion rest for 15 min . after 15 min the surface pressure typically reaches 1 to 2 mn / m . large deviations may indicate poor quality of the following deposition.set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. , 20 mn / m).monitor the development of surface pressure and surface area . wait until the increase of the pressure slows down significantly and the pressure approaches its maximum . the maximum pressure typically reaches 15.0 to 18.0 mn / m , but this is neither an absolute nor a constant value.enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . monitor the surface pressure.remove the wilhelmy plate when the deposition is finished , rinse it with di water and store it in di water again.remove the substrate after it has dried completely.for multilayer deposition , decompose the organic residues from the previous layer . this can be done for example by heating to 600 c in a microwave oven for 30 min or by ultraviolet irradiation for 30 min . repeat the protocol from step 2.2.2 , but do not clean the substrate other than with a blow of nitrogen gas . clean the wilhelmy plate by rinsing with di water and cleaning with oxygen plasma at high energy for at least 3 min for each side . store the wilhelmy plate in di water immediately afterwards . note : if multiple depositions are done successively , the wilhelmy plate does not need to be treated with oxygen plasma every time . clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . make sure the setup is placed on an anti - vibration table to protect against vibrations , and in a box that can be closed during deposition to protect against flowing air and dust . take 50 ml from the upper part of a fresh nanosheet dispersion with a syringe and slowly put it in the trough . . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly . attach the substrate to the holder of the lb setup and give it a final blow with nitrogen gas . note : keep in mind that atomically flat films have to be grown on atomically flat substrates . example data in this report were obtained with silicon substrates , which were cleaned with ethanol , a jet of supercritical co2 for 30 sec and oxygen plasma at high energy for 5 min . take the wilhelmy plate , dip it in the trough and carefully attach it to the spring . remove droplets from the wire of the plate with a piece of paper . lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . set the surface pressure in the software to zero and let the dispersion rest for 15 min . after 15 min the surface pressure typically reaches 1 to 2 mn / m . large deviations may indicate poor quality of the following deposition . set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. , 20 mn / m ) . wait until the increase of the pressure slows down significantly and the pressure approaches its maximum . the maximum pressure typically reaches 15.0 to 18.0 mn / m , but this is neither an absolute nor a constant value . enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . remove the wilhelmy plate when the deposition is finished , rinse it with di water and store it in di water again . this can be done for example by heating to 600 c in a microwave oven for 30 min or by ultraviolet irradiation for 30 min . repeat the protocol from step 2.2.2 , but do not clean the substrate other than with a blow of nitrogen gas . step 1 ) selective etching of srtio3 and dysco3 substrates atomic force microscopy ( afm ) is a straightforward way to obtain an indication about the success of the treatment . the afm image of an srtio3 substrate which had only been flashed to 650 c ( figure 4a ) shows a rough surface , demonstrating the necessity of a high temperature annealing step . the afm data of an annealed substrate ( figures 4a - c ) clearly show two surface terminations , since clear contrast in the friction image is observed , as well as half unit cell height differences in a cross section of the height image . figure 5 shows afm images of tio2 terminated srtio3 substrates , which were treated according to the method described in this protocol . on large scale , straight terrace ledges can be observed ( figure 5a ) . on smaller scale , very smooth terraces are observed , and only unit cell height differences between the terraces are measured , as expected for single terminated surfaces . on substrates with larger terraces , i.e. , with smaller miscut angles , unit cell deep holes are visible near the terrace ledges ( figure 5b ) . these holes disappear when longer annealing times are used , leading to morphologies similar to single terminated substrates with higher miscut angles ( figure 5c ) . the morphology of these holes , as well as the morphology of the terrace ledges , are an important indication of single termination . on single terminated substrates , in contrast , sharp - edged terrace ledges and square holes are visible on double terminated substrates ( see figure 4b ) . another indication of single termination appears in reflection high - energy electron diffraction ( rheed ) images , as shown in figure 6 . in rheed images of as received substrates , streaks appear due to poor crystallinity of the surface . after annealing in oxygen or full treatment of the substrate , the surface is more ordered , as can be seen by the appearance of kikuchi lines and sharp diffraction spots . however , in the case of single terminated substrates , the diffraction spots are even smaller compared to substrates that are only annealed . more important , besides the ( 1x1 ) spots , no additional spots are visible , which are always present in patterns of double terminated substrates in the case of dysco3 , it is more difficult to see whether or not a treatment is successful . no differences can be seen between rheed patterns of annealed double terminated substrates and chemically treated sco2 terminated substrates in figure 7 , afm images of different annealed dysco3 substrates are shown . figure 7e and f show the morphology expected for single terminated substrates , i.e. only 4 steps are visible . due to the limited resolution of the afm , the areas of different terminations are not clearly visible . higher surface roughness in both height and phase images compared to single terminated surfaces are an indication of the presence of both terminations . scanning probe microscopy and surface diffraction techniques are not sufficient to completely determine the success of a treatment . minor regions of the second termination may not be observed with both types of techniques due to limited resolution . however , these minor regions can have a dramatic influence on the quality of the film , as shown in figure 8 . although the afm images of the dysco3 and srtio3 substrates in respectively figure 8c and f seemed to show single terminated surfaces , growth of srruo3 shows that regions of the other termination were still present . in the end , the success of a treatment can only be fully determined considering the quality of the grown film . step 2 ) deposition of ca2nb3o10 nanosheets on arbitrary substrates during nanosheet deposition , the change in surface pressure can be monitored and this gives an indication on how the deposition proceeds . typical plots of the surface pressure during the initial surface area compression and the actual deposition of nanosheets are shown in figure 9 . the pressure generally increases for an increasingly dense packing of floating nanosheets and increases more rapidly as the packing density approaches 100% . the actual deposition should start just before the surface pressure reaches its maximum and this pressure will be maintained throughout the deposition . in case the pressure passes its maximum and ( slightly ) collapses , this could indicate that the high compressing force caused the edges of some nanosheets to overlap each other and create ( partial ) stacks . as long as the pressure does not approach a maximum , the nanosheets are not yet organized into a dense packing . during the actual deposition , the barriers slowly move back and forth to enable local reorganization of the nanosheet monolayer and this causes a saw - like pressure profile . the nanosheet surfaces are smooth and the height difference with adjacent gaps approaches the 1.44 nm crystallographic thickness of ca2nb3o10 layers in their parent compound . a monolayer of nanosheets is fully ( 001 ) oriented in the out - of - plane direction , but has a random in - plane orientation due to the random in - plane ordering of nanosheets . to illustrate their crystal orientations and quality , figure 11 shows an electron backscatter diffraction ( ebsd ) image of epitaxial srruo3 grown on ca2nb3o10 nanosheets with an intermediate layer of srtio3 . the film has an out - of - plane ( 001 ) orientation on all nanosheets and has a single in - plane orientation on individual nanosheets . the surface morphology of such films is illustrated with the afm image in figure 12.the step heights in the continuous parts correspond either with the nanosheet thickness or with the unit cell height of srruo3 , confirming high quality film growth on atomically perfect nanosheets . for an extended report on the properties of epitaxial srruo3 films grown by this approach , please refer to nijland et al . the metal ions a and b are located in , respectively , the corners and center of the unit cell . the oxygen atoms are located at the faces of the cube , forming an octahedron around the b ion . ( b ) schematic representation of a ( 001 ) oriented perovskite substrate . due to a miscut , ( d ) afm image of the surface of a dysco3 substrate after annealing at 1,000 c for 4 hr . the roughness on the terraces is caused by the presence of two surface terminations , as shown in the line profile ( e ) , where not only the 4 unit cell steps , but also 2 height differences are visible . figures a - c are adapted from kleibeuker et al . ion exchange with bulky molecules causes the structure to swell and reduces the interlayer electrostatic forces , allowing the layers to be separated from each other . the nanosheets float towards the surface of the dispersion and are compressed into a dense packing by the barriers moving inward . the substrate is then slowly withdrawn from the dispersion ( a ) afm image of an srtio3 substrate which had been flashed to 650 c . ( b ) afm height and ( c ) friction image of a double terminated srtio3 substrate , showing sharp step edges and terraces with half a unit cell height difference compared to the adjacent terraces , as visible in the line profile of the afm height image shown in ( d ) . the two different terminations cause a clear contrast in the friction image . figure taken with permission from koster et al . d ) is a line profile of ( c ) , showing only unit cell height differences . the circle in ( b ) indicates one of the unit cell deep holes which are visible near the terrace ledges of substrates with low miscut angles . figure taken with permission from koster et al . rheed images of ( a ) an as received srtio3 substrate , ( b ) an annealed substrate and ( c ) a single terminated srtio3 substrate . figure taken with permission from koster et al . figure 7.afm images of annealed dysco3 substrates.(a - d ) show clearly double terminated surfaces . the surfaces of ( e ) and ( f ) look more homogenous , and only unit cell height differences can be measured . however , the resolution of the afm can be too low to measure small areas of a second termination . the films in ( a ) and ( d ) are grown on respectively srtio3 and dysco3 substrates which were treated according to the methods described in this protocol . the films are very smooth , and the corresponding line profiles shown in ( b ) and ( e ) show only unit cell height differences . the films in ( c ) and ( f ) were grown on double terminated annealed substrates . trenches are visible which are in the range of the film thickness . the insets in ( d ) and ( f ) show the substrate before growth . note that both surfaces are very smooth . figure taken with permission from kleibeuker et al . typical plots of the surface pressure during the initial surface area compression and the actual deposition of ca2nb3o10 nanosheets . figure 10.typical afm image and line profile of a monolayer of ca2nb3o10 nanosheets deposited on a silicon substrate . figure 11.ebsd image of epitaxial srruo3grown on ca2nb3o10 nanosheets with an intermediate layer of srtio3 . the film has an out - of - plane ( 001 ) orientation on all nanosheets and has a single in - plane orientation on individual nanosheets . figure 12.afm image and line profile of epitaxial srruo3 grown on ca2nb3o10 nanosheets with an intermediate layer of srtio3 . step heights in the continuous parts match the nanosheet thickness of 1.4 nm and the srruo3 unit cell height of 0.4 nm . the most important aspect of all perovskite oxide substrate treatments is the cleanliness of the work . surface contaminations prevent etching of areas of the substrate , while unwanted reactions during annealing can easily damage the surface . the order of the different steps is important as well . in the treatment of dysco3 , the annealing step should be performed before the etching step , since post - annealing leads to unwanted dy diffusion from the bulk to the surface of the substrate . after etching in the 12 m naoh solution , a 1 m solution should always be used in order to prevent precipitation of dysprosium hydroxide complexes onto the substrate surface . soaking in water is necessary for the srtio3 treatment in order to hydroxylize the sro . in this way , short etching times can be used which prevents damaging of the surface due to uncontrolled etching . this step is simply copied from the standardized srtio3 treatment procedure and is not expected to have any significance in the treatment . the indicated annealing times for dysco3 and srtio3 treatments are times that , on average , lead to well defined step ledges . however , sometimes the annealing time needs to be increased for substrates with a low miscut angle , i.e. , with wider terraces . an increased diffusion length is then required for the surface atoms to find the optimal sites . in the case of srtio3 , a too long annealing time may cause unwanted diffusion of sr atoms from the bulk to the surface . this second termination can be observed in the surface morphology by appearance of straight step edges and square holes , as described in the section on representative results . in that case , the surface treatment can be repeated , but the final annealing step should be performed at 920 c for 30 min . the methods described in this protocol are the most successful methods for ( 001 ) srtio3 and rare earth scandates , but are applicable to these substrates only . this is also required when substrates with other orientations are used , or when a - site instead of b - site termination is desired . an overview of existing treatments can be found in snchez et al . and schlom et al . regarding seed layers of nanosheets , delicate parts of the process are to obtain high quality nanosheet dispersions and to prevent contamination during the deposition . delamination of a layered parent compound into unilamellar nanosheets by addition of bulky organic ions occurs readily , but nanosheets tend to aggregate in dispersion and such aggregates will hinder the deposition of homogeneous monolayers . therefore , it is very important to leave a freshly diluted dispersion at rest for at least 24 hr before use and not to use the lower part of the dispersion . this leaves time for large aggregates to settle and the upper part of the dispersion will become relatively pure . since ongoing aggregation will continuously degrade the dispersion , use within one week after dilution is recommended . please note that the occurring gradient in nanosheet concentration throughout the dispersion volume causes some variations in the surface pressure values during lb deposition , depending on the local nanosheet concentration in the volume taken from the stock dispersion . furthermore , lb deposition is based on surface - active molecules and thus is very sensitive to contaminations and movement . careful cleaning of the setup and wilhelmy plate ( preferably with cleaning tools dedicated to this setup only ) and protection against flowing air and vibrations are very important . the concept of creating a seed layer of nanosheets on arbitrary substrates by lb deposition is a valuable tool in the field of thin film growth . the atomically perfect surface of nanosheets yields high quality epitaxial films of , in principle , any film material with matching lattice parameters . nanosheets can be deposited on virtually any substrate material and thus other materials can replace relatively expensive and size - limited single crystalline substrates . the lb method enables nanosheet deposition in monolayers with a high controllability that generally can not be achieved by other conventional techniques like electrophoretic deposition or flocculation . high film qualities over large areas are required for reliable application in functional devices and to date , this has not been achieved . to deposit nanosheets with a perfect coverage and preferably also to control their in - plane orientation are main challenges in the field . nevertheless , the current state of the art has already proven to be a valuable tool in research .

atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films . in this protocol , two approaches to obtain such surfaces are described . the first approach is the preparation of single terminated perovskite srtio3 ( 001 ) and dysco3 ( 110 ) substrates . wet etching was used to selectively remove one of the two possible surface terminations , while an annealing step was used to increase the smoothness of the surface . the resulting single terminated surfaces allow for the heteroepitaxial growth of perovskite oxide thin films with high crystalline quality and well - defined interfaces between substrate and film . in the second approach , seed layers for epitaxial film growth on arbitrary substrates were created by langmuir - blodgett ( lb ) deposition of nanosheets . as model system ca2nb3o10- nanosheets were used , prepared by delamination of their layered parent compound hca2nb3o10 . a key advantage of creating seed layers with nanosheets is that relatively expensive and size - limited single crystalline substrates can be replaced by virtually any substrate material .

Introduction Protocol 1. Atomically Smooth, Singly Terminated Surfaces 2. Atomically Defined Templates on Arbitrary Substrates Representative Results Discussion Disclosures

lots of research is performed on epitaxial thin films and heterostructures of complex oxides because of the wide range of functional properties that can be obtained by tuning the composition and structure of the materials . besides , epitaxial strain and the creation of heterostructures can be used to obtain new or enhanced properties . in order to grow epitaxial films and heterostructures with the desired properties , substrates with well - defined surfaces are required . this criterion is hard to reach when substrates are used that naturally do not have well - defined surfaces , e.g. almost all metals can be incorporated in the a or b site , which makes it possible to fabricate a wide range of different substrates . however , growth on these substrates is not straightforward due to the ambiguous nature of the perovskite surface , which is especially visible in ( 001 ) oriented substrates . in the ( 001 ) direction , when a ( 001 ) oriented substrate is made by cleaving from a larger crystal , both oxides are present at the surface . since the crystal is never perfectly cleaved along the ( 001 ) plane , a surface forms consisting of terraces with unit cell height differences . it is important to have single terminated perovskite substrates in order to grow a continuous film with homogeneous properties , as has been shown especially for the growth of perovskite oxide films . the first successful method to obtain a single terminated perovskite oxide surface kawasaki et al.introduced a wet etching method , which was later ameliorated by koster et al . subsequent annealing to increase the crystallinity yields an atomically smooth surface were only tio2 is present . later , a method to obtain single terminated rare earth scandates was developed by using the higher solubility of rare earth oxides compared to scandates in basic solution . this method was especially described for the orthorhombic ( 110 ) oriented dysco3 , and it was shown that it is possible to obtain completely scandate terminated surfaces . the methods to obtain these single terminated srtio3 and dysco3 substrates are described in this protocol . though the value of single crystalline perovskite substrates is clear , alternatively , arbitrary substrates without suitable crystal structures can be used for epitaxial film growth as well . substrates that are unsuitable for epitaxial film growth by themselves can be made into suitable templates by covering them with a layer of nanosheets . nanosheets are essentially two - dimensional single crystals , with a thickness of a few nanometers and a lateral size in the micrometer range , and thus possess the ability to direct epitaxial growth of thin films . by depositing a layer of nanosheets on an arbitrary substrate , a seed layer is created for oriented growth of any film material with matching lattice parameters . this approach has been reported successful for the oriented growth of for example zno , tio2 , srtio3 , lanio3 , pb(zr , ti)o3 and srruo3.by using nanosheets , the relatively high prices and size limitations of regular single crystalline substrates can be avoided and nanosheets can be deposited on virtually any substrate material . nanosheets are generally obtained by delamination of a layered parent compound into its discrete layers , with their specific thickness determined by the crystal structure of the parent compound . delamination can be achieved in aqueous environment by exchanging the interlayer metal ions in the parent compound with bulky organic ions , which causes the structure to swell and ultimately delaminate into unilamellar nanosheets . in the present protocol , ca2nb3o10 nanosheets were used as a model system and these can be obtained from the perovskite parent compound hca2nb3o10 . once an aqueous dispersion of nanosheets is obtained , they can be deposited on an arbitrary substrate by langmuir - blodgett ( lb ) deposition . the organic ions surrounding the nanosheets are surface - active molecules and tend to diffuse to the surface of the dispersion , creating a monolayer of floating nanosheets . a schematic representation of the deposition process is shown in figure 3 ; a surface coverage of over 95% is generally achievable and this occurs mainly without stacking of nanosheets or overlapping edges . ca2nb3o10 nanosheets were used as a model system , but the principle of using nanosheets as a seed layer for epitaxial film growth is more widely applicable . furthermore , since nanosheets inherit the composition of their parent compound , various functionalities can be inserted by appropriate design of the parent structure . in addition to their use as seed layer for oriented film growth , a wide variety of nanosheets has proven to be a valuable toolbox in studying fundamental material properties and engineering new functional structures . this protocol shows the experimental procedures to obtain the different types of templates for epitaxial growth oxide thin films . the complete procedures to obtain well - defined single terminated srtio3 and dysco3 substrates are described , as well as the procedure to fabricate ca2nb3o10 nanosheet layers on arbitrary of substrates . cleaning srtio3 and dysco3 substrates immerse the substrates in a beaker filled with acetone ( purity 99.5% ) and place it in an ultrasonic bath ( ub ) for 10 min . in this way , particles that are present in ethanol will not stay on the surface after drying.check the surface with an optical microscope . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step.anneal the samples at 950 c in flowing oxygen ( 150 ml / hr ) for 90 min . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step . note : if multiple depositions are done successively , the wilhelmy plate does not need to be treated with oxygen plasma every time.clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . make sure the setup is placed on an anti - vibration table to protect against vibrations , and in a box that can be closed during deposition to protect against flowing air and dust.take 50 ml from the upper part of a fresh nanosheet dispersion with a syringe and slowly put it in the trough . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly.let the dispersion rest for 15 min.choose an arbitrary substrate compatible with aqueous solutions and clean it properly . remove droplets from the wire of the plate with a piece of paper.lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . make sure that the substrate holder does not touch the nanosheet dispersion.set the surface pressure in the software to zero and let the dispersion rest for 15 min . large deviations may indicate poor quality of the following deposition.set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. the maximum pressure typically reaches 15.0 to 18.0 mn / m , but this is neither an absolute nor a constant value.enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly . lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . set the surface pressure in the software to zero and let the dispersion rest for 15 min . set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . cleaning srtio3 and dysco3 substrates immerse the substrates in a beaker filled with acetone ( purity 99.5% ) and place it in an ultrasonic bath ( ub ) for 10 min . in this way , particles that are present in ethanol will not stay on the surface after drying.check the surface with an optical microscope . in this way , particles that are present in ethanol will not stay on the surface after drying . note that , while this step is used for dysco3 just as a surface roughening step , the selective etching of sro occurs in this step . note : if multiple depositions are done successively , the wilhelmy plate does not need to be treated with oxygen plasma every time.clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . make sure the setup is placed on an anti - vibration table to protect against vibrations , and in a box that can be closed during deposition to protect against flowing air and dust.take 50 ml from the upper part of a fresh nanosheet dispersion with a syringe and slowly put it in the trough . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly.let the dispersion rest for 15 min.choose an arbitrary substrate compatible with aqueous solutions and clean it properly . remove droplets from the wire of the plate with a piece of paper.lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . large deviations may indicate poor quality of the following deposition.set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. clean the langmuir - blodgett trough and the two barriers by rinsing with di water , brushing with ethanol , again rinsing with di water and drying with nitrogen gas . the water surface must be slightly higher than the edges of the trough , to make sure that the barriers can compress the surface properly . lower the substrate until it touches the surface of the nanosheet dispersion , set the height in the software to zero and lower the substrate further until the desired depth . set the surface pressure in the software to zero and let the dispersion rest for 15 min . set the surface pressure in the software to zero again and start the first stage of the deposition by moving the barriers with a rate of 3.0 mm / min to slowly compress the surface . make sure that the value of the target pressure in the software is well above the maximum value expected in step 2.2.10 ( i.e. enter the reached value as target pressure , set the dipper height to the actual value and start the second stage of the deposition by withdrawing the substrate from the dispersion with a rate of 1.0 mm / min . step 1 ) selective etching of srtio3 and dysco3 substrates atomic force microscopy ( afm ) is a straightforward way to obtain an indication about the success of the treatment . the afm data of an annealed substrate ( figures 4a - c ) clearly show two surface terminations , since clear contrast in the friction image is observed , as well as half unit cell height differences in a cross section of the height image . figure 5 shows afm images of tio2 terminated srtio3 substrates , which were treated according to the method described in this protocol . on smaller scale , very smooth terraces are observed , and only unit cell height differences between the terraces are measured , as expected for single terminated surfaces . these holes disappear when longer annealing times are used , leading to morphologies similar to single terminated substrates with higher miscut angles ( figure 5c ) . the morphology of these holes , as well as the morphology of the terrace ledges , are an important indication of single termination . in rheed images of as received substrates , streaks appear due to poor crystallinity of the surface . after annealing in oxygen or full treatment of the substrate , the surface is more ordered , as can be seen by the appearance of kikuchi lines and sharp diffraction spots . however , in the case of single terminated substrates , the diffraction spots are even smaller compared to substrates that are only annealed . higher surface roughness in both height and phase images compared to single terminated surfaces are an indication of the presence of both terminations . although the afm images of the dysco3 and srtio3 substrates in respectively figure 8c and f seemed to show single terminated surfaces , growth of srruo3 shows that regions of the other termination were still present . in the end , the success of a treatment can only be fully determined considering the quality of the grown film . step 2 ) deposition of ca2nb3o10 nanosheets on arbitrary substrates during nanosheet deposition , the change in surface pressure can be monitored and this gives an indication on how the deposition proceeds . typical plots of the surface pressure during the initial surface area compression and the actual deposition of nanosheets are shown in figure 9 . the nanosheet surfaces are smooth and the height difference with adjacent gaps approaches the 1.44 nm crystallographic thickness of ca2nb3o10 layers in their parent compound . a monolayer of nanosheets is fully ( 001 ) oriented in the out - of - plane direction , but has a random in - plane orientation due to the random in - plane ordering of nanosheets . the film has an out - of - plane ( 001 ) orientation on all nanosheets and has a single in - plane orientation on individual nanosheets . the surface morphology of such films is illustrated with the afm image in figure 12.the step heights in the continuous parts correspond either with the nanosheet thickness or with the unit cell height of srruo3 , confirming high quality film growth on atomically perfect nanosheets . ( b ) schematic representation of a ( 001 ) oriented perovskite substrate . due to a miscut , ( d ) afm image of the surface of a dysco3 substrate after annealing at 1,000 c for 4 hr . the roughness on the terraces is caused by the presence of two surface terminations , as shown in the line profile ( e ) , where not only the 4 unit cell steps , but also 2 height differences are visible . the nanosheets float towards the surface of the dispersion and are compressed into a dense packing by the barriers moving inward . ( b ) afm height and ( c ) friction image of a double terminated srtio3 substrate , showing sharp step edges and terraces with half a unit cell height difference compared to the adjacent terraces , as visible in the line profile of the afm height image shown in ( d ) . the two different terminations cause a clear contrast in the friction image . the circle in ( b ) indicates one of the unit cell deep holes which are visible near the terrace ledges of substrates with low miscut angles . rheed images of ( a ) an as received srtio3 substrate , ( b ) an annealed substrate and ( c ) a single terminated srtio3 substrate . the surfaces of ( e ) and ( f ) look more homogenous , and only unit cell height differences can be measured . however , the resolution of the afm can be too low to measure small areas of a second termination . the films in ( a ) and ( d ) are grown on respectively srtio3 and dysco3 substrates which were treated according to the methods described in this protocol . trenches are visible which are in the range of the film thickness . typical plots of the surface pressure during the initial surface area compression and the actual deposition of ca2nb3o10 nanosheets . the film has an out - of - plane ( 001 ) orientation on all nanosheets and has a single in - plane orientation on individual nanosheets . the most important aspect of all perovskite oxide substrate treatments is the cleanliness of the work . surface contaminations prevent etching of areas of the substrate , while unwanted reactions during annealing can easily damage the surface . in the treatment of dysco3 , the annealing step should be performed before the etching step , since post - annealing leads to unwanted dy diffusion from the bulk to the surface of the substrate . in this way , short etching times can be used which prevents damaging of the surface due to uncontrolled etching . an increased diffusion length is then required for the surface atoms to find the optimal sites . in the case of srtio3 , a too long annealing time may cause unwanted diffusion of sr atoms from the bulk to the surface . this second termination can be observed in the surface morphology by appearance of straight step edges and square holes , as described in the section on representative results . in that case , the surface treatment can be repeated , but the final annealing step should be performed at 920 c for 30 min . the methods described in this protocol are the most successful methods for ( 001 ) srtio3 and rare earth scandates , but are applicable to these substrates only . regarding seed layers of nanosheets , delicate parts of the process are to obtain high quality nanosheet dispersions and to prevent contamination during the deposition . delamination of a layered parent compound into unilamellar nanosheets by addition of bulky organic ions occurs readily , but nanosheets tend to aggregate in dispersion and such aggregates will hinder the deposition of homogeneous monolayers . careful cleaning of the setup and wilhelmy plate ( preferably with cleaning tools dedicated to this setup only ) and protection against flowing air and vibrations are very important . the concept of creating a seed layer of nanosheets on arbitrary substrates by lb deposition is a valuable tool in the field of thin film growth . nanosheets can be deposited on virtually any substrate material and thus other materials can replace relatively expensive and size - limited single crystalline substrates .

informed consent was obtained , and studies were approved by review boards in holguin and utah . a total of 382 patients were recruited from the sca2 population in holguin province , cuba from 1955 to 2001 . dates of birth , cag rls , ao , sex familial inheritance , and clinical manifestations were collected . ao was defined as the first sign of ataxia , usually manifested by unsteadiness of gait . ao was determined by 1 interviewer ( n.s . ) using self - report and , whenever possible , report of other family members . usually , individuals and family members recalled specific life stages at which function was impaired , such as ability to perform military service , ability to ride a bicycle , to carry a child , or to handle tools . although muscle cramps are common in the cuban sca2 population , noncerebellar symptoms were not used to determine ao in this sample . faulty estimation of ao in individuals would result in a reduced estimate of familiality and an increased estimate of nonshared environmental components and stochastic factors . all samples were analyzed at cirah ( holguin ) using conditions previously described and confirmed in our laboratory in the united states . analyses were performed using sas / stat software ( sas.com ) with log - transformed ao . we used the genmod procedure to predict ao using rl , taking into account family structure . however , certain aspects of sensitivity analysis were performed by reanalysis of data using different constellations of relatives , parent - offspring and sib pairs . because all relationships were first degree , the model was implemented with an exchangeable correlation matrix . familial correlations were estimated using the pap to control for similarity due to genetic relatedness . solar tests the likelihood of heritability due to multiple gene effects ( a polygenic model ) of ao after taking into account rl . the estimate is an upper bound of genetic effects because only first - degree relatives were in the study cohort , and familial similarity , which is used to estimate heritability , includes both genetic and shared family environmental factors . heritability was computed on all available data , then using only siblings and using only pc pairs to determine the impact of these relationships on the estimates . informed consent was obtained , and studies were approved by review boards in holguin and utah . a total of 382 patients were recruited from the sca2 population in holguin province , cuba from 1955 to 2001 . dates of birth , cag rls , ao , sex familial inheritance , and clinical manifestations were collected . ao was defined as the first sign of ataxia , usually manifested by unsteadiness of gait . ao was determined by 1 interviewer ( n.s . ) using self - report and , whenever possible , report of other family members . usually , individuals and family members recalled specific life stages at which function was impaired , such as ability to perform military service , ability to ride a bicycle , to carry a child , or to handle tools . although muscle cramps are common in the cuban sca2 population , noncerebellar symptoms were not used to determine ao in this sample . faulty estimation of ao in individuals would result in a reduced estimate of familiality and an increased estimate of nonshared environmental components and stochastic factors . all samples were analyzed at cirah ( holguin ) using conditions previously described and confirmed in our laboratory in the united states . analyses were performed using sas / stat software ( sas.com ) with log - transformed ao . we used the genmod procedure to predict ao using rl , taking into account family structure . however , certain aspects of sensitivity analysis were performed by reanalysis of data using different constellations of relatives , parent - offspring and sib pairs . because all relationships were first degree , the model was implemented with an exchangeable correlation matrix . familial correlations were estimated using the pap to control for similarity due to genetic relatedness . solar tests the likelihood of heritability due to multiple gene effects ( a polygenic model ) of ao after taking into account rl . the estimate is an upper bound of genetic effects because only first - degree relatives were in the study cohort , and familial similarity , which is used to estimate heritability , includes both genetic and shared family environmental factors . heritability was computed on all available data , then using only siblings and using only pc pairs to determine the impact of these relationships on the estimates . for the analysis of residual variance and heritability , information from 326 sca2 patients was used to identify 69 sibships and 129 pc pairs ( figure 1 ) . table 1 also shown are correlations between ao and cag rl , both uncorrected and corrected for pedigree structure . box plot of age of disease onset and cag repeat length of the expanded allele . outliers for each cag repeat were included in the analysis and are denoted in red asterisks . age at onset ( ao ) and cag repeat correlations when we restricted the analysis to 205 adult onset samples with an rl from 32 to 40 repeats , we found that the variance in ao explained by rl ( as measured using the r statistic from the model ) was lowered ; the r uncorrected for family structure was 0.38 and 0.22 when corrected . for these rls , the mutation explained slightly less than 25% of the variance when corrected for pedigree structure , indicating that genetic and environmental modifiers played a greater role in patients with the shorter and more common rls . our birth year analysis is based on 382 affected subjects with known ao and year of birth . the overall distribution of ao had a mean of 31.22 years , sd of 16.38 , and a range of 265 years . we observed possible sampling bias resulting from a loss of subjects with longer repeats born before 1960 . these individuals had likely died before being examined , leading to an enrichment in this cohort of individuals with shorter pathologic repeats . this observation also implies that shorter pathologic repeats were underrepresented in the cohort born after 1960 because these subjects had not shown evidence of disease at the time of examination . this ascertainment bias may also produce unusual effects when looking at parental transmissions , as paternal transmissions tend to expand more than maternal . there was no difference in the ao between males ( 33.12 14.47 ) and females ( 33.06 13.34 ) . mean cag repeats for both sexes were virtually identical ( males : 40.28 4.46 ; females 40.12 3.71 ) . however , the corrected r was higher for males than that for females , although this difference did not reach statistical significance ( table 1 ) . the repeat was more unstable in paternal vs maternal transmissions ( figure 2 , a and b ) . on average , the repeat increased by 4.8 5.4 units ( range 1 to 36 units ) , when inherited from the father , but only by 1.6 2.0 units ( range 1 to 11 units ) , when inherited from the mother . of the 6 expansions > 10 units , 5 occurred when the repeat was passed through the father . important for genetic counseling , 2 of the large expansions occurred in parents with only 36 repeats . note the higher instability of repeats seen in paternal vs maternal transmission . in our adjusted analyses , sibship membership accounted for a substantial proportion of variance in ao ( f = 1.73 ; p = 0.001 ; r = 0.23 ) . the entries in the last column of table 1 therefore reflect variance explained by ao above and beyond variance explained by sibship membership . note that the effect of repeat remained considerable in each of the models ; although when rl was smaller , variance explained was reduced . variance explained was also greater for paternal vs maternal transmissions , although this could be due to overall larger repeats observed in paternal transmissions . we examined the effect of rl on ao in different male - female pc combinations . in the models run separately by maternal vs paternal inheritance the cause of this sizable interaction may be the reduction in the differential prediction of repeat on ao in the mother - daughter pairs . effects of imprinting have not been studied in the dominant ataxias except for small samples that could only detect large effects . there was no difference in the mean repeat number or ao between paternal and maternal transmissions in our cohort of 326 subjects in whom the sex of the transmitting parent was known . the proportion of the variance in ao explained by the mutant allele was also similar in both patient groups . familial correlation is defined as the correlation of a trait among family members , used as a tool to investigate genetic influences on a defined relationship . these results show an interesting difference in parent - offspring resemblance such that similarity is stronger in mother - child ( mc ) relationships ( table 2 ) . the test for significance of this difference was obtained in the pap by comparing 2 times the natural log of the likelihood ( 2lnl ) of the model estimating a single pc correlation ( pc model ) with 2lnl of the model allowing for different estimates for father - child and mother - child ( fc , mc model ) . for all traits , the model allowing for different correlation estimates for mc vs fc pairs fits better than the model with a single pc estimate . for both unadjusted ao and rl , mc correlations were higher ( p = 0.05 and p < 0.0001 , respectively ) . a similar trend was observed for ao adjusted for repeat , but the difference did not reach significance . freeing all parent - offspring relationships to test significance of sex of the child ( mother - son , mother - daughter , father - son , and father - daughter relationships ) did not result in any further improvement in the fit of the model for any of the traits . parent - offspring correlations ( estimated using the pedigree analysis package ) to estimate familiality or an upper bound for heritability of residual familial variance ( adjusted ao ) , we used polygenic variance component analysis in solar applied to ao data in the full cohort of 326 individuals . most were in families with a single child , although 44 pairs were in families with 2 siblings , and 25 pairs were in families with 3 or more siblings . we determined an upper bound for heritability of the residual variance of 33% ( p = 0.008 ) . as already suggested by our analyses of specific familial resemblance described above , heritability of the residual variance was higher when estimated only from siblings ( 58% , p = 0.001 ) than that from parent - offspring pairs ( 10% , p = 0.02 ) . alternatively , siblings may share additional family environment over and above parent - offspring pairs . because lower heritability for male - male sibs was observed in hd , we analyzed for differences between male - male vs female - female sib pairs . again , heritability estimates for both subgroups and female sibs showed more than twice the heritability of the residual than male sibs . sibling correlation differences by sex were analyzed in more detail using pap , similar to our pc analyses as described above . these correlations are comparable with the intraclass correlations reported on this data set , although the pap generates maximum likelihood estimates . pap additionally allowed us to test for significant differences by sex ( table 3 ) . male - male pairs showed lower correlations for both unadjusted ao ( p = 0.05 ) and adjusted ao ( p < 0.0001 ) . the adjusted ao correlations among sibling pairs were remarkably similar to those reported in a large venezuelan pedigree . when we restricted the analysis to 205 adult onset samples with an rl from 32 to 40 repeats , we found that the variance in ao explained by rl ( as measured using the r statistic from the model ) was lowered ; the r uncorrected for family structure was 0.38 and 0.22 when corrected . for these rls , the mutation explained slightly less than 25% of the variance when corrected for pedigree structure , indicating that genetic and environmental modifiers played a greater role in patients with the shorter and more common rls . our birth year analysis is based on 382 affected subjects with known ao and year of birth . the overall distribution of ao had a mean of 31.22 years , sd of 16.38 , and a range of 265 years . we observed possible sampling bias resulting from a loss of subjects with longer repeats born before 1960 . these individuals had likely died before being examined , leading to an enrichment in this cohort of individuals with shorter pathologic repeats . this observation also implies that shorter pathologic repeats were underrepresented in the cohort born after 1960 because these subjects had not shown evidence of disease at the time of examination . this ascertainment bias may also produce unusual effects when looking at parental transmissions , as paternal transmissions tend to expand more than maternal . there was no difference in the ao between males ( 33.12 14.47 ) and females ( 33.06 13.34 ) . mean cag repeats for both sexes were virtually identical ( males : 40.28 4.46 ; females 40.12 3.71 ) . however , the corrected r was higher for males than that for females , although this difference did not reach statistical significance ( table 1 ) . the repeat was more unstable in paternal vs maternal transmissions ( figure 2 , a and b ) . on average , the repeat increased by 4.8 5.4 units ( range 1 to 36 units ) , when inherited from the father , but only by 1.6 2.0 units ( range 1 to 11 units ) , when inherited from the mother . of the 6 expansions > 10 units , 5 occurred when the repeat was passed through the father . important for genetic counseling , 2 of the large expansions occurred in parents with only 36 repeats . note the higher instability of repeats seen in paternal vs maternal transmission . in our adjusted analyses , sibship membership accounted for a substantial proportion of variance in ao ( f = 1.73 ; p = 0.001 ; r = 0.23 ) . the entries in the last column of table 1 therefore reflect variance explained by ao above and beyond variance explained by sibship membership . note that the effect of repeat remained considerable in each of the models ; although when rl was smaller , variance explained was reduced . variance explained was also greater for paternal vs maternal transmissions , although this could be due to overall larger repeats observed in paternal transmissions . we examined the effect of rl on ao in different male - female pc combinations . in the models run separately by maternal vs paternal inheritance the cause of this sizable interaction may be the reduction in the differential prediction of repeat on ao in the mother - daughter pairs . effects of imprinting have not been studied in the dominant ataxias except for small samples that could only detect large effects . there was no difference in the mean repeat number or ao between paternal and maternal transmissions in our cohort of 326 subjects in whom the sex of the transmitting parent was known . the proportion of the variance in ao explained by the mutant allele was also similar in both patient groups . familial correlation is defined as the correlation of a trait among family members , used as a tool to investigate genetic influences on a defined relationship . these results show an interesting difference in parent - offspring resemblance such that similarity is stronger in mother - child ( mc ) relationships ( table 2 ) . the test for significance of this difference was obtained in the pap by comparing 2 times the natural log of the likelihood ( 2lnl ) of the model estimating a single pc correlation ( pc model ) with 2lnl of the model allowing for different estimates for father - child and mother - child ( fc , mc model ) . for all traits , the model allowing for different correlation estimates for mc vs fc pairs fits better than the model with a single pc estimate . for both unadjusted ao and rl , mc correlations were higher ( p = 0.05 and p < 0.0001 , respectively ) . a similar trend was observed for ao adjusted for repeat , but the difference did not reach significance . freeing all parent - offspring relationships to test significance of sex of the child ( mother - son , mother - daughter , father - son , and father - daughter relationships ) did not result in any further improvement in the fit of the model for any of the traits . parent - offspring correlations ( estimated using the pedigree analysis package ) to estimate familiality or an upper bound for heritability of residual familial variance ( adjusted ao ) , we used polygenic variance component analysis in solar applied to ao data in the full cohort of 326 individuals . most were in families with a single child , although 44 pairs were in families with 2 siblings , and 25 pairs were in families with 3 or more siblings . we determined an upper bound for heritability of the residual variance of 33% ( p = 0.008 ) . as already suggested by our analyses of specific familial resemblance described above , heritability of the residual variance was higher when estimated only from siblings ( 58% , p = 0.001 ) than that from parent - offspring pairs ( 10% , p = 0.02 ) . alternatively , siblings may share additional family environment over and above parent - offspring pairs . because lower heritability for male - male sibs was observed in hd , we analyzed for differences between male - male vs female - female sib pairs . again , heritability estimates for both subgroups and female sibs showed more than twice the heritability of the residual than male sibs . sibling correlation differences by sex were analyzed in more detail using pap , similar to our pc analyses as described above . these correlations are comparable with the intraclass correlations reported on this data set , although the pap generates maximum likelihood estimates . pap additionally allowed us to test for significant differences by sex ( table 3 ) . male - male pairs showed lower correlations for both unadjusted ao ( p = 0.05 ) and adjusted ao ( p < 0.0001 ) . the adjusted ao correlations among sibling pairs were remarkably similar to those reported in a large venezuelan pedigree . in sca2 and other polyq diseases , the longer the length of the cag repeat , the earlier the age of disease onset . surprisingly , effects of sex , imprinting , and modifier alleles have received little attention in sca2 or in other scas . alleles of the retinoic acid induced gene 1 were identified as ao modifiers for sca2 . we examined the relationship between cag repeat and ao in the cuban sca2 founder population . the study of founder populations has certain inherent advantages , as they tend to be more homogenous both genetically and environmentally , which may facilitate genetic analysis . on the other hand , findings in founder populations may be difficult to generalize to other populations . it is unknown if the common founder haplotype is european , african , or native american , and there is evidence of extensive population admixture in cuba . the presence of founder alleles may explain why our prior evidence for variation in cacna1a as a modifier of ao did not generalize to a european and us american population . detailed comparisons can only be made with 2 studies of hd with large sample sizes . despite the large sample size , therefore , it shares with previous studies inherent biases of referral for genetic testing by medical providers , self - referral in motivated families , and ascertainment bias based on inclusion of gene carriers who become affected first in a given family . our cohort did not include asymptomatic gene carriers or individuals at risk for the disease . in the cuban sca2 population , although this is in good agreement with other studies , it is difficult to perform precise comparisons , as most studies used a simple regression without taking pedigree structure into consideration . variability of ao in sca1 individuals appeared to cluster within families , suggesting the presence of shared environmental and genetic factors on ao . in one study , the effect of the normal allele in sca2 was examined and found to have little influence on ao . the normal allele in cuban sca2 is not highly variable ; only 31 patients carried a 23 cag atxn2 allele preventing us from examining this effect . the concept of familiality encompasses shared environmental and shared genetic factors and may be difficult to differentiate from heritability in human populations . in the strict sense , heritability denotes the proportion of phenotypic variance that can be attributed to additive genetic variance . our estimate that 33% of the residual ao variance in sca2 individuals was familial was lower than those of our previous analyses . however , this discrepancy was explained , when we examined residual heritability separately in sibling and pc pairs . an upper bound estimate of heritability as estimated using solar was higher in siblings , and at 58% , it was close to the previously reported value of 55% using the coefficient of intraclass correlation in sibships to estimate heritability . we can compare our analyses to 1 study of sca2 and 2 studies in hd . in sca2 pedigrees , the cag repeat determined 73% of ao variance and by incorporating familial dependency , the repeat influence actually increased to 80% . in one study of hd , when the size of the normal allele was taken into consideration , this value increased to 68% . variance - components analysis was used to estimate the between and within sibling pair variances allowing for the computation of the sibling intraclass correlation . the estimated sibling correlation for the residual ao was 0.42 , leading to an upper estimate for heritability of 84% . this value is higher than that of our sca2 siblings , who only showed a correlation of 0.33 . both studies found a much lower correlation for pc than sibling pairs , which was 0.10 in both studies . this may point to the presence of recessive modifying alleles but could also point to fewer shared environmental factors across generations than for siblings . female - female sib pairs had much higher correlation than male - male sibs ( table 3 ) . this was noted in the venezuelan pedigrees as well , but the authors were cautious in commenting on them , as the large standard errors associated with these estimates did not result in statistical significance . it is intriguing that our data not only show similar qualitative differences but display virtually identical correlation coefficients for sex effects in sibling pairs . sex effects also extend to pc pairs , as mc pairs had higher correlations than fc pairs . what model may explain the greatly reduced brother - brother correlations of ao after correction for cag repeat size ? of note , imprinting does not explain the observations , as the sca2 repeat has the same effect on ao in females and in males , as well as after passage through the paternal or maternal germline . thus , one needs to search for mechanisms that largely act stochastically , affecting males only or at least preferentially . these could be related to endogenous male factors such as hormonal differences or to male - specific lifestyle choices such as smoking or alcohol use . furthermore , males share x - chromosomes only 50% of the time , whereas females all receive the same x from the father and share the other one 50% of the time . finally , the possibility remains that there is greater somatic instability in males , which would show as a stochastic variance component in our analyses . we did not collect epidemiologic data to analyze whether the environment experienced by males was more variable than that for females . for example , if tobacco or alcohol use were important in sca2 pathogenesis , and women were largely abstinent with highly variable use among men , this might explain reduced heritability in male siblings . the nature of the recently identified modifiers for ao in hd is important with regard to this study . this study found connection between ao and several pathways subgrouped into 3 clusters by gene membership in the following : dna repair , mitochondrial fission , and oxidoreductase activity . several of the single nucleotide variants ( single nucleotide polymorphisms [ snps ] ) were replicated in separate cohorts of hd and sca patients . some of the snps were validated for sca2 in particular . although the 2 studies defined common snps and identified potentially common pathways related to dna repair , the majority of the residual ao variance remained unexplained . this points to the potential presence of rare variation with large effects or to modifiers acting only in a specific setting of gene - environment interactions that is not shared across different cohorts . k.p . figueroa : study concept and design , analysis , interpretation of data , and critical revision of manuscript for intellectual content . pulst : study concept and design , analysis , interpretation of data , and critical revision of manuscript for intellectual content . this work was supported by grants from the nih ( r37 ns33123 to s.m.p . , and r01 mh099134 and r01 mh094400 to h.c . ) , the national ataxia foundation ( s.m.p . ) , and the carmen and louis warschaw endowment for neurology ( s.m.p . ) pulst serves on the editorial boards of journal of cerebellum , neuromolecular medicine , continuum , experimental neurology , neurogenetics , and nature clinical practice neurology and as editor - in - chief of current genomics and of neurology genetics ; and holds patents for the following : nucleic acids encoding ataxin-2 binding proteins ; nucleic acid encoding schwannomin - binding - proteins and products related thereto ; transgenic mouse expressing a polynucleotide encoding a human ataxin-2 polypeptide ; methods of detecting spinocerebellar ataxia-2 nucleic acids ; nucleic acid encoding spinocerebellar ataxia-2 and products related thereto ; shwannomin - binding - proteins ; and compositions and methods for spinocerebellar ataxia . in addition , he receives publishing royalties from churchill livingston , aan press , academic press , and oxford university press ; has served on the speakers ' bureau of athena diagnostics , inc . ; receives research support from nih , target als , national ataxia foundation , and isis pharmaceuticals ; has consulted for ataxion therapeutics ; is a stockholder of progenitor life sciences ; has received license fee payments from cedars - sinai medical center ; has given expert testimony for hall & evans , llc ; and receives an honorarium from the aan as the editor of neurology : genetics .

objective : to examine heritability of the residual variability of spinocerebellar ataxia type 2 ( sca2 ) age at onset ( ao ) after controlling for cag repeat length.methods:from 1955 to 2001 , dates of birth , cag repeat lengths , ao , sex , familial inheritances , and clinical manifestations were collected for a large cuban sca2 cohort of 382 affected individuals , including 129 parent - child pairs and 69 sibships . analyses were performed with log - transformed ao in the genmod procedure to predict ao using repeat length , taking into account family structure . because all relationships were first degree , the model was implemented with an exchangeable correlation matrix . familial correlations were estimated using the pedigree analysis package to control for similarity due to genetic relatedness.results:for the entire sample , the mutant cag repeat allele explained 69% of ao variance . when adjusted for pedigree structure , this decreased to 50% . evidence for imprinting or sex - specific effects of the cag repeat on ao was not found . for the entire sample , we determined an upper bound for heritability of the residual variance of 33% ( p = 0.008 ) . heritability was higher in sib - sib pairs , especially in female sib - sib pairs , than in parent - child pairs.conclusions:we established that a large proportion of ao variance in sca2 was determined by genetic modifiers in addition to cag repeat length . the genetic structure of heritability of the residual ao variance was surprisingly similar to huntington disease , suggesting the presence of recessive modifying alleles and possibly x - chromosome linked modifiers .

METHODS Standard protocol approvals, registrations, and patient consents. Determination of AO. RESULTS Subcohort 1: RL. Subcohort 2: Effect of birth year. Heritability of residual AO variance. DISCUSSION AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

informed consent was obtained , and studies were approved by review boards in holguin and utah . a total of 382 patients were recruited from the sca2 population in holguin province , cuba from 1955 to 2001 . dates of birth , cag rls , ao , sex familial inheritance , and clinical manifestations were collected . ao was determined by 1 interviewer ( n.s . ) although muscle cramps are common in the cuban sca2 population , noncerebellar symptoms were not used to determine ao in this sample . faulty estimation of ao in individuals would result in a reduced estimate of familiality and an increased estimate of nonshared environmental components and stochastic factors . analyses were performed using sas / stat software ( sas.com ) with log - transformed ao . we used the genmod procedure to predict ao using rl , taking into account family structure . however , certain aspects of sensitivity analysis were performed by reanalysis of data using different constellations of relatives , parent - offspring and sib pairs . because all relationships were first degree , the model was implemented with an exchangeable correlation matrix . familial correlations were estimated using the pap to control for similarity due to genetic relatedness . solar tests the likelihood of heritability due to multiple gene effects ( a polygenic model ) of ao after taking into account rl . the estimate is an upper bound of genetic effects because only first - degree relatives were in the study cohort , and familial similarity , which is used to estimate heritability , includes both genetic and shared family environmental factors . heritability was computed on all available data , then using only siblings and using only pc pairs to determine the impact of these relationships on the estimates . informed consent was obtained , and studies were approved by review boards in holguin and utah . a total of 382 patients were recruited from the sca2 population in holguin province , cuba from 1955 to 2001 . dates of birth , cag rls , ao , sex familial inheritance , and clinical manifestations were collected . ao was defined as the first sign of ataxia , usually manifested by unsteadiness of gait . ao was determined by 1 interviewer ( n.s . ) although muscle cramps are common in the cuban sca2 population , noncerebellar symptoms were not used to determine ao in this sample . faulty estimation of ao in individuals would result in a reduced estimate of familiality and an increased estimate of nonshared environmental components and stochastic factors . analyses were performed using sas / stat software ( sas.com ) with log - transformed ao . we used the genmod procedure to predict ao using rl , taking into account family structure . however , certain aspects of sensitivity analysis were performed by reanalysis of data using different constellations of relatives , parent - offspring and sib pairs . because all relationships were first degree , the model was implemented with an exchangeable correlation matrix . familial correlations were estimated using the pap to control for similarity due to genetic relatedness . solar tests the likelihood of heritability due to multiple gene effects ( a polygenic model ) of ao after taking into account rl . the estimate is an upper bound of genetic effects because only first - degree relatives were in the study cohort , and familial similarity , which is used to estimate heritability , includes both genetic and shared family environmental factors . heritability was computed on all available data , then using only siblings and using only pc pairs to determine the impact of these relationships on the estimates . for the analysis of residual variance and heritability , information from 326 sca2 patients was used to identify 69 sibships and 129 pc pairs ( figure 1 ) . table 1 also shown are correlations between ao and cag rl , both uncorrected and corrected for pedigree structure . box plot of age of disease onset and cag repeat length of the expanded allele . outliers for each cag repeat were included in the analysis and are denoted in red asterisks . age at onset ( ao ) and cag repeat correlations when we restricted the analysis to 205 adult onset samples with an rl from 32 to 40 repeats , we found that the variance in ao explained by rl ( as measured using the r statistic from the model ) was lowered ; the r uncorrected for family structure was 0.38 and 0.22 when corrected . for these rls , the mutation explained slightly less than 25% of the variance when corrected for pedigree structure , indicating that genetic and environmental modifiers played a greater role in patients with the shorter and more common rls . our birth year analysis is based on 382 affected subjects with known ao and year of birth . the overall distribution of ao had a mean of 31.22 years , sd of 16.38 , and a range of 265 years . these individuals had likely died before being examined , leading to an enrichment in this cohort of individuals with shorter pathologic repeats . there was no difference in the ao between males ( 33.12 14.47 ) and females ( 33.06 13.34 ) . however , the corrected r was higher for males than that for females , although this difference did not reach statistical significance ( table 1 ) . on average , the repeat increased by 4.8 5.4 units ( range 1 to 36 units ) , when inherited from the father , but only by 1.6 2.0 units ( range 1 to 11 units ) , when inherited from the mother . of the 6 expansions > 10 units , 5 occurred when the repeat was passed through the father . in our adjusted analyses , sibship membership accounted for a substantial proportion of variance in ao ( f = 1.73 ; p = 0.001 ; r = 0.23 ) . the entries in the last column of table 1 therefore reflect variance explained by ao above and beyond variance explained by sibship membership . note that the effect of repeat remained considerable in each of the models ; although when rl was smaller , variance explained was reduced . we examined the effect of rl on ao in different male - female pc combinations . in the models run separately by maternal vs paternal inheritance the cause of this sizable interaction may be the reduction in the differential prediction of repeat on ao in the mother - daughter pairs . effects of imprinting have not been studied in the dominant ataxias except for small samples that could only detect large effects . there was no difference in the mean repeat number or ao between paternal and maternal transmissions in our cohort of 326 subjects in whom the sex of the transmitting parent was known . the proportion of the variance in ao explained by the mutant allele was also similar in both patient groups . these results show an interesting difference in parent - offspring resemblance such that similarity is stronger in mother - child ( mc ) relationships ( table 2 ) . the test for significance of this difference was obtained in the pap by comparing 2 times the natural log of the likelihood ( 2lnl ) of the model estimating a single pc correlation ( pc model ) with 2lnl of the model allowing for different estimates for father - child and mother - child ( fc , mc model ) . for all traits , the model allowing for different correlation estimates for mc vs fc pairs fits better than the model with a single pc estimate . for both unadjusted ao and rl , mc correlations were higher ( p = 0.05 and p < 0.0001 , respectively ) . freeing all parent - offspring relationships to test significance of sex of the child ( mother - son , mother - daughter , father - son , and father - daughter relationships ) did not result in any further improvement in the fit of the model for any of the traits . parent - offspring correlations ( estimated using the pedigree analysis package ) to estimate familiality or an upper bound for heritability of residual familial variance ( adjusted ao ) , we used polygenic variance component analysis in solar applied to ao data in the full cohort of 326 individuals . most were in families with a single child , although 44 pairs were in families with 2 siblings , and 25 pairs were in families with 3 or more siblings . we determined an upper bound for heritability of the residual variance of 33% ( p = 0.008 ) . as already suggested by our analyses of specific familial resemblance described above , heritability of the residual variance was higher when estimated only from siblings ( 58% , p = 0.001 ) than that from parent - offspring pairs ( 10% , p = 0.02 ) . alternatively , siblings may share additional family environment over and above parent - offspring pairs . because lower heritability for male - male sibs was observed in hd , we analyzed for differences between male - male vs female - female sib pairs . again , heritability estimates for both subgroups and female sibs showed more than twice the heritability of the residual than male sibs . male - male pairs showed lower correlations for both unadjusted ao ( p = 0.05 ) and adjusted ao ( p < 0.0001 ) . the adjusted ao correlations among sibling pairs were remarkably similar to those reported in a large venezuelan pedigree . when we restricted the analysis to 205 adult onset samples with an rl from 32 to 40 repeats , we found that the variance in ao explained by rl ( as measured using the r statistic from the model ) was lowered ; the r uncorrected for family structure was 0.38 and 0.22 when corrected . for these rls , the mutation explained slightly less than 25% of the variance when corrected for pedigree structure , indicating that genetic and environmental modifiers played a greater role in patients with the shorter and more common rls . our birth year analysis is based on 382 affected subjects with known ao and year of birth . the overall distribution of ao had a mean of 31.22 years , sd of 16.38 , and a range of 265 years . this observation also implies that shorter pathologic repeats were underrepresented in the cohort born after 1960 because these subjects had not shown evidence of disease at the time of examination . there was no difference in the ao between males ( 33.12 14.47 ) and females ( 33.06 13.34 ) . however , the corrected r was higher for males than that for females , although this difference did not reach statistical significance ( table 1 ) . of the 6 expansions > 10 units , 5 occurred when the repeat was passed through the father . important for genetic counseling , 2 of the large expansions occurred in parents with only 36 repeats . in our adjusted analyses , sibship membership accounted for a substantial proportion of variance in ao ( f = 1.73 ; p = 0.001 ; r = 0.23 ) . variance explained was also greater for paternal vs maternal transmissions , although this could be due to overall larger repeats observed in paternal transmissions . we examined the effect of rl on ao in different male - female pc combinations . in the models run separately by maternal vs paternal inheritance the cause of this sizable interaction may be the reduction in the differential prediction of repeat on ao in the mother - daughter pairs . effects of imprinting have not been studied in the dominant ataxias except for small samples that could only detect large effects . there was no difference in the mean repeat number or ao between paternal and maternal transmissions in our cohort of 326 subjects in whom the sex of the transmitting parent was known . the proportion of the variance in ao explained by the mutant allele was also similar in both patient groups . these results show an interesting difference in parent - offspring resemblance such that similarity is stronger in mother - child ( mc ) relationships ( table 2 ) . the test for significance of this difference was obtained in the pap by comparing 2 times the natural log of the likelihood ( 2lnl ) of the model estimating a single pc correlation ( pc model ) with 2lnl of the model allowing for different estimates for father - child and mother - child ( fc , mc model ) . for all traits , the model allowing for different correlation estimates for mc vs fc pairs fits better than the model with a single pc estimate . for both unadjusted ao and rl , mc correlations were higher ( p = 0.05 and p < 0.0001 , respectively ) . a similar trend was observed for ao adjusted for repeat , but the difference did not reach significance . freeing all parent - offspring relationships to test significance of sex of the child ( mother - son , mother - daughter , father - son , and father - daughter relationships ) did not result in any further improvement in the fit of the model for any of the traits . parent - offspring correlations ( estimated using the pedigree analysis package ) to estimate familiality or an upper bound for heritability of residual familial variance ( adjusted ao ) , we used polygenic variance component analysis in solar applied to ao data in the full cohort of 326 individuals . most were in families with a single child , although 44 pairs were in families with 2 siblings , and 25 pairs were in families with 3 or more siblings . we determined an upper bound for heritability of the residual variance of 33% ( p = 0.008 ) . as already suggested by our analyses of specific familial resemblance described above , heritability of the residual variance was higher when estimated only from siblings ( 58% , p = 0.001 ) than that from parent - offspring pairs ( 10% , p = 0.02 ) . because lower heritability for male - male sibs was observed in hd , we analyzed for differences between male - male vs female - female sib pairs . again , heritability estimates for both subgroups and female sibs showed more than twice the heritability of the residual than male sibs . male - male pairs showed lower correlations for both unadjusted ao ( p = 0.05 ) and adjusted ao ( p < 0.0001 ) . the adjusted ao correlations among sibling pairs were remarkably similar to those reported in a large venezuelan pedigree . in sca2 and other polyq diseases , the longer the length of the cag repeat , the earlier the age of disease onset . surprisingly , effects of sex , imprinting , and modifier alleles have received little attention in sca2 or in other scas . we examined the relationship between cag repeat and ao in the cuban sca2 founder population . the presence of founder alleles may explain why our prior evidence for variation in cacna1a as a modifier of ao did not generalize to a european and us american population . in the cuban sca2 population , although this is in good agreement with other studies , it is difficult to perform precise comparisons , as most studies used a simple regression without taking pedigree structure into consideration . variability of ao in sca1 individuals appeared to cluster within families , suggesting the presence of shared environmental and genetic factors on ao . in one study , the effect of the normal allele in sca2 was examined and found to have little influence on ao . in the strict sense , heritability denotes the proportion of phenotypic variance that can be attributed to additive genetic variance . our estimate that 33% of the residual ao variance in sca2 individuals was familial was lower than those of our previous analyses . an upper bound estimate of heritability as estimated using solar was higher in siblings , and at 58% , it was close to the previously reported value of 55% using the coefficient of intraclass correlation in sibships to estimate heritability . in sca2 pedigrees , the cag repeat determined 73% of ao variance and by incorporating familial dependency , the repeat influence actually increased to 80% . in one study of hd , when the size of the normal allele was taken into consideration , this value increased to 68% . variance - components analysis was used to estimate the between and within sibling pair variances allowing for the computation of the sibling intraclass correlation . the estimated sibling correlation for the residual ao was 0.42 , leading to an upper estimate for heritability of 84% . both studies found a much lower correlation for pc than sibling pairs , which was 0.10 in both studies . this may point to the presence of recessive modifying alleles but could also point to fewer shared environmental factors across generations than for siblings . female - female sib pairs had much higher correlation than male - male sibs ( table 3 ) . this was noted in the venezuelan pedigrees as well , but the authors were cautious in commenting on them , as the large standard errors associated with these estimates did not result in statistical significance . sex effects also extend to pc pairs , as mc pairs had higher correlations than fc pairs . what model may explain the greatly reduced brother - brother correlations of ao after correction for cag repeat size ? of note , imprinting does not explain the observations , as the sca2 repeat has the same effect on ao in females and in males , as well as after passage through the paternal or maternal germline . furthermore , males share x - chromosomes only 50% of the time , whereas females all receive the same x from the father and share the other one 50% of the time . finally , the possibility remains that there is greater somatic instability in males , which would show as a stochastic variance component in our analyses . for example , if tobacco or alcohol use were important in sca2 pathogenesis , and women were largely abstinent with highly variable use among men , this might explain reduced heritability in male siblings . the nature of the recently identified modifiers for ao in hd is important with regard to this study . this study found connection between ao and several pathways subgrouped into 3 clusters by gene membership in the following : dna repair , mitochondrial fission , and oxidoreductase activity . several of the single nucleotide variants ( single nucleotide polymorphisms [ snps ] ) were replicated in separate cohorts of hd and sca patients . although the 2 studies defined common snps and identified potentially common pathways related to dna repair , the majority of the residual ao variance remained unexplained . this points to the potential presence of rare variation with large effects or to modifiers acting only in a specific setting of gene - environment interactions that is not shared across different cohorts . figueroa : study concept and design , analysis , interpretation of data , and critical revision of manuscript for intellectual content . , the national ataxia foundation ( s.m.p . ) pulst serves on the editorial boards of journal of cerebellum , neuromolecular medicine , continuum , experimental neurology , neurogenetics , and nature clinical practice neurology and as editor - in - chief of current genomics and of neurology genetics ; and holds patents for the following : nucleic acids encoding ataxin-2 binding proteins ; nucleic acid encoding schwannomin - binding - proteins and products related thereto ; transgenic mouse expressing a polynucleotide encoding a human ataxin-2 polypeptide ; methods of detecting spinocerebellar ataxia-2 nucleic acids ; nucleic acid encoding spinocerebellar ataxia-2 and products related thereto ; shwannomin - binding - proteins ; and compositions and methods for spinocerebellar ataxia . in addition , he receives publishing royalties from churchill livingston , aan press , academic press , and oxford university press ; has served on the speakers ' bureau of athena diagnostics , inc . ; receives research support from nih , target als , national ataxia foundation , and isis pharmaceuticals ; has consulted for ataxion therapeutics ; is a stockholder of progenitor life sciences ; has received license fee payments from cedars - sinai medical center ; has given expert testimony for hall & evans , llc ; and receives an honorarium from the aan as the editor of neurology : genetics .

anti - aging effect of medicinal plants is as a result of the antioxidant or free radical scavenging properties of their ingredients ( 2 - 4 ) . cinnamon ( cinnamomum zeylanicum ) and clove ( syzygium aromaticum ) are rich in two active ingredients , which are , cinnamaldehyde and eugenol , respectively ( 5 , 6 ) . these two phytochemicals have different biological activities including , cytotoxic and apoptotic effects on the cancer cell lines ( 6 - 8 ) . anti - mutagenicity , anti - oxidant , anti - inflammatory and anti - depressant activities have also been reported for cinnamaldehyde and eugenol ( 5 , 9 - 11 ) . the effect of phytochemicals on telomere length and telomerase interacting proteins of proliferating stem cells have not been reported yet . gene folds and cell characteristic changes could manifest the ultimate effect of any interaction between chemical components and intracellular proteins . recently , we have reported that cinnamaldehyde and eugenol affect the human adipose derived stem cells ( hascs ) viability , doubling time and differentiation ( 12 ) using cheminforma - tics and experimental approaches . in the present work , the effect of cinnamaldehyde and eugenol on hascs telomere length was determined . also , three important telomere/ telomerase interacting proteins ( tips ) including tert , protein kinase b isoform 1 ( akt1 ) and dkc1 gene expressions under cinnamaldehyde and eugenol treatment were assessed . tert and dkc1 gene s products are necessary for whole enzyme telomerase activity ( 13 ) ; protein kinase b phosphorylates and activates the telomerase enzyme ( 14 ) . a probable mechanism of action of herbal ingredients may be the direct binding of phytochemi - cals to the cell signaling molecules ; this has been shown for some phytochemicals , since the last decade ( 15 , 16 ) . further , experimental methods are becoming more complicated , sensitive and accurate , but they are not empirical to study the ligand - protein docking . computational biology tools help in exploring and assessing the probable ligand - protein interactions in a virtual environment . therefore , in the present investigation , using computational biology and docking tools , the probable cinnamaldehyde and eugenol interactions with tips were explored . cinnamaldehyde ( hplc , > 99.9% ) , eugenol ( hplc grade ; > 98% ) , dmso and antibiotics were from sigma - aldrich . the plasmid dna of psicheck-2 , used in telomere length determination , was a gift from dr farideh talebi . human ascs were prepared , after filling a consent form by a 34-year - old woman who was admitted to a hospital for caesarean section . the hascs preparation and stem cell confirmation methods were as described method in our recent publication ( 12 ) . four groups of hascs studied were treated for 48 hr : 2.5 m / ml cinnamaldehyde treated , 0.1 g / ml eugenol treated , 0.01% dmso treated and untreated hascs . cinnamaldehyde and eugenol were primarily dissolved in 0.01% dmso in phosphate buffer , which is the solvent . we have selected this concentrations according to the cheminformatics and experimental information about the best results of doubling time of hascs denoted in recently published work . according to mentioned study , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol exert better cell viability and doubling time on hascs ( 12 ) . treated and untreated hascs were assessed for the telomere length and the expression of tert , akt1 and dkc1 genes . rna extraction ( geneall ) , complementary dna ( cdna ) synthesis ( viogene ) and total dna extraction ( qiagen ) were performed according to the kit instructions . semi - quantitative pcr was done for tert , akt1 , dkc1 and actin - beta gene expression , and quantitative pcr ( qpcr ) was done for telomere length determination . primers and pcr programs are shown in tables 1 and 2 . for absolute telomere length by diploid genome measurement using qpcr , primer pairs used for expression analysis of human tert , akt1 , dkc1 and housekeeping actin - beta genes oligomers used for absolute quantification of hascs telomere length docking study needs 3ds structures of the proteins and ligands . the telomerase interacting proteins ( tips ) were selected according to the telomerase database ( http://telomerase.asu.edu/ ) ( 18 ) . therefore , 3dss were downloaded from pdb or modeled using swissmodel server ( http://swissmodel.expasy.org/ ) ( 19 , 20 ) . table 3 shows the 3ds of modeled tips using swissmodel server . for docking analysis , the mol2 format of cinnamaldehyde and eugenol molegro virtual docker ver.4.2 was used for docking analysis ; this software has about 87% docking accuracy ( 22 ) . the predicted tertiary structures of some tips or unites tr : telomerase enzyme ; rnp : ribonucleoprotein ; tert : telomerase gene the gene folds , absolute quantities of telomere length and docking scores were analyzed by spss software ver.20 . the meansd was compared using analysis of variances ( anova ) and tukey - hsd with a confidence interval of 95% . also , cluster analysis was performed on the docking scores to select the best scores presentation as graphic views of docking . for clustering , the algorithm of nearest neighbor method and squared euclidean distance that was standardized with z score in spss software were used . string database at the url http://string-db.org/ predicts and plots the protein - protein direct or indirect association networks . the string confidence of association net tools was adjusted on the lowest confidence level ( 0.15 ) to cover more associations . cinnamaldehyde ( hplc , > 99.9% ) , eugenol ( hplc grade ; > 98% ) , dmso and antibiotics were from sigma - aldrich . the plasmid dna of psicheck-2 , used in telomere length determination , was a gift from dr farideh talebi . human ascs were prepared , after filling a consent form by a 34-year - old woman who was admitted to a hospital for caesarean section . the hascs preparation and stem cell confirmation methods were as described method in our recent publication ( 12 ) . four groups of hascs studied were treated for 48 hr : 2.5 m / ml cinnamaldehyde treated , 0.1 g / ml eugenol treated , 0.01% dmso treated and untreated hascs . cinnamaldehyde and eugenol were primarily dissolved in 0.01% dmso in phosphate buffer , which is the solvent . we have selected this concentrations according to the cheminformatics and experimental information about the best results of doubling time of hascs denoted in recently published work . according to mentioned study , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol exert better cell viability and doubling time on hascs ( 12 ) . treated and untreated hascs were assessed for the telomere length and the expression of tert , akt1 and dkc1 genes . rna extraction ( geneall ) , complementary dna ( cdna ) synthesis ( viogene ) and total dna extraction ( qiagen ) were performed according to the kit instructions . semi - quantitative pcr was done for tert , akt1 , dkc1 and actin - beta gene expression , and quantitative pcr ( qpcr ) was done for telomere length determination . primers and pcr programs are shown in tables 1 and 2 . for absolute telomere length by diploid genome measurement using qpcr , primer pairs used for expression analysis of human tert , akt1 , dkc1 and housekeeping actin - beta genes oligomers used for absolute quantification of hascs telomere length the telomerase interacting proteins ( tips ) were selected according to the telomerase database ( http://telomerase.asu.edu/ ) ( 18 ) . therefore , 3dss were downloaded from pdb or modeled using swissmodel server ( http://swissmodel.expasy.org/ ) ( 19 , 20 ) . table 3 shows the 3ds of modeled tips using swissmodel server . for docking analysis , the mol2 format of cinnamaldehyde and eugenol were prepared from zinc database ( http://zinc.docking.org ) ( 21 ) . molegro virtual docker ver.4.2 was used for docking analysis ; this software has about 87% docking accuracy ( 22 ) . the predicted tertiary structures of some tips or unites tr : telomerase enzyme ; rnp : ribonucleoprotein ; tert : telomerase gene the gene folds , absolute quantities of telomere length and docking scores were analyzed by spss software ver.20 . the meansd was compared using analysis of variances ( anova ) and tukey - hsd with a confidence interval of 95% . also , cluster analysis was performed on the docking scores to select the best scores presentation as graphic views of docking . for clustering , the algorithm of nearest neighbor method and squared euclidean distance that was standardized with z score in spss software were used . string database at the url http://string-db.org/ predicts and plots the protein - protein direct or indirect association networks . the string confidence of association net tools was adjusted on the lowest confidence level ( 0.15 ) to cover more associations . untreated hascs were compared with 0.01% dmso , 2.5 m / ml cinnamaldehyde or 0.1 g / ml eugenol treated ones . k562 cells , telomerase positive cell lines , were used to confirm whether the designed primers are competent for amplification of detectable tert in qpcr reaction . 0.01% dmso and 2.5 m / ml cinnamaldehyde treated hascs had higher akt1 folds ( p<0.05 ) when compared with the untreated cells . but the akt1 folds did not change in the 0.1 g / ml eugenol treated cells as compared to the untreated hascs ( p>0.05 ) ( figure 1 ) . the 0.01% dmso and 2.5 m / ml cinnamaldehyde significantly increased the dkc1 gene folds in hascs ( p<0.05 ) . but 0.1 g / ml eugenol decreased the dkc1 folds ( p<0.05 ) compared with the untreated cells ( figure 2 ) . untreated hascs has the longest telomere length as compared to 0.01% dmso , 2.5 m / ml cinnamal - dehyde or 0.1 g / ml eugenol treated hascs ( figure 3 ) . there was no statistical difference among 0.01% dmso and untreated or phytochemicals treated cells ( p>0.05 ) . generally , the phytochemicals treated hascs had shortened telomeres as compared to the untreated cells ( p<0.05 ) . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the akt1 expression folds in hascs . nevertheless , the 0.1 g / ml eugenol did not affect akt1 expression significantly tukey - hsd and anova analysis for dkc1 folds comparison . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the dkc1 expression folds in hascs . but the 0.1 g / ml eugenol decreased dkc1 expression significantly telomere length comparison between examined groups . the control hascs , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol significantly decreased the telomere length ( p - value < 0.05 ) but not in comparison with 0.01% dmso group ( p - value > 0.05 ) figure 4 shows the gene name , number of docking sites and the best minimum docking scores of cinnamaldehyde and eugenol with 22 tips . the 3ds prediction was done for human gar1p , nhp2 , nop10 , pinx1 , stau2 , tert and dyskerin that had no 3ds on the protein data bank . dendrogram of cluster analysis for 22 tips plus telomerase ( tert ) ; left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores . clustering for docking scores performed by the nearest neighbor method and the measure of squared euclidian distance . some important docking results are shown in figure 6 - 14 docking scores of both cinnamaldehyde and eugenol were analyzed using cluster analysis ( figure 4 ) and one - way anova ( figure 5 ) . cinnamaldehyde tend to inhabit the hydrophobic parts of the proteins , although in some cases , it formed hydrogen bonds with hydrophilic domains of tips . according to the cluster analysis , the ligand - protein docking scores were divided into 8 distinct clusters . the dendrogram of cluster analysis represents calculated distances between the best phytochemicals - tips docking scores ( figure 4 ) . the cinnamaldehyde and eugenol docked with tips in molegro virtual docker ( mvd ) software . analysis of variance ( anova ) shows there is a significant difference between all scores ( ci= 0.95 ; p= 0.000 ) there was a significant difference between docking scores of tips - phytochemicals ( ci= 0.95 ; p - value= 0.000 ) . as an example of docking results and according to the cluster analysis , only the best interactions from each category are presented in this article ( figure 6 - 13 ) . an interesting result was obtained for eugenol and telomeric dna binding domain of hnrnpa1 protein ( pdb i d : 1pgz ) . the eugenol was exactly docked in the binding site of the telomeric dna purines of hnrnpa1 ( figure 14 ) . docking result of rpl22 ( pdb i d : 3j3a ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . it is the best score of dockings and belong to the category 5 of clustering docking result of tert ( modeled structure ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 6 of clustering docking result of hsp90aa1 ( pdb i d : 3q6n ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 3 of clustering docking result of ku70-ku80 hetero - complex ( pdb i d : 1jeq ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 4 of clustering docking result of 14 - 3 - 3 ( pdb i d : 2br9 ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 1 of clustering docking result of kip ( pdb i d : 1y1a ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 2 of clustering docking result of rnpc2 ( pdb i d : 2cq4 ) protein with cinnamaldehyde ; above : binding site of cinnamaldehyde ( green ) on the protein ; below : binding pocket has not hydrogen bounds . this score belongs to category 8 of clustering docking result of smn1 ( pdb i d : 1g5v ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bound of ligand - protein interaction ( striated line ) in binding site pocket . this score belongs to category 7 of clustering docking result of hnrnpa1 ( pdb i d : 1pgz ) protein with eugenol ; left : binding site of eugenol ( green ) on the protein ; right : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . the molecule eugenol locates in a deep binding pocket of protein figure 15 shows an association network between tips and telomerase ; this is plotted using string data base ( see materials and methods ) . was selected for drawing the association net ; apart from the stau2 protein , all the 30 proteins had at least a weak association . according to the string - db biological function analyzer ( 23 ) , the most important biological pathways , the tips , play roles include : intrinsic apoptosis pathway , intracellular transport , cellular localization , membrane organization , cell organelle organization , protein localization , etc . also , molecular functions affected by tips include : the telomeric dna binding , poly(a ) rna binding , telomerase activity , rna binding , rna - directed dna polymerase activity and telomeric rna binding . besides , protein kinase c inhibitory activity , ion channel binding , enzyme binding and nitric oxide synthase regulatory activity . some abbreviations in the string - db plotted net were not mentioned in the text ; they include : rbm38 equals rnpc1 ; rbm39 equals rnpc2 ; xrcc6 equals ku70 ; xrcc5 equals ku80 ; ncl equals nucleolin ; la equals ssb ; cib1 equals kip ; ywhaq equals 14 - 3 - 3 ; tmed10 equals p23 untreated hascs were compared with 0.01% dmso , 2.5 m / ml cinnamaldehyde or 0.1 g / ml eugenol treated ones . k562 cells , telomerase positive cell lines , were used to confirm whether the designed primers are competent for amplification of detectable tert in qpcr reaction . 0.01% dmso and 2.5 m / ml cinnamaldehyde treated hascs had higher akt1 folds ( p<0.05 ) when compared with the untreated cells . but the akt1 folds did not change in the 0.1 g / ml eugenol treated cells as compared to the untreated hascs ( p>0.05 ) ( figure 1 ) . the 0.01% dmso and 2.5 m / ml cinnamaldehyde significantly increased the dkc1 gene folds in hascs ( p<0.05 ) . but 0.1 g / ml eugenol decreased the dkc1 folds ( p<0.05 ) compared with the untreated cells ( figure 2 ) . untreated hascs has the longest telomere length as compared to 0.01% dmso , 2.5 m / ml cinnamal - dehyde or 0.1 g / ml eugenol treated hascs ( figure 3 ) . there was no statistical difference among 0.01% dmso and untreated or phytochemicals treated cells ( p>0.05 ) . generally , the phytochemicals treated hascs had shortened telomeres as compared to the untreated cells ( p<0.05 ) . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the akt1 expression folds in hascs . nevertheless , the 0.1 g / ml eugenol did not affect akt1 expression significantly tukey - hsd and anova analysis for dkc1 folds comparison . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the dkc1 expression folds in hascs . but the 0.1 g / ml eugenol decreased dkc1 expression significantly telomere length comparison between examined groups . the control hascs , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol significantly decreased the telomere length ( p - value < 0.05 ) but not in comparison with 0.01% dmso group ( p - value > 0.05 ) figure 4 shows the gene name , number of docking sites and the best minimum docking scores of cinnamaldehyde and eugenol with 22 tips . the 3ds prediction was done for human gar1p , nhp2 , nop10 , pinx1 , stau2 , tert and dyskerin that had no 3ds on the protein data bank . dendrogram of cluster analysis for 22 tips plus telomerase ( tert ) ; left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores . clustering for docking scores performed by the nearest neighbor method and the measure of squared euclidian distance . some important docking results are shown in figure 6 - 14 docking scores of both cinnamaldehyde and eugenol were analyzed using cluster analysis ( figure 4 ) and one - way anova ( figure 5 ) . cinnamaldehyde tend to inhabit the hydrophobic parts of the proteins , although in some cases , it formed hydrogen bonds with hydrophilic domains of tips . according to the cluster analysis , the ligand - protein docking scores were divided into 8 distinct clusters . the dendrogram of cluster analysis represents calculated distances between the best phytochemicals - tips docking scores ( figure 4 ) . the cinnamaldehyde and eugenol docked with tips in molegro virtual docker ( mvd ) software . analysis of variance ( anova ) shows there is a significant difference between all scores ( ci= 0.95 ; p= 0.000 ) there was a significant difference between docking scores of tips - phytochemicals ( ci= 0.95 ; p - value= 0.000 ) . as an example of docking results and according to the cluster analysis , only the best interactions from each category are presented in this article ( figure 6 - 13 ) . an interesting result was obtained for eugenol and telomeric dna binding domain of hnrnpa1 protein ( pdb i d : 1pgz ) . the eugenol was exactly docked in the binding site of the telomeric dna purines of hnrnpa1 ( figure 14 ) . docking result of rpl22 ( pdb i d : 3j3a ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . it is the best score of dockings and belong to the category 5 of clustering docking result of tert ( modeled structure ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 6 of clustering docking result of hsp90aa1 ( pdb i d : 3q6n ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 3 of clustering docking result of ku70-ku80 hetero - complex ( pdb i d : 1jeq ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 4 of clustering docking result of 14 - 3 - 3 ( pdb i d : 2br9 ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 1 of clustering docking result of kip ( pdb i d : 1y1a ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 2 of clustering docking result of rnpc2 ( pdb i d : 2cq4 ) protein with cinnamaldehyde ; above : binding site of cinnamaldehyde ( green ) on the protein ; below : binding pocket has not hydrogen bounds . this score belongs to category 8 of clustering docking result of smn1 ( pdb i d : 1g5v ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bound of ligand - protein interaction ( striated line ) in binding site pocket . this score belongs to category 7 of clustering docking result of hnrnpa1 ( pdb i d : 1pgz ) protein with eugenol ; left : binding site of eugenol ( green ) on the protein ; right : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . figure 15 shows an association network between tips and telomerase ; this is plotted using string data base ( see materials and methods ) . was selected for drawing the association net ; apart from the stau2 protein , all the 30 proteins had at least a weak association . according to the string - db biological function analyzer ( 23 ) , the most important biological pathways , the tips , play roles include : intrinsic apoptosis pathway , intracellular transport , cellular localization , membrane organization , cell organelle organization , protein localization , etc . also , molecular functions affected by tips include : the telomeric dna binding , poly(a ) rna binding , telomerase activity , rna binding , rna - directed dna polymerase activity and telomeric rna binding . besides , protein kinase c inhibitory activity , ion channel binding , enzyme binding and nitric oxide synthase regulatory activity . some abbreviations in the string - db plotted net were not mentioned in the text ; they include : rbm38 equals rnpc1 ; rbm39 equals rnpc2 ; xrcc6 equals ku70 ; xrcc5 equals ku80 ; ncl equals nucleolin ; la equals ssb ; cib1 equals kip ; ywhaq equals 14 - 3 - 3 ; tmed10 equals p23 recently , we have reported that cinnamaldehyde and eugenol affect the hascs doubling time and differentiation ( 12 ) . in the current study , cinnamalde - hyde and eugenol did not increase the tert expression to a detectable level in hascs . however , human mesenchymal stem cells had been previously reported to be telomerase negative ( 24 ) . we have used k562 cell lines , as a positive cell line model for tert gene expression during real - time pcr method validation . as far as we know , there is not known chemical material with a definite positive effect on the telomerase expression in adscs . eugenol decreased the akt1 expression but cinnamaldehyde increased or decreased it as compared to the untreated or dmso treated cells , respectively . when 0.01% dmso is also present in the cinnamal - dehyde and eugenol groups , as the solvent of phytochemicals ( see materials and methods ) , the ultimate effect of both phytochemicals on the akt1 gene expression will decrease . this evidence confirms more potent effect of eugenol on the decrease of the akt1 expression . cinnamaldehyde or dmso - treated hascs increased the dkc1 gene expression folds but the eugenol decreased it severely as compared to the untreated cells . these findings suggest that the final targets of cinnamaldehyde and eugenol are different in the intracellular environment . the effect of cinnamal - dehyde and eugenol on some tips or cell aging was reported previously . for example , cinnamaldehyde up - regulates the stau2 expression in mutz-3 cell line and human peripheral blood monocyte - derived dendritic cells ( 25 ) . according to the comparative toxicogenomics database ( http://ctdbase.org/ ) , king and colleagues reported that cinnamaldehyde induces the dna damage in hct116 , a human colon cancer cell line ( 9 ) . the result of the present study on telomere length showed that damage such as telomere shortening may be obvious ( figure 3 ) . the hallmark of some publications is the insignificance of the effect of solvents on the gene expression of treated cells . king and coworkers did not report the effect of dmso on the hct116 cell line . in the current study , it was shown that a low concentration of dmso increases both akt1 and dkc1 gene folds when compared with the untreated cells . the findings of this study infer the importance of the solving agent ( dmso ) effects on the gene expression and cell aging . cinnamaldehyde and eugenol reduced the telomere lengths as compared to the untreated cells notably , but insignificantly when compared with dmso treated hascs . then , a low concentration of dmso had a negative effect on the hascs telomeres , although not significantly ( figure 3 , p - value= 0.110 ) . cinnamaldehyde and eugenol treated hascs telomeres were not meaningfully different from dmso treated cells ( figure 3 , p > 0.05 ) , although the telomere lengths were shortened . it is assumed that the broad ranges of telomere lengths in the studied groups ( see standard deviations ) , are responsible for the results of telomere length comparisons insignificance . absolute quantification of the telo - mere length using quantitative polymerase chain reaction ( pcr ) showed high coefficient of variations percent ( cv % ) . aviv and colleagues described this fact previously when comparing southern blot and qpcr techniques ( 26 ) . however , the qpcr is more rapid , lesser time consuming , with lower costs , without radioactive hazards and acceptable regression with southern blot ( 17 ) . the decreased telomere length , in the presence of phytochemicals , in the present work could be logical when considering the docking results . the docking results showed that cinnamaldehyde and eugenol attached well within the binding pockets on the tips . some herbal ingredients change the gene expression folds of molecular chaperons such as heat shock proteins ; therefore , medicinal plants ingredients are proposed as chaperon - based medications ( 27 ) . based on the docking results , it is assumed that cinnamaldehyde and eugenol interact with chaperons , such hsp90aa1 , or chaperon inducers ; then they change the behaviors of tips . grover and coworkers , using a virtual study , reported that withaferin a , a phytochemical , targets the hsp90/cdc37-chaperone or co - chaperone complex . they proposed that the withaferin a is a hsp90/cdc37 inhibitor and a potent anticancer agent ( 28 ) . in addition , the findings of the present work proposed that cinnamaldehyde and eugenol might exert similar effects on the telomerase and tips . the molecular effects of cinnamaldehyde and eugenol on the hascs were telomere shortening and the gene expression fold changes . logically , such changes did not occur without involving regulatory events of the cell aging . researchers have shown that the active ingredients of some herbs inhibit the telomerase enzyme dose - dependently ( 29 , 30 ) but others increase its gene expression ( 31 , 32 ) . the role of herbal ingredients on the telomere or telomerase dependent anti - aging or aging induction is not well known . the study on direct interaction of ligands - proteins or biomolecules needs certain tools and professional operators . nevertheless , computational biology tools and analysis , such as docking , helps in predicting the probable direct bindings . intracellular environment has dynamic properties ( 33 ) ; therefore , the chemicals interact with biomolecules in a dynamic way . this infers that the best docking scores are of interest , especially if the dynamic binding site conformations change whenever it is necessary . elizabeth h. blackburn is a noble prizewinner for her works on the telomere and telomerase enzyme . she believes the telomerase and the telomere interacting enzymes exert four distinct biochemical effects on the telomeric dna in vivo : 1 . in fusion with another telomere or dna end , it has been shown that using computational tools , the cinnamaldehyde and eugenol may target tips efficiently . after cinnamaldehyde and eugenol docking with tips or regulating proteins , three effects are probable : telomere shortening , lengthening or no length change . figure 16 proposes a subjective map for the in silico and experimental results of this study . according to the map , the probable effects of the cinnamaldehyde and eugenol on the senescence and expected events after the cell treatment . the cinnamaldehyde and eugenol may bind to tips or act on their gene expression and impel the signal transduction pathways . the left gray box shows the gene names of proteins , which have positive effect on the telomere or telomerase . the left gray box shows two proteins with negative effects on the telomere or telomerase . in our work then , the red lines show the probable occurred events in the current work the hnrnpa1 activates the telomerase ( 35 ) , but as shown in figure 14 , it also interacts with telomeric repeats ( 36 ) . the eugenol docking with hnrnpa1 is the most significant result of docking , which occurred exactly in the telomeric dna binding pocket . such result may suggest a competitive inhibition between telomeric repeats of the dna and the eugenol for binding to the hnrnpa1 . same result was derived for cinnamaldehyde and hnrnpa1 docking and exactly in the binding pocket ( the result is not shown ) . they believed that eugenol metabolism products exert toxic effect on the rat hepatocytes dose - dependently ( 37 ) . also , eugenol inhibits the monoamine oxidase a ( maoa ) , a metabolic enzyme , through the covalent binding ( 11 ) . all mentioned studies show negative effect of eugenol on the cellular proteins and are in accordance with the experimental and virtual findings of the present study . weibel and hansen have shown that the cinnamaldehyde binds to the nucleophilic groups of proteins ( 38 ) . python and coworkers evaluated the effect of cinnamaldehyde on 80 gene expression profiles in dendritic cells and mutz-3 cell line . using dna microarray and qpcr , they showed that some genes were down - regulated but others were up - regulated ( 25 ) . all the mentioned studies confirm the present study results of cinnamaldehyde effect on the gene expression changes . in this work , however , in comparison with the only dmso treated hascs , cinnamaldehyde decreased akt1 and increased dkc1 folds . these findings are in accordance with python and coworkers results on the discrepant behavior of cinnamaldehyde in the gene expression folds . a single ligand - protein docking , as cinnamaldehyde or eugenol to any tip , is not enough for interpreting experimental results . in fact , other protein - protein interrelations are important for their impacts on the special pathway members . the ligand - protein interaction may exert direct or indirect effect on protein - protein interactions in a pathway . tips are good examples of such interactions because of the different roles they play in the cell signaling ( 33 , 39 ) . the network shows that the protein - protein interactions may be weak or strong ; therefore , the docking affects the quantities of signaling changes . mount and pandey reported on the importance of the systems biology application for new drug finding . they emphasized the inter - relations between the different biomolecules in an event , such as cancer development ( 40 ) . in fact , such interrelationships between members of a biological network make sequential steps logical . curcumin , the active ingredient of turmeric , makes sequentially , activation or inactivation in the signaling pathways ( 16 ) . according to the in silico analysis of the present study , the cumulative effects of cinnamaldehyde or eugenol therefore , any prediction or estimation on phytochemical targets is not true without considering pre or post events in a signaling pathway . curcumin down - regulates the cell division , apoptosis and metastatic genes in nf - kappa b signaling in a sequential order ( 16 ) . cinnamaldehyde is proposed as a chemopreventive agent and has toxic effect on cancer cell lines ( 8 , 41 ) . altogether , cinnamaldehyde and eugenol proposed as anticancer agents which affect the human cells through the telomere shortening ; they change the gene folds and have many targets , of which some were checked virtually and 3 , experimentally in the present study . combining the in silico and experiments offer a novel view of the phytochemicals effects on the stem cell aging . this study is a novel one as it focused on the phytochemicals effects on hascs . the results of this study revealed that cinnamaldehyde and eugenol might be useful in cancer therapy or chemoprevention because of the induction of stem cell aging characteristics . in silico predictions , confirmed the results of ex - vivo interventional results . the aims of this study were to explore the stem cell anti - aging properties of the phytochemicals but surprisingly , the adverse results were obtained . the cinnamaldehyde and eugenol could be considered as the potential chemothera - peutic agents for cancer prevention via telomere shortening or telomerase blockade in proliferating cells such as cancer stem cells . however , the field is so large for complementary works on the shelterins , regulatory and epigenetics agents . the authors declare that there is no conflict or competing interest for the publication of the present data .

objective(s):to investigate the effect of cinnamaldehyde and eugenol on the telomere - dependent senescence of stem cells . in addition , to search the probable targets of mentioned phytochemicals between human telomere interacting proteins ( tips ) using in silico studies.materials and methods : human adipose derived stem cells ( hascs ) were studied under treatments with 2.5 m / ml cinnamaldehyde , 0.1 g / ml eugenol , 0.01% dmso or any additive . the expression of tert , akt1 and dkc1 genes and the telomere length were assessed over 48-hr treatment . in addition , docking study was conducted to show probable ways through which phytochemicals interact with tips.results:treated and untreated hascs had undetectable tert expression , but they had different akt1 and dkc1 expression levels ( ci=0.95 ; p<0.05 ) . the telomere lengths were reduced in phytochemicals treated with hascs when compared with the untreated cells ( p<0.05 ) . docking results showed that the tips might be the proper targets for cinnamaldehyde and eugenol . data mining showed there are many targets for cinnamaldehyde and eugenol in the intracellular environment.conclusion:the general effect of cinnamaldehyde and eugenol is their induction of stem cell senescence . therefore , they could be applicable as chemo - preventive or antineoplastic agents .

Introduction Materials and Methods Cell culture, gene folds and telomere length Experimental design Protein tertiary structure prediction and docking study Statistical analysis Data mining using protein-protein association search tools Results Gene expression and telomere length Protein tertiary structure modeling and docking results Association network information Discussion Conclusion Conflict of interest

cinnamon ( cinnamomum zeylanicum ) and clove ( syzygium aromaticum ) are rich in two active ingredients , which are , cinnamaldehyde and eugenol , respectively ( 5 , 6 ) . anti - mutagenicity , anti - oxidant , anti - inflammatory and anti - depressant activities have also been reported for cinnamaldehyde and eugenol ( 5 , 9 - 11 ) . the effect of phytochemicals on telomere length and telomerase interacting proteins of proliferating stem cells have not been reported yet . recently , we have reported that cinnamaldehyde and eugenol affect the human adipose derived stem cells ( hascs ) viability , doubling time and differentiation ( 12 ) using cheminforma - tics and experimental approaches . in the present work , the effect of cinnamaldehyde and eugenol on hascs telomere length was determined . also , three important telomere/ telomerase interacting proteins ( tips ) including tert , protein kinase b isoform 1 ( akt1 ) and dkc1 gene expressions under cinnamaldehyde and eugenol treatment were assessed . further , experimental methods are becoming more complicated , sensitive and accurate , but they are not empirical to study the ligand - protein docking . therefore , in the present investigation , using computational biology and docking tools , the probable cinnamaldehyde and eugenol interactions with tips were explored . four groups of hascs studied were treated for 48 hr : 2.5 m / ml cinnamaldehyde treated , 0.1 g / ml eugenol treated , 0.01% dmso treated and untreated hascs . cinnamaldehyde and eugenol were primarily dissolved in 0.01% dmso in phosphate buffer , which is the solvent . according to mentioned study , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol exert better cell viability and doubling time on hascs ( 12 ) . treated and untreated hascs were assessed for the telomere length and the expression of tert , akt1 and dkc1 genes . semi - quantitative pcr was done for tert , akt1 , dkc1 and actin - beta gene expression , and quantitative pcr ( qpcr ) was done for telomere length determination . for absolute telomere length by diploid genome measurement using qpcr , primer pairs used for expression analysis of human tert , akt1 , dkc1 and housekeeping actin - beta genes oligomers used for absolute quantification of hascs telomere length docking study needs 3ds structures of the proteins and ligands . the telomerase interacting proteins ( tips ) were selected according to the telomerase database ( http://telomerase.asu.edu/ ) ( 18 ) . for docking analysis , the mol2 format of cinnamaldehyde and eugenol molegro virtual docker ver.4.2 was used for docking analysis ; this software has about 87% docking accuracy ( 22 ) . four groups of hascs studied were treated for 48 hr : 2.5 m / ml cinnamaldehyde treated , 0.1 g / ml eugenol treated , 0.01% dmso treated and untreated hascs . cinnamaldehyde and eugenol were primarily dissolved in 0.01% dmso in phosphate buffer , which is the solvent . according to mentioned study , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol exert better cell viability and doubling time on hascs ( 12 ) . treated and untreated hascs were assessed for the telomere length and the expression of tert , akt1 and dkc1 genes . semi - quantitative pcr was done for tert , akt1 , dkc1 and actin - beta gene expression , and quantitative pcr ( qpcr ) was done for telomere length determination . for absolute telomere length by diploid genome measurement using qpcr , primer pairs used for expression analysis of human tert , akt1 , dkc1 and housekeeping actin - beta genes oligomers used for absolute quantification of hascs telomere length the telomerase interacting proteins ( tips ) were selected according to the telomerase database ( http://telomerase.asu.edu/ ) ( 18 ) . for docking analysis , the mol2 format of cinnamaldehyde and eugenol were prepared from zinc database ( http://zinc.docking.org ) ( 21 ) . untreated hascs were compared with 0.01% dmso , 2.5 m / ml cinnamaldehyde or 0.1 g / ml eugenol treated ones . 0.01% dmso and 2.5 m / ml cinnamaldehyde treated hascs had higher akt1 folds ( p<0.05 ) when compared with the untreated cells . but the akt1 folds did not change in the 0.1 g / ml eugenol treated cells as compared to the untreated hascs ( p>0.05 ) ( figure 1 ) . the 0.01% dmso and 2.5 m / ml cinnamaldehyde significantly increased the dkc1 gene folds in hascs ( p<0.05 ) . but 0.1 g / ml eugenol decreased the dkc1 folds ( p<0.05 ) compared with the untreated cells ( figure 2 ) . untreated hascs has the longest telomere length as compared to 0.01% dmso , 2.5 m / ml cinnamal - dehyde or 0.1 g / ml eugenol treated hascs ( figure 3 ) . there was no statistical difference among 0.01% dmso and untreated or phytochemicals treated cells ( p>0.05 ) . generally , the phytochemicals treated hascs had shortened telomeres as compared to the untreated cells ( p<0.05 ) . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the akt1 expression folds in hascs . nevertheless , the 0.1 g / ml eugenol did not affect akt1 expression significantly tukey - hsd and anova analysis for dkc1 folds comparison . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the dkc1 expression folds in hascs . but the 0.1 g / ml eugenol decreased dkc1 expression significantly telomere length comparison between examined groups . the control hascs , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol significantly decreased the telomere length ( p - value < 0.05 ) but not in comparison with 0.01% dmso group ( p - value > 0.05 ) figure 4 shows the gene name , number of docking sites and the best minimum docking scores of cinnamaldehyde and eugenol with 22 tips . dendrogram of cluster analysis for 22 tips plus telomerase ( tert ) ; left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores . some important docking results are shown in figure 6 - 14 docking scores of both cinnamaldehyde and eugenol were analyzed using cluster analysis ( figure 4 ) and one - way anova ( figure 5 ) . the cinnamaldehyde and eugenol docked with tips in molegro virtual docker ( mvd ) software . it is the best score of dockings and belong to the category 5 of clustering docking result of tert ( modeled structure ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 2 of clustering docking result of rnpc2 ( pdb i d : 2cq4 ) protein with cinnamaldehyde ; above : binding site of cinnamaldehyde ( green ) on the protein ; below : binding pocket has not hydrogen bounds . the molecule eugenol locates in a deep binding pocket of protein figure 15 shows an association network between tips and telomerase ; this is plotted using string data base ( see materials and methods ) . some abbreviations in the string - db plotted net were not mentioned in the text ; they include : rbm38 equals rnpc1 ; rbm39 equals rnpc2 ; xrcc6 equals ku70 ; xrcc5 equals ku80 ; ncl equals nucleolin ; la equals ssb ; cib1 equals kip ; ywhaq equals 14 - 3 - 3 ; tmed10 equals p23 untreated hascs were compared with 0.01% dmso , 2.5 m / ml cinnamaldehyde or 0.1 g / ml eugenol treated ones . 0.01% dmso and 2.5 m / ml cinnamaldehyde treated hascs had higher akt1 folds ( p<0.05 ) when compared with the untreated cells . but the akt1 folds did not change in the 0.1 g / ml eugenol treated cells as compared to the untreated hascs ( p>0.05 ) ( figure 1 ) . the 0.01% dmso and 2.5 m / ml cinnamaldehyde significantly increased the dkc1 gene folds in hascs ( p<0.05 ) . but 0.1 g / ml eugenol decreased the dkc1 folds ( p<0.05 ) compared with the untreated cells ( figure 2 ) . untreated hascs has the longest telomere length as compared to 0.01% dmso , 2.5 m / ml cinnamal - dehyde or 0.1 g / ml eugenol treated hascs ( figure 3 ) . there was no statistical difference among 0.01% dmso and untreated or phytochemicals treated cells ( p>0.05 ) . generally , the phytochemicals treated hascs had shortened telomeres as compared to the untreated cells ( p<0.05 ) . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the akt1 expression folds in hascs . nevertheless , the 0.1 g / ml eugenol did not affect akt1 expression significantly tukey - hsd and anova analysis for dkc1 folds comparison . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the dkc1 expression folds in hascs . but the 0.1 g / ml eugenol decreased dkc1 expression significantly telomere length comparison between examined groups . the control hascs , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol significantly decreased the telomere length ( p - value < 0.05 ) but not in comparison with 0.01% dmso group ( p - value > 0.05 ) figure 4 shows the gene name , number of docking sites and the best minimum docking scores of cinnamaldehyde and eugenol with 22 tips . dendrogram of cluster analysis for 22 tips plus telomerase ( tert ) ; left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores . some important docking results are shown in figure 6 - 14 docking scores of both cinnamaldehyde and eugenol were analyzed using cluster analysis ( figure 4 ) and one - way anova ( figure 5 ) . the cinnamaldehyde and eugenol docked with tips in molegro virtual docker ( mvd ) software . it is the best score of dockings and belong to the category 5 of clustering docking result of tert ( modeled structure ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 2 of clustering docking result of rnpc2 ( pdb i d : 2cq4 ) protein with cinnamaldehyde ; above : binding site of cinnamaldehyde ( green ) on the protein ; below : binding pocket has not hydrogen bounds . figure 15 shows an association network between tips and telomerase ; this is plotted using string data base ( see materials and methods ) . some abbreviations in the string - db plotted net were not mentioned in the text ; they include : rbm38 equals rnpc1 ; rbm39 equals rnpc2 ; xrcc6 equals ku70 ; xrcc5 equals ku80 ; ncl equals nucleolin ; la equals ssb ; cib1 equals kip ; ywhaq equals 14 - 3 - 3 ; tmed10 equals p23 recently , we have reported that cinnamaldehyde and eugenol affect the hascs doubling time and differentiation ( 12 ) . in the current study , cinnamalde - hyde and eugenol did not increase the tert expression to a detectable level in hascs . when 0.01% dmso is also present in the cinnamal - dehyde and eugenol groups , as the solvent of phytochemicals ( see materials and methods ) , the ultimate effect of both phytochemicals on the akt1 gene expression will decrease . this evidence confirms more potent effect of eugenol on the decrease of the akt1 expression . cinnamaldehyde or dmso - treated hascs increased the dkc1 gene expression folds but the eugenol decreased it severely as compared to the untreated cells . these findings suggest that the final targets of cinnamaldehyde and eugenol are different in the intracellular environment . the effect of cinnamal - dehyde and eugenol on some tips or cell aging was reported previously . the result of the present study on telomere length showed that damage such as telomere shortening may be obvious ( figure 3 ) . the hallmark of some publications is the insignificance of the effect of solvents on the gene expression of treated cells . king and coworkers did not report the effect of dmso on the hct116 cell line . in the current study , it was shown that a low concentration of dmso increases both akt1 and dkc1 gene folds when compared with the untreated cells . cinnamaldehyde and eugenol reduced the telomere lengths as compared to the untreated cells notably , but insignificantly when compared with dmso treated hascs . cinnamaldehyde and eugenol treated hascs telomeres were not meaningfully different from dmso treated cells ( figure 3 , p > 0.05 ) , although the telomere lengths were shortened . it is assumed that the broad ranges of telomere lengths in the studied groups ( see standard deviations ) , are responsible for the results of telomere length comparisons insignificance . the decreased telomere length , in the presence of phytochemicals , in the present work could be logical when considering the docking results . the docking results showed that cinnamaldehyde and eugenol attached well within the binding pockets on the tips . based on the docking results , it is assumed that cinnamaldehyde and eugenol interact with chaperons , such hsp90aa1 , or chaperon inducers ; then they change the behaviors of tips . in addition , the findings of the present work proposed that cinnamaldehyde and eugenol might exert similar effects on the telomerase and tips . the molecular effects of cinnamaldehyde and eugenol on the hascs were telomere shortening and the gene expression fold changes . the role of herbal ingredients on the telomere or telomerase dependent anti - aging or aging induction is not well known . intracellular environment has dynamic properties ( 33 ) ; therefore , the chemicals interact with biomolecules in a dynamic way . elizabeth h. blackburn is a noble prizewinner for her works on the telomere and telomerase enzyme . she believes the telomerase and the telomere interacting enzymes exert four distinct biochemical effects on the telomeric dna in vivo : 1 . in fusion with another telomere or dna end , it has been shown that using computational tools , the cinnamaldehyde and eugenol may target tips efficiently . after cinnamaldehyde and eugenol docking with tips or regulating proteins , three effects are probable : telomere shortening , lengthening or no length change . according to the map , the probable effects of the cinnamaldehyde and eugenol on the senescence and expected events after the cell treatment . the cinnamaldehyde and eugenol may bind to tips or act on their gene expression and impel the signal transduction pathways . the left gray box shows two proteins with negative effects on the telomere or telomerase . in our work then , the red lines show the probable occurred events in the current work the hnrnpa1 activates the telomerase ( 35 ) , but as shown in figure 14 , it also interacts with telomeric repeats ( 36 ) . same result was derived for cinnamaldehyde and hnrnpa1 docking and exactly in the binding pocket ( the result is not shown ) . all mentioned studies show negative effect of eugenol on the cellular proteins and are in accordance with the experimental and virtual findings of the present study . weibel and hansen have shown that the cinnamaldehyde binds to the nucleophilic groups of proteins ( 38 ) . python and coworkers evaluated the effect of cinnamaldehyde on 80 gene expression profiles in dendritic cells and mutz-3 cell line . all the mentioned studies confirm the present study results of cinnamaldehyde effect on the gene expression changes . in this work , however , in comparison with the only dmso treated hascs , cinnamaldehyde decreased akt1 and increased dkc1 folds . these findings are in accordance with python and coworkers results on the discrepant behavior of cinnamaldehyde in the gene expression folds . the network shows that the protein - protein interactions may be weak or strong ; therefore , the docking affects the quantities of signaling changes . according to the in silico analysis of the present study , the cumulative effects of cinnamaldehyde or eugenol therefore , any prediction or estimation on phytochemical targets is not true without considering pre or post events in a signaling pathway . altogether , cinnamaldehyde and eugenol proposed as anticancer agents which affect the human cells through the telomere shortening ; they change the gene folds and have many targets , of which some were checked virtually and 3 , experimentally in the present study . combining the in silico and experiments offer a novel view of the phytochemicals effects on the stem cell aging . the results of this study revealed that cinnamaldehyde and eugenol might be useful in cancer therapy or chemoprevention because of the induction of stem cell aging characteristics . the cinnamaldehyde and eugenol could be considered as the potential chemothera - peutic agents for cancer prevention via telomere shortening or telomerase blockade in proliferating cells such as cancer stem cells .

conversion and storage of sunlight into renewable fuels using ceria - based solar thermochemical redox cycles has gained considerable interest in recent years . ceria and its solid solutions are promising redox materials because of their ability to show high oxygen storage and release capacities , fast redox kinetics , single - phase stability at elevated temperatures , and high nonstoichiometries and abundant existence in earth s ore deposits . two - step thermochemical redox cycling comprises ( 1 ) an endothermic reduction of ceria driven by concentrated solar energy at elevated temperatures ( tred > 1673 k ) and low oxygen partial pressures and ( 2 ) an exothermic oxidation of reduced ceria by splitting h2o and co2 into h2 and co ( syngas ) at lower temperatures ( t < tred ) . if desired , syngas can further be processed to liquid fuels with well - developed catalytic conversion procedures , viz . by fischer in general , solar - to - fuel energy conversion efficiency relies on the oxygen storage and release capacity , the radiative heat absorptivity , and kinetics of ceria toward co2 . various studies have investigated reduction and oxidation kinetics of pure and doped ceria solid solutions . using the weight relaxation methodology , ackermann et al . determined ambipolar diffusion coefficients of pure ceria pellets at high temperatures ( t < 1773 k ) . based on the results , reduction reaction times less than 10 s are estimated for bulk diffusion length scales of 0.4 mm and less . because of such rapid reaction rates and relatively small characteristic diffusion lengths , reduction within solar reactors is most likely limited by the heating rate rather than chemical kinetics . oxidation rates , in contrast , are shown to be primarily dictated by the chemical kinetics of splitting co2 and h2o . for example , gopal and haile used an electrical conductivity relaxation technique for determination of bulk oxygen diffusivity within sm - doped ceria and reported 40 times larger surface reaction rate constants for splitting co2 compared to h2o . examined activation energies and determined the average fuel production rates ( h2o / co2 = 2:1 ) by measuring temporal downstream gas concentrations for nonstoichiometric porous sm - doped ceria placed in a packed bed reactor . in contrast to gopal and haile , h2 production rates were generally higher compared to co production rates , and a lower activation energy for dissociation of h2o was observed compared to co2 . furler et al . reported an increase in co2 splitting rates when surface area is increased by using structures containing micrometer - sized interconnected pores . similar observations were confirmed by venstrom et al . and petkovich et al . with structures consisting of three - dimensionally ordered and interconnected submicrometer - sized pores . additionally , furler et al . showed decreasing rates with increasing temperature beyond 1000 k because of a strong influence of the backward reaction with product gas co. srensen reported changes in oxidation rates and characteristic activation energies with co2 for different subphases formed at high reduction extents by using quasi - isothermal thermogravimetry at very low oxygen partial pressures ( po2 < 10 atm ) . to date , such phase changes have not been considered for thermochemical fuel conversion applications . in summary , there is a general consensus that oxidation rates are limited by the specific surface area , whereas reduction rates are typically limited by the rate of heat transfer because of rapid ambipolar diffusion at such elevated temperatures . despite the promising proof - of - principles in applying the material in a reactor and initial kinetics studies , investigation of the splitting kinetics and associated other than the work by srensen , there is little work that has investigated the effect of nonstoichiometry on oxidation rates , yet this data is critical for the design of solar reactors . therefore , within the framework of this investigation , we elucidate co2 reduction rates over nonstoichiometric ceria for fine powder and sintered pellets , a very broad range of temperatures and co2 concentrations , and a large variation of nonstoichiometries . to gain insight into morphology , structure , and chemistry with respect to nonstoichiometry and co2 reactivity , scanning electron microscopy ( sem ) , raman spectroscopy , and thermogravimetry are utilized . ceria samples with different length scales were used , namely , fine powder ( sigma - aldrich , < 5 m , 99.9% trace metals basis ) and sintered pellets made from the same powder or 10 mol % gd doped ceria ( sigma - aldrich , 510 nm , ce0.9gd0.1o1.95 ) . the pellets have a diameter and thickness of 8.8 mm and 1.5 mm , respectively , and were uniaxially cold pressed at 8 tons . the pellets were sintered for 5 h at 1873 k under atmospheric conditions resulting in a porosity < 0.05 . the grain size was 2050 m and 310 m for the undoped and gd - doped ceria pellets , respectively . the mass of the ceria pellets and powder samples were 600 mg and 25100 mg , respectively . isothermal oxidation experiments were conducted using a thermogravimetric analysis instrument ( tga , netzsch sta 409 cd ) . the ceria sample was reduced at elevated temperatures and then cooled at a rate of 20 k / min to the desired oxidation temperature followed by isothermal oxidation with co2 . two different methods were applied to reduce ceria : ( 1 ) thermal reduction at 1773 k at low oxygen partial pressure 10 atm ( pellets ) and ( 2 ) chemical reduction at 1373 k enhanced with ph2 = 0.02 atm ( powder and pellets ) to prevent sintering . from the absolute sample mass , ms , and temporal weight loss , m(t ) , nonstoichiometry , (t ) , was calculated as shown in eq 1.1where mceo2 and mo are the molar mass of ceria and oxygen , respectively . the nonstoichiometry right before starting oxidation with co2 is denoted as the initial nonstoichiometry , i , as schematically shown in figure 1 . it is changed by varying reduction temperature , reduction duration , and gas flow composition ( low po2 or ph2 = 0.02 atm ) . rate of co production was evaluated at constant nonstoichiometry , denoted as eval , which represents an averaged value for the sample according to eq 1 , and is necessarily smaller than i ( see figure 1 ) . buoyancy effects were corrected with blank experiments using equivalent masses of inert zirconium(iv ) oxide powder ( sigma - aldrich , 5 m , 99% trace metals basis ) . additionally , ceria powder oxidation rates were corrected for rate contributions from small oxygen levels present in the system . rate of co production was evaluated at constant nonstoichiometries , eval , for oxidations that start from different initial nonstoichiometries , i , where i = f(tred , t , po2 , ph2 ) . a hitachi tm-1000 and zeiss supra vp55 sem instrument were used to optically characterize the samples . the spectra were generated with an excitation wavelength of 532 nm by taking the average of three repetitions with an integration time of 20 s. all samples described in this section were reduced for 30 min at 1373 k with ph2 = 0.02 atm in argon at a total volumetric flow rate of 320 ml min , leading to an average i of 0.1 . figure 2 shows versus time during oxidation for two different powder masses , 26.8 mg ( white triangles ) and 54.6 mg ( black circles ) for different temperatures and co2 concentrations . from the visual similarity of the weight relaxation curves , isothermal weight relaxation curves of ceria oxidation for two different sample masses of 26.8 mg ( white triangles ) and 54.6 mg ( black circles ) at different temperatures and co2 concentrations . figure 3a shows relative mass changes as a function of time during oxidation for various temperatures ranging from 693 to 773 k and a constant pco2 ( 0.02 atm ) . in general , oxidation rates increase with increasing temperature , and the oxidation time decreases from 25 to 3 min . at higher temperatures where the reaction is more rapid , however , external mass - transfer limitations and/or the onset of the reverse reaction is eventually observed . as seen in figure 3b for t > 783 k , rates are less dependent on temperature and eventually decrease with increasing temperature . for example , oxidation rates between 793 and 853 k are nearly identical with an oxidation time of 2 min . further kinetic analysis of the powder samples is limited to temperatures below 773 k where these limitations are not observed . isothermal oxidation of ceria powder between ( a ) 693 and 773 k with pco2 = 0.02 atm and ( b ) 793 and 853 k with pco2 = 0.02 atm . additional experiments were conducted for pco2 = 0.005 , 0.01 , and 0.04 atm , as well as for mixtures of ceria powder and inert zirconia powder with mass loadings ( mceo2/ms ) of 50% , 75% , or 100% for pco2 = 0.02 atm . in figure 4 , the natural logarithm of co production rates ( rco ) evaluated at eval = 0.04 is shown versus inverse temperature . rco increases with increasing temperature and co2 concentration as expected ( see least - squares fitted trend lines ) . there is little dependence on the inert particle mass , giving us further confidence that data are not limited by external mass transfer . rates of co production over oxidizing ceria powder plotted as a function of the inverse temperature for different co2 concentrations at a constant nonstoichiometry . orange circles and blue diamonds are results from ceria powder mixed with inert zirconia powder with a ceria mass loading of 75 and 50% , respectively . activation energy , ea , was extracted using a simplified rate equation for a more detailed analysis of data . the simplified kinetic rate expression for the case of co2 reduction over nonstoichiometric ceria , rco , was defined as the product of the reaction rate constant , k(t ) , the reaction mechanism , f( ) , and the co2 concentration , cco2 , with reaction order , n , as shown in eq 2.2 the co2 concentration is related to the pco2 through the following relationship:3 for constant composition , the slope of the natural logarithm of kf as a function of inverse temperature yields the activation energy of the reaction . figure 5a shows natural logarithm of kf as a function of inverse temperature for several pco2 data sets at eval = 0.015 and 0.06 calculated from rate data according to eqs 2 and 3 . the data confirms arrhenius type dependency and shows no dependency on co2 concentration for n 0.45 ( numerically determined through a least - squares minimization ) . it should be noted that the function used to describe the reaction mechanism has no bearing on the determination of activation energy because it is evaluated at constant composition . figure 5b shows ea , extracted from the data sets presented in figure 4 , as a function of eval . in general , ea stays almost constant over all eval for lower pco2 values , with a mean value of 145 kj mol . for pco2 = 0.04 , ea increases with increasing eval from 155 to 190 kj mol . one may hypothesize that in this case , where reactions are most rapid , there is insufficient heat dissipation leading to increased activation energy , in accordance with ref ( 5 ) . in effect , measured temperatures are probably lower than those observed locally . an additional measurement error is related to the small mass changes due to oxidation in comparison to the natural drifts of the system with time . furler et al . reported activation energies from 90 up to 136 kj mol for pure ceria at eval = 0.015 . however , oxidation rates were determined from highly porous structures and may be partly affected by pore diffusion limitations resulting in a lower apparent activation energy . ( a ) arrhenius plot of oxidizing ceria powder for several co2 concentrations at constant nonstoichiometries ; ( b ) activation energy of oxidizing ceria powder as a function of nonstoichiometry ( see figure 4 for legend ) . to investigate oxidation behavior for t > 783 k , isothermal weight relaxation experiments were conducted with dense ceria pellets where oxidation rates were much slower than for the particles . experimental errors associated with small mass changes during oxidation could be suppressed because substantially larger sample masses were utilized . initially , pellets were reduced to different nonstoichiometries and subsequently oxidized at different temperatures . figure 6a shows rco ( eval = 0.04 ) in log space as a function of inverse temperature ( 913 t 1053 k , pco2 = 0.2 atm , eval = 0.04 ) for four different initial nonstoichiometries , namely , i = 0.07 , 0.154 , 0.19 , and 0.214 . interestingly , rates were strongly dependent on the initial nonstoichiometry , increasing by orders of magnitude with decreasing i for the lowest temperatures . at higher temperatures here there is not a linear increase of rates with increasing temperature , as was observed for lower temperatures , presumably because of the onset of the reverse reaction . figure 6b shows the same data presented in figure 6a , but as a function of i for varying temperature . here , the transition in rates at high nonstoichiometries is more easily observed . co production rates for pco2 = 0.2 atm evaluated at eval = 0.04 : ( a ) as a function of inverse temperature for several i ; ( b ) as a function of i for several temperatures . such behavior may be based on a phase change and is a typical explanation for a sudden transition in a physical property of solid oxides . normally , ceria is assumed to be a single - phase material for the purpose of thermochemical applications ; however , several changes in local near order have been reported . for example , bevan and kordis used sudden changes in thermodynamic properties of ceria to construct a phase diagram between temperatures of 909 and 1442 k and between 0 and 0.5 . subphase changes have also been hypothesized by sorenson to explain sudden changes in the slope of partial gibbs free energies at high nonstoichiometries . several ordered intermediate phases were proposed within the -phase region , each consisting of its own apparent nonstoichiometric single phase . in fact , the rate transition region shown in figure 6b is found to be very close to a phase boundary , as shown in figure 7 . here , the phase diagram of ceo2 , determined from specific heat measurements reported by riess and co - workers with y = 2 , is plotted alongside colored dots representing the transition region extracted from figure 6b at 943 , 993 , 1043 , and 1093 k. each point is shifted slightly to lower i , yet parallel to a phase boundary . one explanation for a shift toward lower compared to the boundary might be because the samples were not fully equilibrated . thus , the surface where the reaction with co2 takes place might be reduced to nonstoichiometries above the phase boundary , whereas some of the bulk remains below . we do not believe the hindered rates are due to carbonate formation because the mass balance between released o2 and produced co is shown to be closed in this work and under similar reaction conditions in other works . additionally , li et al . showed with fourier transform infrared spectroscopy that carbonates thermally desorb for t > 773 k. furthermore , we saw no evidence of carbonate formation from unreasonable mass changes observed during oxidation . colored points represent initial nonstoichiometries of pellets where transition for drastic decrease in co production rate is observed with tga measurements ( see figure 6b ) . cationic changes for the ceria pellets with respect to the oxygen nonstoichiometry . for this , we compare ( i ) a freshly sintered oxidized pellet , ( ii ) two chemically reduced pellets with different reduction durations at 1373 k using ph2 = 0.02 atm , and ( iii ) two thermally reduced samples with different reduction durations at 1773 k at low oxygen partial pressures as shown in table 1 . during cooling , chemically and thermally reduced samples were held under pure argon for 15 min at 993 and 1073 k , respectively . chemical reduction at 1373 k for 120 min resulted in i 0.253 , which is clearly above the transition point of drastic decrease in co production rate ( see figure 6b ) . for a given cubic fluorite crystal structure such as in undoped ceria , there is only a single characteristic cationic anionic stretching vibration mode with raman activity , namely , the f2 g triply degenerated raman mode , which is viewed as a symmetric breathing mode of the oxygen ions around each cerium cation in the lattice , see refs ( 3537 ) for details . property changes such as oxygen nonstoichiometry bond strength changes , namely , the f2 g mode shifts in wavenumber with respect to the nonstoichiometry of the material . figure 8a shows the normalized raman intensity as a function of wavelength for all samples . no additional bands were detected , clearly confirming the single - phase nature of the ceria independent of the oxygen nonstoichiometry over the range of i = 00.253 . cationic breathing raman f2 g mode and is in accordance with earlier literature references by mcbride et al . extraction of the f2 g peak position and full width at half - maximum as a function of i was obtained through fitting data with a sixth - order polynomial function . increasing the oxygen nonstoichiometry lowers the f2 g raman peak position by half a wavenumber from 465 cm ( i 0 ) to 464.5 cm ( i 0.253 ) , figure 8b . this trend is accompanied by a broadening of full width at half - maximum with increasing oxygen nonstoichiometry from 7 to 9.5 cm for i 0 to 0.253 , respectively , figure 8c . conventionally , one would expect that with increasing oxygen nonstoichiometry and lattice expansion of the ceria , the f2 g signature peak would shift up to higher wavenumbers and peak width would broaden because of mixing of the band with second - order phonon scatter effects , see mcbride et al . for details . in contrast , we report a lowering of the f2 g mode in its wavenumber upon reduction of the ceria for the rather unusually high oxygen nonstoichiometries of 0.253 . cation near order and ceria lattice . in agreement with the ceria phase diagram , this is close to the phase transition regime of face - centered cubic cells with long - range defect defect interactions ( -phase ) to intermediate rhombohedral cell structures ( -phase ) . at this stage , the material may develop a phase instability that thermodynamically benefits an increased association energy of the reduced ce ions ( cece ) with oxygen vacancies ( vo ) to form dimer associates ( cece/vo ) . very recently , marrocchelli et al . highlighted how changes in chemical expansion may significantly alter lattice bond strength by atomic scale simulations in the ceria system ; for example , compressively straining the lattice can lead to shifts in the association energies and thereby result in oxygen anionic cationic bond compression , in agreement with refs ( 38 , and 4244 ) . raman spectroscopy of ceria pellet surface reduced to different i : ( a ) single f2 g peak at 465 cm ; ( b ) decreasing peak wavelength position with increasing i ; ( c ) increasing full width at half - maximum intensity with increasing i . to study oxidation behavior following thermal reduction , sintered ceria pellets were thermally reduced at 1773 k and po2 2.5 10 atm for different reduction times . compared to reduction with h2 , these slower rates are attributed to the fact that fewer small cracks are induced . this can be seen in figure 9 where we show sem pictures of the surface of the thermally reduced pellet in the top panel ( i = 0.034 ) and the chemically reduced in the bottom panel ( i = 0.128 ) . teller ( bet ) surface area without destroying their morphology and therefore could not quantify the change in bet surface area induced by cracks . sem pictures of ( top panel ) grain surface of a ceria pellet thermally reduced to i = 0.034 and ( bottom panel ) grain surface of a ceria pellet chemically reduced to i = 0.128 . figure 10a shows rco in log space at eval = 0.02 as a function of inverse temperature for 973 t 1273 k for co2 concentrations ranging from 0.1 to 0.4 atm . increasing rates are observed with increasing co2 concentration , whereas a clear turnover of rco with increasing temperature is observed for all co2 concentrations . similar behavior was observed after chemical reduction at these elevated temperatures , and is likely due to a shift in the chemical equilibrium . increasing co2 concentrations shift the transition to higher temperatures because higher co2 concentrations favor the forward reaction . oxidation rates were also strongly dependent on the initial nonstoichiometry and increased with i over the range of nonstoichiometries probed ( i < 0.06 ) . this is shown in figure 10b , in which the oxidation rates are shown at t = 1073 k for initial nonstoichiometries increasing from 0.031 to 0.052 . for reference , the rate versus inverse temperature is also shown for a single i , extracted from figure 10a . during chemical reduction in this reduction range , there was no observable effect on oxidation rates . the rates were affected only at higher , but in a contrasting way , that is , rates increased with increasing initial nonstoichiometry . ( a ) co production rates at eval = 0.02 for 973 t 1273 k and 0.1 pco2 0.4 atm concentrations for i = 0.048 ; ( b ) increasing co production rate with increasing i at t = 1073 k and pco2 = 0.1 atm . lnrco increases almost linearly with i for a range of operating conditions , as shown in figure 11 . here , rco is shown in log space as a function of i for eval = 0.02 , pco2 = 0.1 and 0.2 atm , and t = 1123 and 1173 k. rates at i 0.055 are almost twice as large as rates at i 0.035 . on the basis of the raman results and phase diagram discussed above , this behavior is probably not due to near - order phase changes . one possible explanation is an increase in charge - driven defect interactions caused by increasing electron and vacancy concentrations . this would necessarily affect the dominant transport processes occurring in the lattice . in an effort to test this hypothesis , similar experiments were performed with a 10 mol % gd doped ceria pellet that is expected to have less defect interactions under the conditions investigated . in fact , for ce0.9gd0.1o1.95 an ideal solution model accounting only for doubly ionized oxygen vacancies and localized electrons on the cerium lattice sites has been shown to be valid above 1073 k for < 0.04 . in the case of pure ceria , such a model is not applicable except in the case of very low nonstoichiometries ( < 0.01 ) . results are also shown in figure 11 , and in this case oxidation rates are mostly constant for i < 0.04 . initial nonstoichiometries greater than i = 0.04 were not possible in this system because of limitations in the operating temperature and baseline oxygen level . co production rates as a function of increasing initial nonstoichiometry for different temperatures and co2 concentrations for ceria and 10 mol % gd doped ceria . another possible explanation for increased rates with nonstoichiometry may be related to the increase in chemical expansion with increasing i . prior to reduction , the grain sizes within the pure ceria pellet are larger ( 2050 m ) compared to the grain sizes of 10 mol % gd doped ceria ( 310 m ) , and no cracks are visible ( figure 12a , c ) . however , following reduction , cracks are formed within pure ceria grains ( figure 12b ) which probably lead to an increased surface area . no such small intragrain cracks can be observed on grain surface of the reduced 10 mol % gd doped ceria pellet , as shown in figure 12d , probably because the small grains can accommodate chemical expansion more easily than larger grains . unfortunately , because the same degree of crack propagation does not occur in gd - doped ceria as in pure ceria , it is not possible to point toward a likely explanation for increasing oxidation rates with nonstoichiometry . therefore , we can only hypothesize that it is due to either increasing defect interactions or increasing surface area caused by chemical expansion . sem images : ( a ) grain surface of a stoichiometric pure ceria pellet ; ( b ) grain surface of pure ceria pellet reduced to i = 0.034 ; ( c ) grain surface of a stoichiometric 10 mol % gd doped ceria pellet ; and ( d ) grain surface of a stoichiometric 10 mol % gd doped ceria pellet reduced to i = 0.03 . it is shown that ceria oxidation rates are not only dependent on temperature and co2 concentration but also strongly dependent on reduction extent and surface morphology of the sample . for sintered ceria pellets reduced with h2 to extreme nonstoichiometries ( > 0.2 ) , a transition toward very slow oxidation rates is observed . specifically , as nonstoichiometry increased , the oxidation rate for a fixed oxygen concentration ( constant ) decreased . the nonstoichiometry where the transition occurs slightly increases with temperature and agrees well with a near - order phase change reported in literature . results are further supported with raman spectroscopy results that indicate an active compression of the oxygen anion < 0.06 , achieved during higher - temperature thermal reduction with low oxygen partial pressure , the opposite behavior is observed , and oxidation rates increase almost linearly with increasing nonstoichiometry for pure ceria . we attribute this trend to one of two phenomena : ( 1 ) the formation of defect clusters that affect bulk transport , primarily because such behavior is not observed for 10 mol % gd doped ceria where defect interactions are known to be less prevalent or ( 2 ) slight changes in morphology due to chemical expansion . while these results shed new light on oxidation rates of ceria under a broad range of operating conditions , further work is required to obtain more quantitative information about the rates and their relationship to nonstoichiometry so that they may ultimately be applied in system models to improve overall performance . for example , it will be important to decouple intrinsic reaction rates from any changes in subtle morphology during reduction ( i.e. , expansion , crack propagation ) . already , extensive data exists describing the chemical and thermal expansion behavior of these materials , but it will also be important to quantify how cracks propagate as a function of grain size and nonstoichiometry .

the kinetics of co2 reduction over nonstoichimetric ceria , ceo2 , a material of high potential for thermochemical conversion of sunlight to fuel , has been investigated for a wide range of nonstoichiometries ( 0.02 0.25 ) , temperatures ( 693 t 1273 k ) , and co2 concentrations ( 0.005 pco2 0.4 atm ) . samples were reduced thermally at 1773 k to probe low nonstoichiometries ( < 0.05 ) and chemically at lower temperatures in a h2 atmosphere to prevent particle sintering and probe the effect of higher nonstoichiometries ( < 0.25 ) . for extents greater than = 0.2 , oxidation rates at a given nonstoichiometry are hindered for the duration of the reaction , presumably because of near - order changes , such as lattice compression , as confirmed via raman spectroscopy . importantly , this behavior is reversible and oxidation rates are not affected at lower . following thermal reduction at very low , however , oxidation rates are an order of magnitude slower than those of chemically reduced samples , and rates monotonically increase with the initial nonstoichiometry ( up to = 0.05 ) . this dependence may be attributed to the formation of stable defect complexes formed between oxygen vacancies and polarons . when the same experiments are performed with 10 mol % gd3 + doped ceria , in which defect complexes are less prevalent than in pure ceria , this dependence is not observed .

Introduction Experimental Section Results and Discussion Conclusion

ceria and its solid solutions are promising redox materials because of their ability to show high oxygen storage and release capacities , fast redox kinetics , single - phase stability at elevated temperatures , and high nonstoichiometries and abundant existence in earth s ore deposits . two - step thermochemical redox cycling comprises ( 1 ) an endothermic reduction of ceria driven by concentrated solar energy at elevated temperatures ( tred > 1673 k ) and low oxygen partial pressures and ( 2 ) an exothermic oxidation of reduced ceria by splitting h2o and co2 into h2 and co ( syngas ) at lower temperatures ( t < tred ) . various studies have investigated reduction and oxidation kinetics of pure and doped ceria solid solutions . determined ambipolar diffusion coefficients of pure ceria pellets at high temperatures ( t < 1773 k ) . oxidation rates , in contrast , are shown to be primarily dictated by the chemical kinetics of splitting co2 and h2o . examined activation energies and determined the average fuel production rates ( h2o / co2 = 2:1 ) by measuring temporal downstream gas concentrations for nonstoichiometric porous sm - doped ceria placed in a packed bed reactor . showed decreasing rates with increasing temperature beyond 1000 k because of a strong influence of the backward reaction with product gas co. srensen reported changes in oxidation rates and characteristic activation energies with co2 for different subphases formed at high reduction extents by using quasi - isothermal thermogravimetry at very low oxygen partial pressures ( po2 < 10 atm ) . to date , such phase changes have not been considered for thermochemical fuel conversion applications . in summary , there is a general consensus that oxidation rates are limited by the specific surface area , whereas reduction rates are typically limited by the rate of heat transfer because of rapid ambipolar diffusion at such elevated temperatures . despite the promising proof - of - principles in applying the material in a reactor and initial kinetics studies , investigation of the splitting kinetics and associated other than the work by srensen , there is little work that has investigated the effect of nonstoichiometry on oxidation rates , yet this data is critical for the design of solar reactors . therefore , within the framework of this investigation , we elucidate co2 reduction rates over nonstoichiometric ceria for fine powder and sintered pellets , a very broad range of temperatures and co2 concentrations , and a large variation of nonstoichiometries . to gain insight into morphology , structure , and chemistry with respect to nonstoichiometry and co2 reactivity , scanning electron microscopy ( sem ) , raman spectroscopy , and thermogravimetry are utilized . ceria samples with different length scales were used , namely , fine powder ( sigma - aldrich , < 5 m , 99.9% trace metals basis ) and sintered pellets made from the same powder or 10 mol % gd doped ceria ( sigma - aldrich , 510 nm , ce0.9gd0.1o1.95 ) . the pellets were sintered for 5 h at 1873 k under atmospheric conditions resulting in a porosity < 0.05 . the ceria sample was reduced at elevated temperatures and then cooled at a rate of 20 k / min to the desired oxidation temperature followed by isothermal oxidation with co2 . two different methods were applied to reduce ceria : ( 1 ) thermal reduction at 1773 k at low oxygen partial pressure 10 atm ( pellets ) and ( 2 ) chemical reduction at 1373 k enhanced with ph2 = 0.02 atm ( powder and pellets ) to prevent sintering . from the absolute sample mass , ms , and temporal weight loss , m(t ) , nonstoichiometry , (t ) , was calculated as shown in eq 1.1where mceo2 and mo are the molar mass of ceria and oxygen , respectively . the nonstoichiometry right before starting oxidation with co2 is denoted as the initial nonstoichiometry , i , as schematically shown in figure 1 . it is changed by varying reduction temperature , reduction duration , and gas flow composition ( low po2 or ph2 = 0.02 atm ) . the spectra were generated with an excitation wavelength of 532 nm by taking the average of three repetitions with an integration time of 20 s. all samples described in this section were reduced for 30 min at 1373 k with ph2 = 0.02 atm in argon at a total volumetric flow rate of 320 ml min , leading to an average i of 0.1 . figure 2 shows versus time during oxidation for two different powder masses , 26.8 mg ( white triangles ) and 54.6 mg ( black circles ) for different temperatures and co2 concentrations . from the visual similarity of the weight relaxation curves , isothermal weight relaxation curves of ceria oxidation for two different sample masses of 26.8 mg ( white triangles ) and 54.6 mg ( black circles ) at different temperatures and co2 concentrations . figure 3a shows relative mass changes as a function of time during oxidation for various temperatures ranging from 693 to 773 k and a constant pco2 ( 0.02 atm ) . in general , oxidation rates increase with increasing temperature , and the oxidation time decreases from 25 to 3 min . at higher temperatures where the reaction is more rapid , however , external mass - transfer limitations and/or the onset of the reverse reaction is eventually observed . further kinetic analysis of the powder samples is limited to temperatures below 773 k where these limitations are not observed . isothermal oxidation of ceria powder between ( a ) 693 and 773 k with pco2 = 0.02 atm and ( b ) 793 and 853 k with pco2 = 0.02 atm . additional experiments were conducted for pco2 = 0.005 , 0.01 , and 0.04 atm , as well as for mixtures of ceria powder and inert zirconia powder with mass loadings ( mceo2/ms ) of 50% , 75% , or 100% for pco2 = 0.02 atm . rates of co production over oxidizing ceria powder plotted as a function of the inverse temperature for different co2 concentrations at a constant nonstoichiometry . the simplified kinetic rate expression for the case of co2 reduction over nonstoichiometric ceria , rco , was defined as the product of the reaction rate constant , k(t ) , the reaction mechanism , f( ) , and the co2 concentration , cco2 , with reaction order , n , as shown in eq 2.2 the co2 concentration is related to the pco2 through the following relationship:3 for constant composition , the slope of the natural logarithm of kf as a function of inverse temperature yields the activation energy of the reaction . an additional measurement error is related to the small mass changes due to oxidation in comparison to the natural drifts of the system with time . reported activation energies from 90 up to 136 kj mol for pure ceria at eval = 0.015 . however , oxidation rates were determined from highly porous structures and may be partly affected by pore diffusion limitations resulting in a lower apparent activation energy . ( a ) arrhenius plot of oxidizing ceria powder for several co2 concentrations at constant nonstoichiometries ; ( b ) activation energy of oxidizing ceria powder as a function of nonstoichiometry ( see figure 4 for legend ) . to investigate oxidation behavior for t > 783 k , isothermal weight relaxation experiments were conducted with dense ceria pellets where oxidation rates were much slower than for the particles . figure 6a shows rco ( eval = 0.04 ) in log space as a function of inverse temperature ( 913 t 1053 k , pco2 = 0.2 atm , eval = 0.04 ) for four different initial nonstoichiometries , namely , i = 0.07 , 0.154 , 0.19 , and 0.214 . interestingly , rates were strongly dependent on the initial nonstoichiometry , increasing by orders of magnitude with decreasing i for the lowest temperatures . at higher temperatures here there is not a linear increase of rates with increasing temperature , as was observed for lower temperatures , presumably because of the onset of the reverse reaction . co production rates for pco2 = 0.2 atm evaluated at eval = 0.04 : ( a ) as a function of inverse temperature for several i ; ( b ) as a function of i for several temperatures . such behavior may be based on a phase change and is a typical explanation for a sudden transition in a physical property of solid oxides . normally , ceria is assumed to be a single - phase material for the purpose of thermochemical applications ; however , several changes in local near order have been reported . here , the phase diagram of ceo2 , determined from specific heat measurements reported by riess and co - workers with y = 2 , is plotted alongside colored dots representing the transition region extracted from figure 6b at 943 , 993 , 1043 , and 1093 k. each point is shifted slightly to lower i , yet parallel to a phase boundary . one explanation for a shift toward lower compared to the boundary might be because the samples were not fully equilibrated . thus , the surface where the reaction with co2 takes place might be reduced to nonstoichiometries above the phase boundary , whereas some of the bulk remains below . for this , we compare ( i ) a freshly sintered oxidized pellet , ( ii ) two chemically reduced pellets with different reduction durations at 1373 k using ph2 = 0.02 atm , and ( iii ) two thermally reduced samples with different reduction durations at 1773 k at low oxygen partial pressures as shown in table 1 . during cooling , chemically and thermally reduced samples were held under pure argon for 15 min at 993 and 1073 k , respectively . for a given cubic fluorite crystal structure such as in undoped ceria , there is only a single characteristic cationic anionic stretching vibration mode with raman activity , namely , the f2 g triply degenerated raman mode , which is viewed as a symmetric breathing mode of the oxygen ions around each cerium cation in the lattice , see refs ( 3537 ) for details . property changes such as oxygen nonstoichiometry bond strength changes , namely , the f2 g mode shifts in wavenumber with respect to the nonstoichiometry of the material . no additional bands were detected , clearly confirming the single - phase nature of the ceria independent of the oxygen nonstoichiometry over the range of i = 00.253 . conventionally , one would expect that with increasing oxygen nonstoichiometry and lattice expansion of the ceria , the f2 g signature peak would shift up to higher wavenumbers and peak width would broaden because of mixing of the band with second - order phonon scatter effects , see mcbride et al . in contrast , we report a lowering of the f2 g mode in its wavenumber upon reduction of the ceria for the rather unusually high oxygen nonstoichiometries of 0.253 . in agreement with the ceria phase diagram , this is close to the phase transition regime of face - centered cubic cells with long - range defect defect interactions ( -phase ) to intermediate rhombohedral cell structures ( -phase ) . highlighted how changes in chemical expansion may significantly alter lattice bond strength by atomic scale simulations in the ceria system ; for example , compressively straining the lattice can lead to shifts in the association energies and thereby result in oxygen anionic cationic bond compression , in agreement with refs ( 38 , and 4244 ) . to study oxidation behavior following thermal reduction , sintered ceria pellets were thermally reduced at 1773 k and po2 2.5 10 atm for different reduction times . compared to reduction with h2 , these slower rates are attributed to the fact that fewer small cracks are induced . this can be seen in figure 9 where we show sem pictures of the surface of the thermally reduced pellet in the top panel ( i = 0.034 ) and the chemically reduced in the bottom panel ( i = 0.128 ) . sem pictures of ( top panel ) grain surface of a ceria pellet thermally reduced to i = 0.034 and ( bottom panel ) grain surface of a ceria pellet chemically reduced to i = 0.128 . figure 10a shows rco in log space at eval = 0.02 as a function of inverse temperature for 973 t 1273 k for co2 concentrations ranging from 0.1 to 0.4 atm . oxidation rates were also strongly dependent on the initial nonstoichiometry and increased with i over the range of nonstoichiometries probed ( i < 0.06 ) . this is shown in figure 10b , in which the oxidation rates are shown at t = 1073 k for initial nonstoichiometries increasing from 0.031 to 0.052 . ( a ) co production rates at eval = 0.02 for 973 t 1273 k and 0.1 pco2 0.4 atm concentrations for i = 0.048 ; ( b ) increasing co production rate with increasing i at t = 1073 k and pco2 = 0.1 atm . lnrco increases almost linearly with i for a range of operating conditions , as shown in figure 11 . here , rco is shown in log space as a function of i for eval = 0.02 , pco2 = 0.1 and 0.2 atm , and t = 1123 and 1173 k. rates at i 0.055 are almost twice as large as rates at i 0.035 . on the basis of the raman results and phase diagram discussed above , this behavior is probably not due to near - order phase changes . in an effort to test this hypothesis , similar experiments were performed with a 10 mol % gd doped ceria pellet that is expected to have less defect interactions under the conditions investigated . in fact , for ce0.9gd0.1o1.95 an ideal solution model accounting only for doubly ionized oxygen vacancies and localized electrons on the cerium lattice sites has been shown to be valid above 1073 k for < 0.04 . in the case of pure ceria , such a model is not applicable except in the case of very low nonstoichiometries ( < 0.01 ) . results are also shown in figure 11 , and in this case oxidation rates are mostly constant for i < 0.04 . initial nonstoichiometries greater than i = 0.04 were not possible in this system because of limitations in the operating temperature and baseline oxygen level . co production rates as a function of increasing initial nonstoichiometry for different temperatures and co2 concentrations for ceria and 10 mol % gd doped ceria . prior to reduction , the grain sizes within the pure ceria pellet are larger ( 2050 m ) compared to the grain sizes of 10 mol % gd doped ceria ( 310 m ) , and no cracks are visible ( figure 12a , c ) . however , following reduction , cracks are formed within pure ceria grains ( figure 12b ) which probably lead to an increased surface area . no such small intragrain cracks can be observed on grain surface of the reduced 10 mol % gd doped ceria pellet , as shown in figure 12d , probably because the small grains can accommodate chemical expansion more easily than larger grains . unfortunately , because the same degree of crack propagation does not occur in gd - doped ceria as in pure ceria , it is not possible to point toward a likely explanation for increasing oxidation rates with nonstoichiometry . sem images : ( a ) grain surface of a stoichiometric pure ceria pellet ; ( b ) grain surface of pure ceria pellet reduced to i = 0.034 ; ( c ) grain surface of a stoichiometric 10 mol % gd doped ceria pellet ; and ( d ) grain surface of a stoichiometric 10 mol % gd doped ceria pellet reduced to i = 0.03 . it is shown that ceria oxidation rates are not only dependent on temperature and co2 concentration but also strongly dependent on reduction extent and surface morphology of the sample . for sintered ceria pellets reduced with h2 to extreme nonstoichiometries ( > 0.2 ) , a transition toward very slow oxidation rates is observed . specifically , as nonstoichiometry increased , the oxidation rate for a fixed oxygen concentration ( constant ) decreased . the nonstoichiometry where the transition occurs slightly increases with temperature and agrees well with a near - order phase change reported in literature . results are further supported with raman spectroscopy results that indicate an active compression of the oxygen anion < 0.06 , achieved during higher - temperature thermal reduction with low oxygen partial pressure , the opposite behavior is observed , and oxidation rates increase almost linearly with increasing nonstoichiometry for pure ceria . we attribute this trend to one of two phenomena : ( 1 ) the formation of defect clusters that affect bulk transport , primarily because such behavior is not observed for 10 mol % gd doped ceria where defect interactions are known to be less prevalent or ( 2 ) slight changes in morphology due to chemical expansion .

does the radiology report , radiology s most conspicuous and permanent product , in its present form , meet the expectations of referring clinicians ? and if not , how can the report , and in a broader sense the communication between radiologists and referring clinicians , be improved ? one of our recent projects at the university of antwerp is cover ( clinicians opinions , views and expectations concerning the radiology report ) , a bi - national survey in the netherlands and flanders , the dutch - speaking part of belgium . the quantitative results of these surveys have been reported recently . at the end of the cover questionnaire , respondents were able to make suggestions on how to improve the radiology report . in this article , we present a synthesis of the suggestions of referring clinicians , both hospital specialists and general practitioners . we are well aware of the fact that radiologists are clinicians too but for practical purposes we will use the term clinicians as shorthand for referring clinicians in this paper . permission for the cover internet survey was obtained from the institutional review board of the authors medical center . informed consent and hipaa compliance were waived by the ethics committee , as no patient health data were used in the surveys . the medical director of each hospital sent an e - mail to all clinical specialists who routinely prescribe imaging examinations to invite them to participate . the e - mail addresses of general practitioners ( gps ) were found in the 2008 address book of the order of physicians of the province of antwerp , and all those who had such an address received the e - mail directly from the investigators . by clicking a hyperlink in the e - mail , responders were referred to a digital questionnaire prepared using surveymonkey , a commercial tool for internet surveys ( surveymonkey , portland , or , usa ) . the main part of the survey consisted of a set of 46 statements on the radiology report , for which respondents were asked to indicate their level of agreement according to a five - tiered likert scale . at the end of this section the survey was performed in two university and two community hospitals in flanders , one university and one community hospital in the netherlands , and among the general practitioners ( gps ) in the province of antwerp , flanders . it took place in two consecutive waves of 2 weeks in the course of the second half of 2008 or the first half of 2009 , according to the institution . after these two waves of 2 weeks , the survey was closed and the results were downloaded . the investigators identified , examined , ordered , counted , compared and synthesised subjects and themes by means of qualitative analysis software ( qsr nvivo 8 , qsr international , doncaster , vic , australia ) . of a total of 3,884 invitations to participate , we received 735 response forms from clinicians ( 18.9% ) . the rate of completed response forms with suggestions ranged from 29.1 to 37.5% , according to the institution or target group ( average 31.7% , i.e. 233 out of 735 ) ( table 1 ) . of the clinicians who made suggestions , 148 ( 63.5% ) were male and 85 ( 36.5% ) were female . their ages ranged from 26 to 78 years ( average 46.4 , median 46 , standard deviation 10.7 ) . an overview of the medical specialty of the responders can be found in table 2 . table 1response rates of clinicians and rate of completed questionnaires with suggestions in covercentre or groupsentresponded ( % ) with suggestions ( % ) community hospital 1 nl5916 ( 28.1)6 ( 37.5)university hospital 1 nl1,391163 ( 11.7)59 ( 36.2)community hospital 2 fl11976 ( 63.9)23 ( 30.3)community hospital 3 fl4525 ( 55.6)8 ( 32.0)university hospital 2 fl35965 ( 18.1)21 ( 32.3)university hospital 3 fl590108 ( 18.3)34 ( 31.5)total clinical specialists2,561453 ( 17.7)144 ( 31.8)total gps1,323282 ( 21.3)82 ( 29.1)grand total3,884735 ( 18.9)233 ( 31.7)nl the netherlands , fl flanders , belgiumtable 2specialities represented among responders with suggestions , absolute number and percentage of totalspecialitynumberpercentagegeneral practice8235.2paediatrics156.4surgery156.4internal medicine ( general)125.2other ( not listed)125.2oncology114.7gynaecology114.7anaesthesiology114.7cardiology93.9orthopaedics83.4gastroenterology - hepatology83.4physical and rehabilitation medicine62.6ear , nose and throat medicine62.6pulmonology52.1urology41.7neurology41.7psychiatry31.3ophthalmology31.3dermatology20.9endocrinology - diabetology20.9emergency medicine10.4rheumatology10.4nephrology10.4haematology10.4total233100.0 response rates of clinicians and rate of completed questionnaires with suggestions in cover nl the netherlands , fl flanders , belgium specialities represented among responders with suggestions , absolute number and percentage of total fifty - one different themes were identified , and suggestions were linked to one or several of these themes ( table 3 ) . we only present these numbers as an indication of the relative importance of the themes to the respondents . as we do not report the results of a quantitative study , we do not further specify the number of respondents who put forward each and every idea we present in this section . table 3frequency of themes mentioned three times or more in clinicians suggestions for improving the reportsubjectnumber of referencessubjectnumber of referencesclinical information and the clinical question94accessibility of the report10conclusion / impression of the report55multidisciplinary rounds8structured reports37content7communicating directly to the clinician25descriptive part of the report7completeness19report as training update for clinicians7integrating images or referring to images19satisfaction with the report7relevant findings outside of the clinical question19competence of the radiologist6mentioning a diagnosis / differential diagnosis19irrelevant reports6concise reporting17narrative reports5electronic patient record ( epr)16terminology5vague reports / the hedge16variable quality or approach5suggestions for further examinations16probability of a diagnosis4use of abbreviations14quality of the report4use of a standard lexicon14performing the right / inadequate examinations4measuring lesions13language3proofreading reports12preformatted text3 frequency of themes mentioned three times or more in clinicians suggestions for improving the report in the following sections , we present a synthesis of these suggestions , as well as a selection of quotes . each quote is accompanied by a note stating specialty , age , gender and place of activity ( fl flanders , belgium , nl the netherlands ) of the person quoted . many respondents thought it is the duty of the referring clinician to provide the radiologist with useful clinical information and a clear and unequivocal clinical question . it was felt that the clinical questions provided in the request form should be addressed in the report . pneumonia? , not just : no abnormal findings but also : no signs of pneumonia , so we know the radiologist has looked at the patient with the eye of a clinician . ( anesthesiologist , m , 41 , nl ) answering questions with new questions was considered inappropriate . several clinicians , gps especially , were convinced that the radiologist does not always read the clinical question on the request . so can i suppose they haven't been read ? ( gp , m , 40 , fl ) one of the respondents wrote if radiologists would present themselves credibly as imaging clinicians , referring clinicians would be more prone to provide useful information . mentioning the absence of clinical information was deemed unjust towards the referring clinician : a note saying : clinical information : none suggests that the referring clinician has not transmitted any information , while the request may have been lost , may have been removed by the technician , the patient was unable to present it , etc . ( ear , nose and throat specialist , m , 54 , fl ) one colleague was convinced that radiologists make too little use of their right to read the electronic patient record ( epr ) of inpatients for useful information . another thought that in the case of complex clinical questions , the radiologist should see the patient before and after the examination . clinicians were well aware of their responsibility to request the most appropriate examination . if the radiologist does not agree , she / he should not perform the examination and should contact the clinician . but : when a clinical specialist requests an examination , the radiologist has to check if the examination can answer the clinical question , but not question the indications for the examination . ( surgeon , m , 43 , nl ) the selection of an examination protocol should be done by a radiologist himself , not by a roentgen technician . even with extensive clinical information , i often see routine sequences without sagittal t2-weighted images in case of hydrocephalus ( aqueduct stenosis ? ) or without contrast when a brain tumor is suspected . ( non - listed specialist , m , 51 , fl ) radiologists could complement a tentative diagnosis by giving a degree of certainty . on the other hand : no conclusions like infiltrate can not be excluded. no test has a sensitivity of 100% . ( nephrologist , m , 35 , nl ) where appropriate , the conclusion should contain advice to the gp for follow - up examinations or for referral to a dedicated specialist . in reports of complex examinations such as ultrasound , the radiologist should describe accurately what has been seen , not just note that the examination is normal . pathological findings outside the scope of the examination however , e.g. a bone lesion on a chest x - ray , should always be mentioned . we sometimes have to go back three or four chest x - rays to know what the condition was ( anesthesiologist , f , 40 , fl ) if a diagnosis can not be made , a differential diagnosis should be presented . in case several diagnoses are possible , it would help if the radiologist could give an idea of the probability of each one . ( internist , m , 55 , fl ) radiologists should stick their necks out and take responsibility , rather than hiding themselves behind vague and ambiguous statements . the umbrella effect ( tumor can not be excluded ) does not help us in any way . ( gp , f , 49 , fl ) radiologists should always be aware of the specialty of the referring clinician and tailor their reports accordingly . requesting x - rays is part of my routine as a cardiologist but reports invariably tell me there is no pulmonary infiltrate . i get the impression that the radiologist does nt know anything about heart configuration , volume and so on . ( cardiologist , m , 66 , fl ) radiologists should only describe what they know : no comments on the quality and position of prostheses , as they are wrong most of the time . ( orthopaedic surgeon , m , 38 , nl ) although radiologists may have a strong interest in the clinical side of medicine , their judgement should be based on the results of imaging procedures . i notice more and more often clinical diagnoses that can not be made on the basis of imaging alone . [ ] radiologists have to distinguish diagnosis from suspicion . ( pulmonologist , m , 34 , nl ) how about measurements ? in case of a tumor , always measure ! the same in follow - up examinations , instead of talking about further diminution ( pediatrician , f , 49 , fl ) and : measurements would be more instructive if the standard deviation would be given . liver too big for age is less informative than liver volume too big , + 3.5 sd. ( neurologist , f , 36 , nl ) measurements too should be linked to specific images . radiology reports should be much shorter , schematic and concise , some of the respondents thought . so one can see in the wink of an eye where something abnormal was seen . reports should at least contain the clinical question , the examination technique , a description of the findings , a conclusion and sometimes advice . other tips : important findings should be highlighted . providing digital images ( which are not compatible with our mac ! ) is a waste of time unless they specify which images we need to look at . ( gp , f , 50 , fl ) it would be interesting if the radiologist would use an arrow to indicate pathology like a hernia , a cyst on us and especially in case of ct or mri ( gp , f , 56 , fl ) the conclusion was considered an essential part of the report by many , especially gps . it was felt that the conclusion should only contain relevant results , and more specifically the answer to the clinical question . the use of templates to promote reporting consistency was advised by some but rejected by others . ready - to - use reports need to be avoided , they cause too many errors . ( surgeon , m , 56 , fl ) no computer - generated reports in which just a few words have been changed . ( gp , f , 49 , fl ) the importance of applying guidelines and criteria for follow - up was emphasised . when evaluating scans in case of multiple sclerosis , use the barkhof criteria . ( neurologist , f , 36 , nl ) the contents need to comply with international guidelines written with the help of radiologists . in oncology , ( oncologist , m , 38 , nl ) several respondents objected to unusual abbreviations or eponyms . it 's a nuisance having to call the radiologist to ask for an explanation . ( gp , f , 33 , fl ) what about the length of the report ? according to some , reports of simple examinations can consist of one or two words , but reports of complex examinations should be substantial . however , several thought that long and tedious radiology reports often cover up the radiologist s incompetence to answer the clinical question . and even when long reports are indicated , the conclusion should be short and clear . the idea that easy communication between clinician and radiologist is in the interest of the patient and can prevent vague conclusions came up several times . if the radiologist sees something unexpected , or if an examination fails or causes adverse effects , he / she must call the clinician . clinicians mentioned the need to look at the images themselves , so they can give feedback to the radiologist . the direct phone number of the reporting radiologist should always be present , so they can be contacted in case something is not clear . ( gp , m , 30 , fl ) and : call centers are there for patients , not for doctors . ( internist , f , 50 , fl ) the report should also contain a schedule of the hours the radiologist can be reached directly . it was suggested that exceptionally complex examinations should be analysed together by radiologist and clinician . if a radiologist on call , e.g. a resident - in - training , has reported an examination orally , what has been said should be quoted in the report . if the final report contradicts these preliminary conclusions , that should be mentioned too . quite often a supervisor will add something to a report that was made during the night , with dramatic consequences for its conclusion . ( surgeon , m , 36 , nl ) if they have a choice , clinicians would rather work with radiologists they know and trust . some radiologists give very vague descriptions , but we most often know which ones do so . ( gp , f , 43 , fl ) radiologists do not have an easy job but in general they perform quite well , some clinicians thought . their s is a great contribution to our work and a strong support for what we do . ( gp , m , 55 , fl ) if an examination has failed because the patient has moved or contrast medium administration was omitted , it is not enough to state this in the report . ( orthopedic surgeon , m , 38 , nl ) in the case of ercp examinations , the report often only refers to the results of endoscopy . imaging studies should be subjected to a double blind protocol , according to one respondent . reports can have an educational side : good reports stimulate gps to study the images and can become a refresher course for their knowledge of anatomy . ( gp , m , 64 , fl ) the flawless use of their mother tongue is dear to some clinicians . there is a need for terminological consistency : standardization of terms like bulging , ( gp , f , 50 , fl ) speech recognition is still a matter of dispute : reports made using speech recognition are not corrected . . the result may be grammatically and semantically correct , but what about , for example , left many respondents thought it is the duty of the referring clinician to provide the radiologist with useful clinical information and a clear and unequivocal clinical question . it was felt that the clinical questions provided in the request form should be addressed in the report . pneumonia? , not just : no abnormal findings but also : no signs of pneumonia , so we know the radiologist has looked at the patient with the eye of a clinician . ( anesthesiologist , m , 41 , nl ) answering questions with new questions was considered inappropriate . several clinicians , gps especially , were convinced that the radiologist does not always read the clinical question on the request . so can i suppose they haven't been read ? ( gp , m , 40 , fl ) one of the respondents wrote if radiologists would present themselves credibly as imaging clinicians , referring clinicians would be more prone to provide useful information . mentioning the absence of clinical information was deemed unjust towards the referring clinician : a note saying : clinical information : none suggests that the referring clinician has not transmitted any information , while the request may have been lost , may have been removed by the technician , the patient was unable to present it , etc . ( ear , nose and throat specialist , m , 54 , fl ) one colleague was convinced that radiologists make too little use of their right to read the electronic patient record ( epr ) of inpatients for useful information . another thought that in the case of complex clinical questions , the radiologist should see the patient before and after the examination . if the radiologist does not agree , she / he should not perform the examination and should contact the clinician . but : when a clinical specialist requests an examination , the radiologist has to check if the examination can answer the clinical question , but not question the indications for the examination . ( surgeon , m , 43 , nl ) the selection of an examination protocol should be done by a radiologist himself , not by a roentgen technician . even with extensive clinical information , i often see routine sequences without sagittal t2-weighted images in case of hydrocephalus ( aqueduct stenosis ? ) or without contrast when a brain tumor is suspected . radiologists could complement a tentative diagnosis by giving a degree of certainty . on the other hand : no conclusions like infiltrate can not be excluded. no test has a sensitivity of 100% . ( nephrologist , m , 35 , nl ) where appropriate , the conclusion should contain advice to the gp for follow - up examinations or for referral to a dedicated specialist . in reports of complex examinations such as ultrasound , the radiologist should describe accurately what has been seen , not just note that the examination is normal . pathological findings outside the scope of the examination however , e.g. a bone lesion on a chest x - ray , should always be mentioned . describing normal findings we sometimes have to go back three or four chest x - rays to know what the condition was ( anesthesiologist , f , 40 , fl ) if a diagnosis can not be made , a differential diagnosis should be presented . in case several diagnoses are possible , it would help if the radiologist could give an idea of the probability of each one . ( internist , m , 55 , fl ) radiologists should stick their necks out and take responsibility , rather than hiding themselves behind vague and ambiguous statements . the umbrella effect ( tumor can not be excluded ) does not help us in any way . ( gp , f , 49 , fl ) radiologists should always be aware of the specialty of the referring clinician and tailor their reports accordingly . requesting x - rays is part of my routine as a cardiologist but reports invariably tell me there is no pulmonary infiltrate . i get the impression that the radiologist does nt know anything about heart configuration , volume and so on . ( cardiologist , m , 66 , fl ) radiologists should only describe what they know : no comments on the quality and position of prostheses , as they are wrong most of the time . ( orthopaedic surgeon , m , 38 , nl ) although radiologists may have a strong interest in the clinical side of medicine , their judgement should be based on the results of imaging procedures . i notice more and more often clinical diagnoses that can not be made on the basis of imaging alone . [ ] radiologists have to distinguish diagnosis from suspicion . ( pulmonologist , m , 34 , nl ) how about measurements ? in case of a tumor , always measure ! the same in follow - up examinations , instead of talking about further diminution ( pediatrician , f , 49 , fl ) and : measurements would be more instructive if the standard deviation would be given . liver too big for age is less informative than liver volume too big , + 3.5 sd. ( neurologist , f , 36 , nl ) measurements too should be linked to specific images . radiology reports should be much shorter , schematic and concise , some of the respondents thought . so one can see in the wink of an eye where something abnormal was seen . reports should at least contain the clinical question , the examination technique , a description of the findings , a conclusion and sometimes advice . other tips : important findings should be highlighted . providing digital images ( which are not compatible with our mac ! ) is a waste of time unless they specify which images we need to look at . ( gp , f , 50 , fl ) it would be interesting if the radiologist would use an arrow to indicate pathology like a hernia , a cyst on us and especially in case of ct or mri ( gp , f , 56 , fl ) the conclusion was considered an essential part of the report by many , especially gps . it was felt that the conclusion should only contain relevant results , and more specifically the answer to the clinical question . the use of templates to promote reporting consistency was advised by some but rejected by others . ready - to - use reports need to be avoided , they cause too many errors . ( surgeon , m , 56 , fl ) no computer - generated reports in which just a few words have been changed . ( gp , f , 49 , fl ) the importance of applying guidelines and criteria for follow - up was emphasised . when evaluating scans in case of multiple sclerosis , use the barkhof criteria . ( neurologist , f , 36 , nl ) the contents need to comply with international guidelines written with the help of radiologists . in oncology , ( oncologist , m , 38 , nl ) several respondents objected to unusual abbreviations or eponyms . it 's a nuisance having to call the radiologist to ask for an explanation . ( gp , f , 33 , fl ) what about the length of the report ? according to some , reports of simple examinations can consist of one or two words , but reports of complex examinations should be substantial . however , several thought that long and tedious radiology reports often cover up the radiologist s incompetence to answer the clinical question . and even when long reports are indicated , the conclusion should be short and clear . radiologists could complement a tentative diagnosis by giving a degree of certainty . on the other hand : no conclusions like infiltrate can not be excluded. no test has a sensitivity of 100% . ( nephrologist , m , 35 , nl ) where appropriate , the conclusion should contain advice to the gp for follow - up examinations or for referral to a dedicated specialist . in reports of complex examinations such as ultrasound , the radiologist should describe accurately what has been seen , not just note that the examination is normal . pathological findings outside the scope of the examination however , e.g. a bone lesion on a chest x - ray , should always be mentioned . describing normal findings we sometimes have to go back three or four chest x - rays to know what the condition was ( anesthesiologist , f , 40 , fl ) if a diagnosis can not be made , a differential diagnosis should be presented . in case several diagnoses are possible , it would help if the radiologist could give an idea of the probability of each one . ( internist , m , 55 , fl ) radiologists should stick their necks out and take responsibility , rather than hiding themselves behind vague and ambiguous statements . the umbrella effect ( tumor can not be excluded ) does not help us in any way . ( gp , f , 49 , fl ) radiologists should always be aware of the specialty of the referring clinician and tailor their reports accordingly . requesting x - rays is part of my routine as a cardiologist but reports invariably tell me there is no pulmonary infiltrate . i get the impression that the radiologist does nt know anything about heart configuration , volume and so on . ( cardiologist , m , 66 , fl ) radiologists should only describe what they know : no comments on the quality and position of prostheses , as they are wrong most of the time . ( orthopaedic surgeon , m , 38 , nl ) although radiologists may have a strong interest in the clinical side of medicine , their judgement should be based on the results of imaging procedures . i notice more and more often clinical diagnoses that can not be made on the basis of imaging alone . [ ] radiologists have to distinguish diagnosis from suspicion . ( pulmonologist , m , 34 , nl ) how about measurements ? in case of a tumor , always measure ! the same in follow - up examinations , instead of talking about further diminution ( pediatrician , f , 49 , fl ) and : measurements would be more instructive if the standard deviation would be given . liver too big for age is less informative than liver volume too big , + 3.5 sd. ( neurologist , f , 36 , nl ) measurements too should be linked to specific images . radiology reports should be much shorter , schematic and concise , some of the respondents thought . so one can see in the wink of an eye where something abnormal was seen . reports should at least contain the clinical question , the examination technique , a description of the findings , a conclusion and sometimes advice . other tips : important findings should be highlighted . providing digital images ( which are not compatible with our mac ! ) is a waste of time unless they specify which images we need to look at . ( gp , f , 50 , fl ) it would be interesting if the radiologist would use an arrow to indicate pathology like a hernia , a cyst on us and especially in case of ct or mri ( gp , f , 56 , fl ) the conclusion was considered an essential part of the report by many , especially gps . it was felt that the conclusion should only contain relevant results , and more specifically the answer to the clinical question . the use of templates to promote reporting consistency was advised by some but rejected by others . ready - to - use reports need to be avoided , they cause too many errors . ( surgeon , m , 56 , fl ) no computer - generated reports in which just a few words have been changed . ( gp , f , 49 , fl ) the importance of applying guidelines and criteria for follow - up was emphasised . when evaluating scans in case of multiple sclerosis , use the barkhof criteria . ( neurologist , f , 36 , nl ) the contents need to comply with international guidelines written with the help of radiologists . in oncology , ( oncologist , m , 38 , nl ) several respondents objected to unusual abbreviations or eponyms . it 's a nuisance having to call the radiologist to ask for an explanation . ( gp , f , 33 , fl ) what about the length of the report ? according to some , reports of simple examinations can consist of one or two words , but reports of complex examinations should be substantial . however , several thought that long and tedious radiology reports often cover up the radiologist s incompetence to answer the clinical question . and even when long reports are indicated , the conclusion should be short and clear . the idea that easy communication between clinician and radiologist is in the interest of the patient and can prevent vague conclusions came up several times . if the radiologist sees something unexpected , or if an examination fails or causes adverse effects , he / she must call the clinician . clinicians mentioned the need to look at the images themselves , so they can give feedback to the radiologist . the direct phone number of the reporting radiologist should always be present , so they can be contacted in case something is not clear . ( gp , m , 30 , fl ) and : call centers are there for patients , not for doctors . ( internist , f , 50 , fl ) the report should also contain a schedule of the hours the radiologist can be reached directly . it was suggested that exceptionally complex examinations should be analysed together by radiologist and clinician . if a radiologist on call , e.g. a resident - in - training , has reported an examination orally , what has been said should be quoted in the report . if the final report contradicts these preliminary conclusions , that should be mentioned too . quite often a supervisor will add something to a report that was made during the night , with dramatic consequences for its conclusion . ( surgeon , m , 36 , nl ) if they have a choice , clinicians would rather work with radiologists they know and trust . some radiologists give very vague descriptions , but we most often know which ones do so . ( gp , f , 43 , fl ) radiologists do not have an easy job but in general they perform quite well , some clinicians thought . their s is a great contribution to our work and a strong support for what we do . if an examination has failed because the patient has moved or contrast medium administration was omitted , it is not enough to state this in the report . ( orthopedic surgeon , m , 38 , nl ) in the case of ercp examinations , the report often only refers to the results of endoscopy . imaging studies should be subjected to a double blind protocol , according to one respondent . reports can have an educational side : good reports stimulate gps to study the images and can become a refresher course for their knowledge of anatomy . ( gp , m , 64 , fl ) the flawless use of their mother tongue is dear to some clinicians . there is a need for terminological consistency : standardization of terms like bulging , ( gp , f , 50 , fl ) speech recognition is still a matter of dispute : reports made using speech recognition are not corrected . . the result may be grammatically and semantically correct , but what about , for example , left right mistakes , one colleague said . do radiologists make reports that fulfill the needs of the referring clinicians ? in our study , many referring clinicians saw radiologists as peers who , in the interests of the diagnostic outcome , are entitled to receive as much clinical information as necessary . in exchange , clinicians expected radiologists to amend preset imaging protocols if these were inadequate or incomplete . in the past , several studies have shown that the availability of clinical information and a pertinent examination question can indeed improve the diagnostic accuracy of the imaging process [ 3 , 4 ] . if the requested examination is totally unfit to answer the clinical question , the radiologist is to contact the referring clinician . in a recent retrospective study , 26% of ct and mri examination requests by primary care physicians were considered inappropriate . one respondent emphasised that handwritten clinical information should be readable . trying to decipher handwriting can be a frustrating task . calling the referring clinician is time - consuming , as is reading an inpatient s electronic record for clinical information . electronic examination requests may offer a solution , but this will not solve the problem of imperfect requests by gps , made while on a house call . these are often brief , if not minimal , and gps can be notoriously difficult to reach for additional information during office hours . however , as portable gp information systems are already widely available , online requests by gps may gradually replace ad hoc handwritten requests . non - technical abbreviations in a radiology report give an air of informality and imply that the radiologist was in a hurry ; technical abbreviations run the risk of not being understood . while they should be avoided as much as possible in radiology reports , abbreviations in examination requests can cause misunderstandings too , as well as a loss of precious time . radiologists serve several tens of departments , some of which may use the same abbreviations for different conditions . especially residents - in - training in rotating schedules may lack the experience to decipher all . therefore , if their departments can not provide a list of the abbreviations they use , clinicians ought to refrain from using them at all . as for the conclusion , the acr 2005 practice guideline for communication of diagnostic imaging findings states that unless the report is brief , each report should contain an impression section ; a precise diagnosis should be given when possible ; a differential diagnosis should be provided when appropriate ; follow - up or additional diagnostic studies to clarify or confirm the impression should be suggested when appropriate ; and any significant patient reaction should be reported . standardised computer - generated template reports that satisfy these criteria are considered to conform to these guidelines . recently , the european society of radiology ( esr ) issued its own guidelines on good practice for radiological reporting . these state that it can be argued that concise , consistent ordering of the report both reduces variation among reports and makes it easier for referrers who become familiar with the format to assimilate information . the broad categories that esr identifies are clinical referral , technique , findings , conclusion and advice . for esr , further study is required before they fully support structured reporting ( sr ) . most of the acr and esr guidelines are reflected in the suggestions in our survey . radiologists are expected to be competent enough to produce a diagnosis or a suitably ordered differential diagnosis . vague conclusions are rejected by clinicians , as are other expressions of a perceived refusal to take responsibility . the supposition of one respondent that many clinicians only read the conclusion of the report was not supported in the quantitative part of the cover survey : only 2.4% of the clinicians entirely agreed with it , and just 20.7% partially , 23.1% in all . it is remarkable that none of the respondents suggested concrete measures to improve the quality and efficiency of the conclusion , such as putting the conclusion before the descriptive part or putting the most relevant parts of the conclusion first . this confirms our hypothesis that respondents mainly made suggestions based on their own daily experience . some suggestions were conflicting , e.g. concerning sr . in line with the results of quantitative surveys [ 2 , 9 , 10 ] , there seems to be a demand for easily accessible , neatly arranged reports . the use of templates was approved by some clinicians in this survey but questioned by others . while few suggestions are in favour of reporting in free text , the rigorous application of predetermined templates and a standard lexicon may be experienced as tedious and boring , and lacking an element of diversity , of entertainment . however , if international guidelines for the content of reports in specific areas of interest exist , such as in the follow - up of tumours , they should be routinely applied . supervision by radiologists of reports made by residents can be considered a form of second opinion , although it will seldom be double - blind . considering the lack of radiologists in many countries and the high cost involved , in most medical centers double reading will be restricted to random samples in the interest of quality control . suggestions that radiologists should refrain from mentioning a histological diagnosis may reflect a lack of familiarity with today s advanced imaging techniques . using a combination of magnetic resonance imaging , contrast - enhanced multislice ct and/or contrast - enhanced ultrasound , it is perfectly possible to characterise , for example , a number of soft tissue tumours . radiologists have to communicate about this more effectively , which can be done by actively engaging in continuing medical education initiatives for both gps and specialists . it would indeed be practical if the report would mention explicitly how and where the radiologist can be reached . on the other hand , mentioning phone numbers in reports carries the risk of patients using the same communication lines . although some authors believe that reporting in the future will imply direct communication between radiologists and patients , in the interest of efficiency and privacy the two tracks need to remain separate . efficient communication lines between radiologists and clinicians also imply reciprocity . especially in large institutions with many residents - in - training , the direct phone number or the pager of the referring physician should be on every request . another important element is that clinicians apparently want to know where exactly abnormal findings can be seen . while digital imaging should have made such markings much easier ( and less messy ) , their application seems to have largely faded away . radiologists should consider readopting a modern version of the old habit . despite a clear demand for more on behalf of some gps , the radiology report is there to provide adequate and accessible diagnostic information , not to replace a refresher course on radioanatomy . in the course of the last decade , several authors have published quantitative results of surveys among referring clinicians on the radiology report [ 2 , 9 , 10 , 13 , 14 ] . such quantitative research can demonstrate the relative weight of predetermined expressions within the research population . that is why when designing the cover study we reserved space for suggestions in free text . open questions lead to open answers . freed from the chains of preset forms and phrases , some respondents in our survey tended to stray off the subject . many suggestions therefore had less to do with the radiology report as such , and more to do with communication between radiologists and clinicians in a broader sense . topics presented in the quantitative part of the cover survey may have had some influence on the spontaneous suggestions made afterwards . this could have been avoided by putting the suggestions section first , but we were afraid that too many potential participants would have dropped out if the survey had started with open questions . one can not expect respondents to express an overarching view of the radiology report in a few lines . most likely , they will express views that spring to mind most easily , maybe due to their emotional value or to recent personal experiences . some of these suggestions may reflect local circumstances with little or no significance elsewhere , and suggestions from different respondents may even be conflicting . therefore , we believe that they can contribute to a better insight into the ideas and emotions of referring clinicians and have to be taken into account when rethinking the report . the fact that a considerable number of respondents of the cover trial , 233 out of 735 ( 31.7% ) , made concrete suggestions for its improvement certainly shows that radiologists need to look critically at their end product . the most frequent suggestions however pertained to the position of the radiologist as a well - informed clinical specialist , and this implies an engagement from both sides . how can the report , and in a broader sense the communication between radiologists and referring clinicians , be improved ? there seems to be a need for well - structured , concise but complete reports without verbosity . both radiologists and clinicians should be more easily available to one another for consultation . in specific situations such as follow - up of solid tumours we do not pretend to provide the definitive answer to all questions concerning the quality of the radiology report . however , although the spontaneous suggestions in this study were erratic and sometimes contradictory , they do give an overview of the ideas as well as the emotions of these clients of the radiology department . we therefore believe it is highly advisable to take them into account when developing new ways of reporting .

objectiveto investigate what referring clinicians suggest when asked how the quality of radiology reports can be improved.methodsat the end of the questionnaire of the cover survey , a bi - national quantitative survey on the radiology report among referring physicians , clinical specialists and general practitioners were able to freely enter suggestions with regard to improving the quality of the report . these suggestions were isolated from the quantitative results . subjects and themes were identified , examined , ordered , counted , compared and analysed.resultsof a total of 3,884 invitations to participate , we received 735 response forms from clinicians ( 18.9% ) , 233 ( 31.7% ) of which contained suggestions . issues mentioned most frequently were the need for clinical information and a clinical question , for a conclusion , structuring , communicating directly with the clinician , completeness , integrating images or referring to images , mentioning relevant findings outside of the clinical question , mentioning a diagnosis or suitable differential diagnosis , and concise reporting.conclusionalthough these spontaneous suggestions are erratic and sometimes contradictory , they summarise the ideas as well as the emotions of these clients of the radiology department . therefore it is advisable to take them into account when developing new ways of reporting .

Introduction Materials and methods Results The radiologist as a clinical specialist Responsibilities of the referring physician and the radiologist Content and structure of the radiology report Content Structure Collaboration between radiologists and referring clinicians Casual remarks Discussion Conclusion

does the radiology report , radiology s most conspicuous and permanent product , in its present form , meet the expectations of referring clinicians ? and if not , how can the report , and in a broader sense the communication between radiologists and referring clinicians , be improved ? one of our recent projects at the university of antwerp is cover ( clinicians opinions , views and expectations concerning the radiology report ) , a bi - national survey in the netherlands and flanders , the dutch - speaking part of belgium . the quantitative results of these surveys have been reported recently . at the end of the cover questionnaire , respondents were able to make suggestions on how to improve the radiology report . in this article , we present a synthesis of the suggestions of referring clinicians , both hospital specialists and general practitioners . we are well aware of the fact that radiologists are clinicians too but for practical purposes we will use the term clinicians as shorthand for referring clinicians in this paper . permission for the cover internet survey was obtained from the institutional review board of the authors medical center . the medical director of each hospital sent an e - mail to all clinical specialists who routinely prescribe imaging examinations to invite them to participate . the e - mail addresses of general practitioners ( gps ) were found in the 2008 address book of the order of physicians of the province of antwerp , and all those who had such an address received the e - mail directly from the investigators . by clicking a hyperlink in the e - mail , responders were referred to a digital questionnaire prepared using surveymonkey , a commercial tool for internet surveys ( surveymonkey , portland , or , usa ) . the main part of the survey consisted of a set of 46 statements on the radiology report , for which respondents were asked to indicate their level of agreement according to a five - tiered likert scale . at the end of this section the survey was performed in two university and two community hospitals in flanders , one university and one community hospital in the netherlands , and among the general practitioners ( gps ) in the province of antwerp , flanders . the investigators identified , examined , ordered , counted , compared and synthesised subjects and themes by means of qualitative analysis software ( qsr nvivo 8 , qsr international , doncaster , vic , australia ) . of a total of 3,884 invitations to participate , we received 735 response forms from clinicians ( 18.9% ) . the rate of completed response forms with suggestions ranged from 29.1 to 37.5% , according to the institution or target group ( average 31.7% , i.e. of the clinicians who made suggestions , 148 ( 63.5% ) were male and 85 ( 36.5% ) were female . an overview of the medical specialty of the responders can be found in table 2 . table 1response rates of clinicians and rate of completed questionnaires with suggestions in covercentre or groupsentresponded ( % ) with suggestions ( % ) community hospital 1 nl5916 ( 28.1)6 ( 37.5)university hospital 1 nl1,391163 ( 11.7)59 ( 36.2)community hospital 2 fl11976 ( 63.9)23 ( 30.3)community hospital 3 fl4525 ( 55.6)8 ( 32.0)university hospital 2 fl35965 ( 18.1)21 ( 32.3)university hospital 3 fl590108 ( 18.3)34 ( 31.5)total clinical specialists2,561453 ( 17.7)144 ( 31.8)total gps1,323282 ( 21.3)82 ( 29.1)grand total3,884735 ( 18.9)233 ( 31.7)nl the netherlands , fl flanders , belgiumtable 2specialities represented among responders with suggestions , absolute number and percentage of totalspecialitynumberpercentagegeneral practice8235.2paediatrics156.4surgery156.4internal medicine ( general)125.2other ( not listed)125.2oncology114.7gynaecology114.7anaesthesiology114.7cardiology93.9orthopaedics83.4gastroenterology - hepatology83.4physical and rehabilitation medicine62.6ear , nose and throat medicine62.6pulmonology52.1urology41.7neurology41.7psychiatry31.3ophthalmology31.3dermatology20.9endocrinology - diabetology20.9emergency medicine10.4rheumatology10.4nephrology10.4haematology10.4total233100.0 response rates of clinicians and rate of completed questionnaires with suggestions in cover nl the netherlands , fl flanders , belgium specialities represented among responders with suggestions , absolute number and percentage of total fifty - one different themes were identified , and suggestions were linked to one or several of these themes ( table 3 ) . table 3frequency of themes mentioned three times or more in clinicians suggestions for improving the reportsubjectnumber of referencessubjectnumber of referencesclinical information and the clinical question94accessibility of the report10conclusion / impression of the report55multidisciplinary rounds8structured reports37content7communicating directly to the clinician25descriptive part of the report7completeness19report as training update for clinicians7integrating images or referring to images19satisfaction with the report7relevant findings outside of the clinical question19competence of the radiologist6mentioning a diagnosis / differential diagnosis19irrelevant reports6concise reporting17narrative reports5electronic patient record ( epr)16terminology5vague reports / the hedge16variable quality or approach5suggestions for further examinations16probability of a diagnosis4use of abbreviations14quality of the report4use of a standard lexicon14performing the right / inadequate examinations4measuring lesions13language3proofreading reports12preformatted text3 frequency of themes mentioned three times or more in clinicians suggestions for improving the report in the following sections , we present a synthesis of these suggestions , as well as a selection of quotes . each quote is accompanied by a note stating specialty , age , gender and place of activity ( fl flanders , belgium , nl the netherlands ) of the person quoted . many respondents thought it is the duty of the referring clinician to provide the radiologist with useful clinical information and a clear and unequivocal clinical question . it was felt that the clinical questions provided in the request form should be addressed in the report . several clinicians , gps especially , were convinced that the radiologist does not always read the clinical question on the request . ( gp , m , 40 , fl ) one of the respondents wrote if radiologists would present themselves credibly as imaging clinicians , referring clinicians would be more prone to provide useful information . if the radiologist does not agree , she / he should not perform the examination and should contact the clinician . but : when a clinical specialist requests an examination , the radiologist has to check if the examination can answer the clinical question , but not question the indications for the examination . on the other hand : no conclusions like infiltrate can not be excluded. pathological findings outside the scope of the examination however , e.g. we sometimes have to go back three or four chest x - rays to know what the condition was ( anesthesiologist , f , 40 , fl ) if a diagnosis can not be made , a differential diagnosis should be presented . in case several diagnoses are possible , it would help if the radiologist could give an idea of the probability of each one . ( cardiologist , m , 66 , fl ) radiologists should only describe what they know : no comments on the quality and position of prostheses , as they are wrong most of the time . ( orthopaedic surgeon , m , 38 , nl ) although radiologists may have a strong interest in the clinical side of medicine , their judgement should be based on the results of imaging procedures . radiology reports should be much shorter , schematic and concise , some of the respondents thought . reports should at least contain the clinical question , the examination technique , a description of the findings , a conclusion and sometimes advice . ( gp , f , 50 , fl ) it would be interesting if the radiologist would use an arrow to indicate pathology like a hernia , a cyst on us and especially in case of ct or mri ( gp , f , 56 , fl ) the conclusion was considered an essential part of the report by many , especially gps . it was felt that the conclusion should only contain relevant results , and more specifically the answer to the clinical question . ( neurologist , f , 36 , nl ) the contents need to comply with international guidelines written with the help of radiologists . ( gp , f , 33 , fl ) what about the length of the report ? however , several thought that long and tedious radiology reports often cover up the radiologist s incompetence to answer the clinical question . the idea that easy communication between clinician and radiologist is in the interest of the patient and can prevent vague conclusions came up several times . the direct phone number of the reporting radiologist should always be present , so they can be contacted in case something is not clear . ( internist , f , 50 , fl ) the report should also contain a schedule of the hours the radiologist can be reached directly . ( gp , m , 55 , fl ) if an examination has failed because the patient has moved or contrast medium administration was omitted , it is not enough to state this in the report . the result may be grammatically and semantically correct , but what about , for example , left many respondents thought it is the duty of the referring clinician to provide the radiologist with useful clinical information and a clear and unequivocal clinical question . it was felt that the clinical questions provided in the request form should be addressed in the report . , not just : no abnormal findings but also : no signs of pneumonia , so we know the radiologist has looked at the patient with the eye of a clinician . several clinicians , gps especially , were convinced that the radiologist does not always read the clinical question on the request . ( gp , m , 40 , fl ) one of the respondents wrote if radiologists would present themselves credibly as imaging clinicians , referring clinicians would be more prone to provide useful information . but : when a clinical specialist requests an examination , the radiologist has to check if the examination can answer the clinical question , but not question the indications for the examination . even with extensive clinical information , i often see routine sequences without sagittal t2-weighted images in case of hydrocephalus ( aqueduct stenosis ? ) pathological findings outside the scope of the examination however , e.g. describing normal findings we sometimes have to go back three or four chest x - rays to know what the condition was ( anesthesiologist , f , 40 , fl ) if a diagnosis can not be made , a differential diagnosis should be presented . ( cardiologist , m , 66 , fl ) radiologists should only describe what they know : no comments on the quality and position of prostheses , as they are wrong most of the time . ( orthopaedic surgeon , m , 38 , nl ) although radiologists may have a strong interest in the clinical side of medicine , their judgement should be based on the results of imaging procedures . radiology reports should be much shorter , schematic and concise , some of the respondents thought . reports should at least contain the clinical question , the examination technique , a description of the findings , a conclusion and sometimes advice . ( gp , f , 50 , fl ) it would be interesting if the radiologist would use an arrow to indicate pathology like a hernia , a cyst on us and especially in case of ct or mri ( gp , f , 56 , fl ) the conclusion was considered an essential part of the report by many , especially gps . it was felt that the conclusion should only contain relevant results , and more specifically the answer to the clinical question . ( gp , f , 33 , fl ) what about the length of the report ? however , several thought that long and tedious radiology reports often cover up the radiologist s incompetence to answer the clinical question . pathological findings outside the scope of the examination however , e.g. describing normal findings we sometimes have to go back three or four chest x - rays to know what the condition was ( anesthesiologist , f , 40 , fl ) if a diagnosis can not be made , a differential diagnosis should be presented . ( cardiologist , m , 66 , fl ) radiologists should only describe what they know : no comments on the quality and position of prostheses , as they are wrong most of the time . ( orthopaedic surgeon , m , 38 , nl ) although radiologists may have a strong interest in the clinical side of medicine , their judgement should be based on the results of imaging procedures . radiology reports should be much shorter , schematic and concise , some of the respondents thought . reports should at least contain the clinical question , the examination technique , a description of the findings , a conclusion and sometimes advice . ( gp , f , 50 , fl ) it would be interesting if the radiologist would use an arrow to indicate pathology like a hernia , a cyst on us and especially in case of ct or mri ( gp , f , 56 , fl ) the conclusion was considered an essential part of the report by many , especially gps . it was felt that the conclusion should only contain relevant results , and more specifically the answer to the clinical question . ( gp , f , 33 , fl ) what about the length of the report ? however , several thought that long and tedious radiology reports often cover up the radiologist s incompetence to answer the clinical question . the idea that easy communication between clinician and radiologist is in the interest of the patient and can prevent vague conclusions came up several times . the direct phone number of the reporting radiologist should always be present , so they can be contacted in case something is not clear . ( internist , f , 50 , fl ) the report should also contain a schedule of the hours the radiologist can be reached directly . their s is a great contribution to our work and a strong support for what we do . if an examination has failed because the patient has moved or contrast medium administration was omitted , it is not enough to state this in the report . the result may be grammatically and semantically correct , but what about , for example , left right mistakes , one colleague said . do radiologists make reports that fulfill the needs of the referring clinicians ? in our study , many referring clinicians saw radiologists as peers who , in the interests of the diagnostic outcome , are entitled to receive as much clinical information as necessary . in the past , several studies have shown that the availability of clinical information and a pertinent examination question can indeed improve the diagnostic accuracy of the imaging process [ 3 , 4 ] . if the requested examination is totally unfit to answer the clinical question , the radiologist is to contact the referring clinician . calling the referring clinician is time - consuming , as is reading an inpatient s electronic record for clinical information . these are often brief , if not minimal , and gps can be notoriously difficult to reach for additional information during office hours . while they should be avoided as much as possible in radiology reports , abbreviations in examination requests can cause misunderstandings too , as well as a loss of precious time . therefore , if their departments can not provide a list of the abbreviations they use , clinicians ought to refrain from using them at all . as for the conclusion , the acr 2005 practice guideline for communication of diagnostic imaging findings states that unless the report is brief , each report should contain an impression section ; a precise diagnosis should be given when possible ; a differential diagnosis should be provided when appropriate ; follow - up or additional diagnostic studies to clarify or confirm the impression should be suggested when appropriate ; and any significant patient reaction should be reported . recently , the european society of radiology ( esr ) issued its own guidelines on good practice for radiological reporting . these state that it can be argued that concise , consistent ordering of the report both reduces variation among reports and makes it easier for referrers who become familiar with the format to assimilate information . radiologists are expected to be competent enough to produce a diagnosis or a suitably ordered differential diagnosis . the supposition of one respondent that many clinicians only read the conclusion of the report was not supported in the quantitative part of the cover survey : only 2.4% of the clinicians entirely agreed with it , and just 20.7% partially , 23.1% in all . it is remarkable that none of the respondents suggested concrete measures to improve the quality and efficiency of the conclusion , such as putting the conclusion before the descriptive part or putting the most relevant parts of the conclusion first . some suggestions were conflicting , e.g. while few suggestions are in favour of reporting in free text , the rigorous application of predetermined templates and a standard lexicon may be experienced as tedious and boring , and lacking an element of diversity , of entertainment . using a combination of magnetic resonance imaging , contrast - enhanced multislice ct and/or contrast - enhanced ultrasound , it is perfectly possible to characterise , for example , a number of soft tissue tumours . it would indeed be practical if the report would mention explicitly how and where the radiologist can be reached . on the other hand , mentioning phone numbers in reports carries the risk of patients using the same communication lines . despite a clear demand for more on behalf of some gps , the radiology report is there to provide adequate and accessible diagnostic information , not to replace a refresher course on radioanatomy . in the course of the last decade , several authors have published quantitative results of surveys among referring clinicians on the radiology report [ 2 , 9 , 10 , 13 , 14 ] . that is why when designing the cover study we reserved space for suggestions in free text . many suggestions therefore had less to do with the radiology report as such , and more to do with communication between radiologists and clinicians in a broader sense . topics presented in the quantitative part of the cover survey may have had some influence on the spontaneous suggestions made afterwards . one can not expect respondents to express an overarching view of the radiology report in a few lines . some of these suggestions may reflect local circumstances with little or no significance elsewhere , and suggestions from different respondents may even be conflicting . therefore , we believe that they can contribute to a better insight into the ideas and emotions of referring clinicians and have to be taken into account when rethinking the report . the fact that a considerable number of respondents of the cover trial , 233 out of 735 ( 31.7% ) , made concrete suggestions for its improvement certainly shows that radiologists need to look critically at their end product . the most frequent suggestions however pertained to the position of the radiologist as a well - informed clinical specialist , and this implies an engagement from both sides . how can the report , and in a broader sense the communication between radiologists and referring clinicians , be improved ? in specific situations such as follow - up of solid tumours we do not pretend to provide the definitive answer to all questions concerning the quality of the radiology report . however , although the spontaneous suggestions in this study were erratic and sometimes contradictory , they do give an overview of the ideas as well as the emotions of these clients of the radiology department . we therefore believe it is highly advisable to take them into account when developing new ways of reporting .

all m. smegmatis strains were isogenic to mc155 and were grown at 37 c in lb supplemented with 0.5% dextrose , 0.5% glycerol , and 0.05% tween 80 ( broth ) . for immunoprecipitation of card , rnap , and rnap , a card merodiploid strain was produced by integrating pmsg430smcard - ha ( constitutively expresses m. smegmatis c - terminal ha tagged card , kanamycin resistant ) into the attb site of m. smegmatis mc155 . allelic exchange experiments were performed with the card merodiploid strain using a dna donor sequence with homology to mc155 nucleotides 6141480 to 6142268 and 6140266 to 6141010 to delete all of the card gene except the nucleotides encoding the first 10 and last 3 amino acids from the endogenous locus , generating card attb::tetcard - ha . for immunoprecipitation of unfused ha peptide as a control , mc155 was transformed with pmsg431 , which integrates into the attb site of the genome and constitutively expresses ha peptide . cultures of m. smegmatis card attb::tetcard - ha and mc155 attb::pmsg431 strains were grown to late log phase ( od600 = ~ 1 ) before adding a final concentration of 2% formaldehyde and shaking at room temperature for 30 min to crosslink dna and proteins . the crosslinking was quenched by the addition of 0.25 ml of 2.5 m glycine per 5 ml of culture and incubated 5 min at 25 c with shaking . 5 ml ( ~ 2.5 10 mycobacterial cells ) of each culture was then collected by centrifugation . the cells were washed once with te and resuspended in 100 l of te supplemented with roche complete protease inhibitor cocktail . the cell suspension was lysed using a covaris focused - ultrasonicator so that the genomic dna was sheared into ~ 100 base pair ( bp ) fragments , as assessed by dna gel electrophoresis . the use of the covaris focused - ultrasonicator was critical for this step and other sonicator systems were unable to yield a comparable consistency and homogeneity of dna fragment distribution . the cell debris was spun down and the lysate was added to 400 l chip lysis buffer ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 1% triton x-100 ) plus roche complete protease inhibitor cocktail . protein nucleic acid complexes containing card - ha were immunoprecipitated from the m. smegmatis mc155 card attb::tetcard - ha strain cell lysate by adding 50 l of anti - ha agarose ( sigma ) . complexes containing unfused ha were immunoprecipitated from the mc155 attb::pmsg431 strain with the same anti - ha agarose . rnap and were immunoprecipitated from card attb::tetcard - ha with monoclonal antibodies specific for these subunits ( neoclone ; 8rb13 for , 2g10 for ) immobilized on gammabind g sepharose ( ge healthcare life sciences ) . each immunoprecipitation was performed in duplicate from two separate cultures , thus comprising two biological replicates . however , one of the rnap samples was lost during library preparation and , therefore , there is only data for one rnap replicate . the antibody matrix was washed 2 with chip lysis buffer , 2 with chip lysis buffer plus an additional 360 mm nacl , 2 with chip wash buffer ( 10 mm tris - hcl ph 8.0 , 250 mm licl , 0.5% np-40 , 0.5% sodium deoxycholate , 1 mm edta ) , and 2 with te , each time by rotating for 10 min at 4 c . complexes that co - precipitated with the respective antibody matrix were eluted twice by adding 100 l of chip elution buffer ( 50 mm tris - hcl ph 8.0 , 10 mm edta , 1% sds ) , incubating for 10 min at 65 c with agitation , spinning down the antibody matrix , and transferring the eluate to a new tube . wash and elution buffers were all supplemented with roche complete protease inhibitor cocktail . to reverse the crosslinks 15 l of each sample was removed for western blot analysis of proteins , while 100 g / ml of proteinase k was added to the rest of each sample and incubated at 37 c for 2 h before isolating nucleic acid by chloroform phenol extracting 2 times , ethanol precipitating and resuspending the dna pellet in 34 l of water . co - precipitated dna was sequenced using an ab solid 4 high - throughput genome sequencer ( life technologies ) and a 50 bp read length , which provided sufficient reads for over 100-fold coverage of the genome in each sample , wherein the m. smegmatis genome is 6,988,209 bp in length and the coverage of each sequencing reaction was over 800 mbp . table 1 shows the total number of reads and number of mapped reads for each sample . specifically , the normalized coverage ( or counts ) was determined by multiplying the raw ( sequenced ) coverage ( or counts ) in each sample by that sample 's size factor . the size factors are determined by taking the median of the ratios of observed counts . the normalized number of sequence reads per base pair was then expressed as a log2 value . if a read mapped with equal quality at multiple loci ( but not more than 3 ) , its contribution was distributed evenly among them . for example , the sequences of the 16s , 23s , and 5s ribosomal rna are identical in the m. smegmatis rrna and rrnb operons . therefore , the total number of reads for those sequences was split equally between the operons . if the number of mapping loci was higher than 3 , the read was discarded . the normalized number of reads for each base pair was saved as a wig file for each sample . we first determined how well replicate samples of the distribution of a given protein correlated to each other and how well the distribution of card correlated to the distributions of rnap and rnap ( table 2 , table 3 ) . the correlations were obtained by computing the pearson correlation of the genomic coverage profiles of each pair of samples . the coverage profiles were computed by summing the contributions of all mapped fragments , assuming they were 100 bp long , and then , in 20-bp steps along the entire genome , computing the average coverage of the surrounding 100-bp window . these data showed that individual replicates for a single immunoprecipitation condition correlated highly with one another ( bolded in table 2 ) and indicated that the distribution of card - ha or rnap was consistent between biological replicates . this consistency between replicates allowed us to average the pearson correlation values for each comparison to simplify the comparisons between immunoprecipitation conditions ( table 3 ) . the correlation between the distribution of card - ha and the distribution of rnap ( bolded in table 3 ) was almost as high as the correlation between the two card - ha replicates , indicating that the distribution of card - ha is very similar to that of rnap . to directly compare the genome distributions of card - ha , rnap , and rnap , the reads per base pair from the unfused ha peptide sample served as the background control and were subtracted from the other datasets . the rationale for this control was that as a non - dna binding protein , the ha peptide should be diffusely localized throughout the cell and serve as a readout for the background levels of nonspecific crosslinking to the dna . the normalized , background - corrected log2 reads per base pair were then smoothed over a 20-bp window and rnap and card - ha peaks were identified as described previously , . briefly , maxima and minima were assigned as inflection points where the values 10 bp were both lower or both higher , respectively . maxima within 20 bp were merged with the peak location assigned to the maximum with the highest absolute signal value . peaks were divided into 0.1 interval bins of peak heights with a lower cutoff of peak height of 0.4 log2 reads per base pair . starting with the lowest bin , we then calculated the distance of each peak to the nearest gene start and compared these distances to those computed using genome coordinates arbitrarily rotated 1 10 bp around the m. smegmatis genome . using the wilcoxon mann whitney ranksum test for nonsimilarity of distributions , , rnap peaks in the 1.11.2 peak - height bins and card - ha peaks in the 0.50.6 peak - height bins were statistically significant ( p values for similarity of the distributions < 0.0001 ) . in other words , for each peak - height bin , the two lists of peak - to - gene start distances ( actual and rotated by 1 10 bp ) were tested for whether they were from different populations using the wilcoxon mann peaks in the lowest peak - height bin for which peak - to - gene start distances differed from random with a p value of < 0.0001 plus all peaks with greater heights were then used as the statistically significant peaks . we then identified rnap peaks associated with each gene as the closest rnap peak upstream from the gene start and card - ha peaks associated with the rnap peaks as the closest card - ha peak to each selected rnap peak . to calculate average chip signals for the aggregate profiles ( fig . 1 ) , we selected a subset of 62 genes meeting the following criteria : ( i ) 300 bp in gene length , ( ii ) average rnap log2 chip signal 1.6/bp , ( iii ) associated with an rnap peak with log2 chip signal 3/bp , ( iv ) absence of other rnap peaks within 500 bp upstream or 1000 bp downstream of the associated rnap peak , ( v ) absence of an oppositely oriented gene with an average rnap log2 chip signal 1 upstream from the gene ( because an oppositely oriented gene could create a divergent promoter region with potential for overlapping rnap and card - ha chip signals ) , and ( vi ) absence of an upstream gene with average rnap log2 chip signal > 0 within 100 bp upstream from the gene ( because such an arrangement would indicate the gene is an internal member of an operon ) . the rnap , card - ha , and rnap signals from the 62 genes were then averaged using the distance from the center of the associated peaks to align the genes ( fig . 1 ) . for the gene alignments , the distance from the center of the associated peak served as a proxy for the transcriptional start site , since most transcriptional start sites are not mapped in m. smegmatis . this analysis showed that whereas rnap was found throughout transcribed regions of the genome , card - ha and rnap were primarily associated with promoter regions . these data matched the high correlation calculated for the distribution of card - ha and rnap ( table 2 , table 3 ) . levels of both card - ha and rnap dropped off immediately following the promoter sequences , suggesting that these proteins are lost from the rnap elongating complex after transcription initiation . the colocalization of rnap and card - ha led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . further analysis of the dataset also revealed that card was never present on the genome in the absence of rnap , suggesting that it may be targeted to the genome through its interaction with rnap . all m. smegmatis strains were isogenic to mc155 and were grown at 37 c in lb supplemented with 0.5% dextrose , 0.5% glycerol , and 0.05% tween 80 ( broth ) . for immunoprecipitation of card , rnap , and rnap , a card merodiploid strain was produced by integrating pmsg430smcard - ha ( constitutively expresses m. smegmatis c - terminal ha tagged card , kanamycin resistant ) into the attb site of m. smegmatis mc155 . allelic exchange experiments were performed with the card merodiploid strain using a dna donor sequence with homology to mc155 nucleotides 6141480 to 6142268 and 6140266 to 6141010 to delete all of the card gene except the nucleotides encoding the first 10 and last 3 amino acids from the endogenous locus , generating card attb::tetcard - ha . for immunoprecipitation of unfused ha peptide as a control , mc155 was transformed with pmsg431 , which integrates into the attb site of the genome and constitutively expresses ha peptide . cultures of m. smegmatis card attb::tetcard - ha and mc155 attb::pmsg431 strains were grown to late log phase ( od600 = ~ 1 ) before adding a final concentration of 2% formaldehyde and shaking at room temperature for 30 min to crosslink dna and proteins . the crosslinking was quenched by the addition of 0.25 ml of 2.5 m glycine per 5 ml of culture and incubated 5 min at 25 c with shaking . 5 ml ( ~ 2.5 10 mycobacterial cells ) of each culture was then collected by centrifugation . the cells were washed once with te and resuspended in 100 l of te supplemented with roche complete protease inhibitor cocktail . the cell suspension was lysed using a covaris focused - ultrasonicator so that the genomic dna was sheared into ~ 100 base pair ( bp ) fragments , as assessed by dna gel electrophoresis . the use of the covaris focused - ultrasonicator was critical for this step and other sonicator systems were unable to yield a comparable consistency and homogeneity of dna fragment distribution . the cell debris was spun down and the lysate was added to 400 l chip lysis buffer ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 1% triton x-100 ) plus roche complete protease inhibitor cocktail . protein nucleic acid complexes containing card - ha were immunoprecipitated from the m. smegmatis mc155 card attb::tetcard - ha strain cell lysate by adding 50 l of anti - ha agarose ( sigma ) . complexes containing unfused ha were immunoprecipitated from the mc155 attb::pmsg431 strain with the same anti - ha agarose . rnap and were immunoprecipitated from card attb::tetcard - ha with monoclonal antibodies specific for these subunits ( neoclone ; 8rb13 for , 2g10 for ) immobilized on gammabind g sepharose ( ge healthcare life sciences ) . each immunoprecipitation was performed in duplicate from two separate cultures , thus comprising two biological replicates . however , one of the rnap samples was lost during library preparation and , therefore , there is only data for one rnap replicate . the antibody matrix was washed 2 with chip lysis buffer , 2 with chip lysis buffer plus an additional 360 mm nacl , 2 with chip wash buffer ( 10 mm tris - hcl ph 8.0 , 250 mm licl , 0.5% np-40 , 0.5% sodium deoxycholate , 1 mm edta ) , and 2 with te , each time by rotating for 10 min at 4 c . complexes that co - precipitated with the respective antibody matrix were eluted twice by adding 100 l of chip elution buffer ( 50 mm tris - hcl ph 8.0 , 10 mm edta , 1% sds ) , incubating for 10 min at 65 c with agitation , spinning down the antibody matrix , and transferring the eluate to a new tube . wash and elution buffers were all supplemented with roche complete protease inhibitor cocktail . to reverse the crosslinks , 15 l of each sample was removed for western blot analysis of proteins , while 100 g / ml of proteinase k was added to the rest of each sample and incubated at 37 c for 2 h before isolating nucleic acid by chloroform phenol extracting 2 times , ethanol precipitating and resuspending the dna pellet in 34 l of water . co - precipitated dna was sequenced using an ab solid 4 high - throughput genome sequencer ( life technologies ) and a 50 bp read length , which provided sufficient reads for over 100-fold coverage of the genome in each sample , wherein the m. smegmatis genome is 6,988,209 bp in length and the coverage of each sequencing reaction was over 800 mbp . table 1 shows the total number of reads and number of mapped reads for each sample . specifically , the normalized coverage ( or counts ) was determined by multiplying the raw ( sequenced ) coverage ( or counts ) in each sample by that sample 's size factor . the size factors are determined by taking the median of the ratios of observed counts . the normalized number of sequence reads per base pair was then expressed as a log2 value . if a read mapped with equal quality at multiple loci ( but not more than 3 ) , its contribution was distributed evenly among them . for example , the sequences of the 16s , 23s , and 5s ribosomal rna are identical in the m. smegmatis rrna and rrnb operons . therefore , the total number of reads for those sequences was split equally between the operons . if the number of mapping loci was higher than 3 , the read was discarded . the normalized number of reads for each base pair was saved as a wig file for each sample . we first determined how well replicate samples of the distribution of a given protein correlated to each other and how well the distribution of card correlated to the distributions of rnap and rnap ( table 2 , table 3 ) . the correlations were obtained by computing the pearson correlation of the genomic coverage profiles of each pair of samples . the coverage profiles were computed by summing the contributions of all mapped fragments , assuming they were 100 bp long , and then , in 20-bp steps along the entire genome , computing the average coverage of the surrounding 100-bp window . these data showed that individual replicates for a single immunoprecipitation condition correlated highly with one another ( bolded in table 2 ) and indicated that the distribution of card - ha or rnap was consistent between biological replicates . this consistency between replicates allowed us to average the pearson correlation values for each comparison to simplify the comparisons between immunoprecipitation conditions ( table 3 ) . the correlation between the distribution of card - ha and the distribution of rnap ( bolded in table 3 ) was almost as high as the correlation between the two card - ha replicates , indicating that the distribution of card - ha is very similar to that of rnap . to directly compare the genome distributions of card - ha , rnap , and rnap , the reads per base pair from the unfused ha peptide sample served as the background control and were subtracted from the other datasets . the rationale for this control was that as a non - dna binding protein , the ha peptide should be diffusely localized throughout the cell and serve as a readout for the background levels of nonspecific crosslinking to the dna . the normalized , background - corrected log2 reads per base pair were then smoothed over a 20-bp window and rnap and card - ha peaks were identified as described previously , . briefly , maxima and minima were assigned as inflection points where the values 10 bp were both lower or both higher , respectively . maxima within 20 bp were merged with the peak location assigned to the maximum with the highest absolute signal value . peaks were divided into 0.1 interval bins of peak heights with a lower cutoff of peak height of 0.4 log2 reads per base pair . starting with the lowest bin , we then calculated the distance of each peak to the nearest gene start and compared these distances to those computed using genome coordinates arbitrarily rotated 1 10 bp around the m. smegmatis genome . using the wilcoxon mann whitney ranksum test for nonsimilarity of distributions , , rnap peaks in the 1.11.2 peak - height bins and card - ha peaks in the 0.50.6 peak - height bins were statistically significant ( p values for similarity of the distributions < 0.0001 ) . in other words , for each peak - height bin , the two lists of peak - to - gene start distances ( actual and rotated by 1 10 bp ) were tested for whether they were from different populations using the wilcoxon mann peaks in the lowest peak - height bin for which peak - to - gene start distances differed from random with a p value of < 0.0001 plus all peaks with greater heights were then used as the statistically significant peaks . we then identified rnap peaks associated with each gene as the closest rnap peak upstream from the gene start and card - ha peaks associated with the rnap peaks as the closest card - ha peak to each selected rnap peak . to calculate average chip signals for the aggregate profiles ( fig . 1 ) , we selected a subset of 62 genes meeting the following criteria : ( i ) 300 bp in gene length , ( ii ) average rnap log2 chip signal 1.6/bp , ( iii ) associated with an rnap peak with log2 chip signal 3/bp , ( iv ) absence of other rnap peaks within 500 bp upstream or 1000 bp downstream of the associated rnap peak , ( v ) absence of an oppositely oriented gene with an average rnap log2 chip signal 1 upstream from the gene ( because an oppositely oriented gene could create a divergent promoter region with potential for overlapping rnap and card - ha chip signals ) , and ( vi ) absence of an upstream gene with average rnap log2 chip signal > 0 within 100 bp upstream from the gene ( because such an arrangement would indicate the gene is an internal member of an operon ) . the rnap , card - ha , and rnap signals from the 62 genes were then averaged using the distance from the center of the associated peaks to align the genes ( fig the distance from the center of the associated peak served as a proxy for the transcriptional start site , since most transcriptional start sites are not mapped in m. smegmatis . this analysis showed that whereas rnap was found throughout transcribed regions of the genome , card - ha and rnap were primarily associated with promoter regions . these data matched the high correlation calculated for the distribution of card - ha and rnap ( table 2 , table 3 ) . levels of both card - ha and rnap dropped off immediately following the promoter sequences , suggesting that these proteins are lost from the rnap elongating complex after transcription initiation . the colocalization of rnap and card - ha led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . further analysis of the dataset also revealed that card was never present on the genome in the absence of rnap , suggesting that it may be targeted to the genome through its interaction with rnap . card modulates transcription through its direct interaction with rnap , . to determine at which stage of the transcription cycle ( initiation , elongation , or termination ) card acts , we used chip - seq to survey the distribution of card throughout the m. smegmatis chromosome . our data shows that card is localized to promoters throughout the m. smegmatis genome , indicating that card functions during transcription initiation . despite the previous finding that card has sequence non - specific dna binding activity , the chip - seq experiments also revealed that card was never present on the genome in the absence of rnap or rnap , suggesting that card is targeted to the genome through its interaction with rnap . the chip - seq data for the distribution of rnap also serves as a map of potential promoter elements throughout the m. smegmatis genome , which has never before been experimentally examined . compilation of the chip - seq data and previous microarray expression profiling analyses indicates that card is broadly distributed on promoters of most transcription units regardless of whether they were deregulated during card depletion . there is also the striking correlation between the distributions of card and rnap on the genome , despite the fact that no direct interaction between these proteins has been reported . the factors contributing to the enrichment of card at rnap containing holoenzymes as opposed to elongating rnap core complexes remain unknown and will be a topic of future study . all together , results from these experiments have provided invaluable information that will help direct the ongoing efforts in determining the mechanism of transcription regulation by card . in addition , this work serves as a framework for further investigations into rnap function in mycobacteria .

card is an essential mycobacterial protein that binds the rna polymerase ( rnap ) and affects the transcriptional profile of mycobacterium smegmatis and mycobacterium tuberculosis [ 6 ] . we predicted that card was directly regulating rnap function but our prior experiments had not determined at what stage of transcription card was functioning and at which genes card interacted with the rnap . to begin to address these open questions , we performed chromatin immunoprecipitation sequencing ( chip - seq ) to survey the distribution of card throughout the m. smegmatis chromosome . the distribution of rnap subunits and a were also profiled . we expected that rnap would be present throughout transcribed regions and rnap a would be predominantly enriched at promoters based on work in escherichia coli [ 3 ] , however this had yet to be determined in mycobacteria . the chip - seq analyses revealed that card was never present on the genome in the absence of rnap , was primarily associated with promoter regions , and was highly correlated with the distribution of rnap a . the colocalization of a and card led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . here we describe in detail the data from the chip - seq experiments associated with the study published by srivastava and colleagues in the proceedings of the national academy of science in 2013 [ 5 ] as well as discuss the findings from this dataset in relation to both card and mycobacterial transcription as a whole.the chip - seq data have been deposited in the gene expression omnibus ( geo ) database , www.ncbi.nlm.nih.gov/geo ( accession no . gse48164 ) .

Direct link to deposited data Experimental design, materials and methods Bacterial strains and culture conditions Chromatin immunoprecipitation Sequencing Normalization Data analysis Discussion

all m. smegmatis strains were isogenic to mc155 and were grown at 37 c in lb supplemented with 0.5% dextrose , 0.5% glycerol , and 0.05% tween 80 ( broth ) . for immunoprecipitation of card , rnap , and rnap , a card merodiploid strain was produced by integrating pmsg430smcard - ha ( constitutively expresses m. smegmatis c - terminal ha tagged card , kanamycin resistant ) into the attb site of m. smegmatis mc155 . allelic exchange experiments were performed with the card merodiploid strain using a dna donor sequence with homology to mc155 nucleotides 6141480 to 6142268 and 6140266 to 6141010 to delete all of the card gene except the nucleotides encoding the first 10 and last 3 amino acids from the endogenous locus , generating card attb::tetcard - ha . for immunoprecipitation of unfused ha peptide as a control , mc155 was transformed with pmsg431 , which integrates into the attb site of the genome and constitutively expresses ha peptide . cultures of m. smegmatis card attb::tetcard - ha and mc155 attb::pmsg431 strains were grown to late log phase ( od600 = ~ 1 ) before adding a final concentration of 2% formaldehyde and shaking at room temperature for 30 min to crosslink dna and proteins . the cell debris was spun down and the lysate was added to 400 l chip lysis buffer ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 1% triton x-100 ) plus roche complete protease inhibitor cocktail . protein nucleic acid complexes containing card - ha were immunoprecipitated from the m. smegmatis mc155 card attb::tetcard - ha strain cell lysate by adding 50 l of anti - ha agarose ( sigma ) . complexes containing unfused ha were immunoprecipitated from the mc155 attb::pmsg431 strain with the same anti - ha agarose . however , one of the rnap samples was lost during library preparation and , therefore , there is only data for one rnap replicate . the antibody matrix was washed 2 with chip lysis buffer , 2 with chip lysis buffer plus an additional 360 mm nacl , 2 with chip wash buffer ( 10 mm tris - hcl ph 8.0 , 250 mm licl , 0.5% np-40 , 0.5% sodium deoxycholate , 1 mm edta ) , and 2 with te , each time by rotating for 10 min at 4 c . complexes that co - precipitated with the respective antibody matrix were eluted twice by adding 100 l of chip elution buffer ( 50 mm tris - hcl ph 8.0 , 10 mm edta , 1% sds ) , incubating for 10 min at 65 c with agitation , spinning down the antibody matrix , and transferring the eluate to a new tube . co - precipitated dna was sequenced using an ab solid 4 high - throughput genome sequencer ( life technologies ) and a 50 bp read length , which provided sufficient reads for over 100-fold coverage of the genome in each sample , wherein the m. smegmatis genome is 6,988,209 bp in length and the coverage of each sequencing reaction was over 800 mbp . the size factors are determined by taking the median of the ratios of observed counts . for example , the sequences of the 16s , 23s , and 5s ribosomal rna are identical in the m. smegmatis rrna and rrnb operons . the normalized number of reads for each base pair was saved as a wig file for each sample . we first determined how well replicate samples of the distribution of a given protein correlated to each other and how well the distribution of card correlated to the distributions of rnap and rnap ( table 2 , table 3 ) . the correlations were obtained by computing the pearson correlation of the genomic coverage profiles of each pair of samples . the coverage profiles were computed by summing the contributions of all mapped fragments , assuming they were 100 bp long , and then , in 20-bp steps along the entire genome , computing the average coverage of the surrounding 100-bp window . these data showed that individual replicates for a single immunoprecipitation condition correlated highly with one another ( bolded in table 2 ) and indicated that the distribution of card - ha or rnap was consistent between biological replicates . the correlation between the distribution of card - ha and the distribution of rnap ( bolded in table 3 ) was almost as high as the correlation between the two card - ha replicates , indicating that the distribution of card - ha is very similar to that of rnap . to directly compare the genome distributions of card - ha , rnap , and rnap , the reads per base pair from the unfused ha peptide sample served as the background control and were subtracted from the other datasets . the rationale for this control was that as a non - dna binding protein , the ha peptide should be diffusely localized throughout the cell and serve as a readout for the background levels of nonspecific crosslinking to the dna . the normalized , background - corrected log2 reads per base pair were then smoothed over a 20-bp window and rnap and card - ha peaks were identified as described previously , . starting with the lowest bin , we then calculated the distance of each peak to the nearest gene start and compared these distances to those computed using genome coordinates arbitrarily rotated 1 10 bp around the m. smegmatis genome . using the wilcoxon mann whitney ranksum test for nonsimilarity of distributions , , rnap peaks in the 1.11.2 peak - height bins and card - ha peaks in the 0.50.6 peak - height bins were statistically significant ( p values for similarity of the distributions < 0.0001 ) . in other words , for each peak - height bin , the two lists of peak - to - gene start distances ( actual and rotated by 1 10 bp ) were tested for whether they were from different populations using the wilcoxon mann peaks in the lowest peak - height bin for which peak - to - gene start distances differed from random with a p value of < 0.0001 plus all peaks with greater heights were then used as the statistically significant peaks . we then identified rnap peaks associated with each gene as the closest rnap peak upstream from the gene start and card - ha peaks associated with the rnap peaks as the closest card - ha peak to each selected rnap peak . 1 ) , we selected a subset of 62 genes meeting the following criteria : ( i ) 300 bp in gene length , ( ii ) average rnap log2 chip signal 1.6/bp , ( iii ) associated with an rnap peak with log2 chip signal 3/bp , ( iv ) absence of other rnap peaks within 500 bp upstream or 1000 bp downstream of the associated rnap peak , ( v ) absence of an oppositely oriented gene with an average rnap log2 chip signal 1 upstream from the gene ( because an oppositely oriented gene could create a divergent promoter region with potential for overlapping rnap and card - ha chip signals ) , and ( vi ) absence of an upstream gene with average rnap log2 chip signal > 0 within 100 bp upstream from the gene ( because such an arrangement would indicate the gene is an internal member of an operon ) . the rnap , card - ha , and rnap signals from the 62 genes were then averaged using the distance from the center of the associated peaks to align the genes ( fig . for the gene alignments , the distance from the center of the associated peak served as a proxy for the transcriptional start site , since most transcriptional start sites are not mapped in m. smegmatis . this analysis showed that whereas rnap was found throughout transcribed regions of the genome , card - ha and rnap were primarily associated with promoter regions . these data matched the high correlation calculated for the distribution of card - ha and rnap ( table 2 , table 3 ) . levels of both card - ha and rnap dropped off immediately following the promoter sequences , suggesting that these proteins are lost from the rnap elongating complex after transcription initiation . the colocalization of rnap and card - ha led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . further analysis of the dataset also revealed that card was never present on the genome in the absence of rnap , suggesting that it may be targeted to the genome through its interaction with rnap . all m. smegmatis strains were isogenic to mc155 and were grown at 37 c in lb supplemented with 0.5% dextrose , 0.5% glycerol , and 0.05% tween 80 ( broth ) . for immunoprecipitation of card , rnap , and rnap , a card merodiploid strain was produced by integrating pmsg430smcard - ha ( constitutively expresses m. smegmatis c - terminal ha tagged card , kanamycin resistant ) into the attb site of m. smegmatis mc155 . allelic exchange experiments were performed with the card merodiploid strain using a dna donor sequence with homology to mc155 nucleotides 6141480 to 6142268 and 6140266 to 6141010 to delete all of the card gene except the nucleotides encoding the first 10 and last 3 amino acids from the endogenous locus , generating card attb::tetcard - ha . for immunoprecipitation of unfused ha peptide as a control , mc155 was transformed with pmsg431 , which integrates into the attb site of the genome and constitutively expresses ha peptide . the cell debris was spun down and the lysate was added to 400 l chip lysis buffer ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 1% triton x-100 ) plus roche complete protease inhibitor cocktail . protein nucleic acid complexes containing card - ha were immunoprecipitated from the m. smegmatis mc155 card attb::tetcard - ha strain cell lysate by adding 50 l of anti - ha agarose ( sigma ) . complexes containing unfused ha were immunoprecipitated from the mc155 attb::pmsg431 strain with the same anti - ha agarose . however , one of the rnap samples was lost during library preparation and , therefore , there is only data for one rnap replicate . complexes that co - precipitated with the respective antibody matrix were eluted twice by adding 100 l of chip elution buffer ( 50 mm tris - hcl ph 8.0 , 10 mm edta , 1% sds ) , incubating for 10 min at 65 c with agitation , spinning down the antibody matrix , and transferring the eluate to a new tube . co - precipitated dna was sequenced using an ab solid 4 high - throughput genome sequencer ( life technologies ) and a 50 bp read length , which provided sufficient reads for over 100-fold coverage of the genome in each sample , wherein the m. smegmatis genome is 6,988,209 bp in length and the coverage of each sequencing reaction was over 800 mbp . the size factors are determined by taking the median of the ratios of observed counts . for example , the sequences of the 16s , 23s , and 5s ribosomal rna are identical in the m. smegmatis rrna and rrnb operons . we first determined how well replicate samples of the distribution of a given protein correlated to each other and how well the distribution of card correlated to the distributions of rnap and rnap ( table 2 , table 3 ) . the coverage profiles were computed by summing the contributions of all mapped fragments , assuming they were 100 bp long , and then , in 20-bp steps along the entire genome , computing the average coverage of the surrounding 100-bp window . these data showed that individual replicates for a single immunoprecipitation condition correlated highly with one another ( bolded in table 2 ) and indicated that the distribution of card - ha or rnap was consistent between biological replicates . this consistency between replicates allowed us to average the pearson correlation values for each comparison to simplify the comparisons between immunoprecipitation conditions ( table 3 ) . the correlation between the distribution of card - ha and the distribution of rnap ( bolded in table 3 ) was almost as high as the correlation between the two card - ha replicates , indicating that the distribution of card - ha is very similar to that of rnap . to directly compare the genome distributions of card - ha , rnap , and rnap , the reads per base pair from the unfused ha peptide sample served as the background control and were subtracted from the other datasets . the rationale for this control was that as a non - dna binding protein , the ha peptide should be diffusely localized throughout the cell and serve as a readout for the background levels of nonspecific crosslinking to the dna . the normalized , background - corrected log2 reads per base pair were then smoothed over a 20-bp window and rnap and card - ha peaks were identified as described previously , . maxima within 20 bp were merged with the peak location assigned to the maximum with the highest absolute signal value . starting with the lowest bin , we then calculated the distance of each peak to the nearest gene start and compared these distances to those computed using genome coordinates arbitrarily rotated 1 10 bp around the m. smegmatis genome . using the wilcoxon mann whitney ranksum test for nonsimilarity of distributions , , rnap peaks in the 1.11.2 peak - height bins and card - ha peaks in the 0.50.6 peak - height bins were statistically significant ( p values for similarity of the distributions < 0.0001 ) . in other words , for each peak - height bin , the two lists of peak - to - gene start distances ( actual and rotated by 1 10 bp ) were tested for whether they were from different populations using the wilcoxon mann peaks in the lowest peak - height bin for which peak - to - gene start distances differed from random with a p value of < 0.0001 plus all peaks with greater heights were then used as the statistically significant peaks . we then identified rnap peaks associated with each gene as the closest rnap peak upstream from the gene start and card - ha peaks associated with the rnap peaks as the closest card - ha peak to each selected rnap peak . 1 ) , we selected a subset of 62 genes meeting the following criteria : ( i ) 300 bp in gene length , ( ii ) average rnap log2 chip signal 1.6/bp , ( iii ) associated with an rnap peak with log2 chip signal 3/bp , ( iv ) absence of other rnap peaks within 500 bp upstream or 1000 bp downstream of the associated rnap peak , ( v ) absence of an oppositely oriented gene with an average rnap log2 chip signal 1 upstream from the gene ( because an oppositely oriented gene could create a divergent promoter region with potential for overlapping rnap and card - ha chip signals ) , and ( vi ) absence of an upstream gene with average rnap log2 chip signal > 0 within 100 bp upstream from the gene ( because such an arrangement would indicate the gene is an internal member of an operon ) . the rnap , card - ha , and rnap signals from the 62 genes were then averaged using the distance from the center of the associated peaks to align the genes ( fig the distance from the center of the associated peak served as a proxy for the transcriptional start site , since most transcriptional start sites are not mapped in m. smegmatis . this analysis showed that whereas rnap was found throughout transcribed regions of the genome , card - ha and rnap were primarily associated with promoter regions . these data matched the high correlation calculated for the distribution of card - ha and rnap ( table 2 , table 3 ) . levels of both card - ha and rnap dropped off immediately following the promoter sequences , suggesting that these proteins are lost from the rnap elongating complex after transcription initiation . the colocalization of rnap and card - ha led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . further analysis of the dataset also revealed that card was never present on the genome in the absence of rnap , suggesting that it may be targeted to the genome through its interaction with rnap . card modulates transcription through its direct interaction with rnap , . to determine at which stage of the transcription cycle ( initiation , elongation , or termination ) card acts , we used chip - seq to survey the distribution of card throughout the m. smegmatis chromosome . our data shows that card is localized to promoters throughout the m. smegmatis genome , indicating that card functions during transcription initiation . despite the previous finding that card has sequence non - specific dna binding activity , the chip - seq experiments also revealed that card was never present on the genome in the absence of rnap or rnap , suggesting that card is targeted to the genome through its interaction with rnap . the chip - seq data for the distribution of rnap also serves as a map of potential promoter elements throughout the m. smegmatis genome , which has never before been experimentally examined . compilation of the chip - seq data and previous microarray expression profiling analyses indicates that card is broadly distributed on promoters of most transcription units regardless of whether they were deregulated during card depletion . there is also the striking correlation between the distributions of card and rnap on the genome , despite the fact that no direct interaction between these proteins has been reported . in addition , this work serves as a framework for further investigations into rnap function in mycobacteria .

insertion of external ventricular drains ( evds ) is one of the most common neurosurgical procedures performed in neurosurgery today , with over 20,000 evds inserted annually in the usa alone . the most widely practised surgical technique for insertion of evds involves the insertion of a ventricular catheter into the ventricle with tunnelling of the distal end a short distance away from the incision , a short - tunnelled evd . the complication rate from short - tunnelled evds is high and includes infection , csf leak , blockage , misplacement of the ventricular catheter at the time of surgery and the inadvertent migration of the ventricular catheter following surgery . the presumed mechanism of infection is bacterial entry at the exit site on the skin with subsequent ascending colonisation of the catheter . there are data suggesting that increasing the distance from the evd exit site to the burr hole reduces infection [ 911 ] . we therefore hypothesised that insertion of a long - tunnelled evd ( ltevd ) would reduce the rate of novel infection and , since ltevds require the incorporation of a reservoir , would also reduce the rate of inadvertent migration of the catheter out of the ventricle . we have defined a ltevd as a ventricular access device comprising a ventricular catheter , a reservoir and a distal catheter that is externalised at the level of the abdomen or anterior chest wall . this paper is a retrospective audit of ltevd insertions at a single paediatric neurosurgical institution , specifically addressing infection and mechanical complications . a retrospective review was conducted in all the patients who had a ltevd inserted from 2 january 2008 to 9 march 2012 . the policy of the neurosurgical department throughout the course of this study was to insert all evds in the operating theatre . a local guideline for insertion of ltevds was in place for the duration of this study . this comprised antibiotic prophylaxis ( flucloxacillin and amikacin ) at induction of anaesthesia and for a further period of 24 h , clipping of hair at least 2 cm away from the wound and preparation of the field with povidone iodine solution and/or chlorhexidine ( 0.5 % in 70 % alcohol ) . bactiseal antibiotic - impregnated ventricular and peritoneal catheters were connected to a miethke ventricular access device . subcutaneous tunnelling was performed from the cranial wound to the chest wall or abdomen , allowing the distal catheter to be passed . a purse - string suture was applied to the exit site and covered with a waterproof dressing . the peritoneal catheter was not cut , allowing a long length of catheter to extend beyond the exit site . post - operatively , surgical and nursing staff followed the external ventricular drain clinical guideline of great ormond street hospital . drain management involved hourly checks of the amount and colour of csf drained , exit site condition and the patients neurological condition . redness , inflammation , oozing of blood and csf leakage at the exit site were all documented . the post - operative dressing was changed at 24 h using 2 % chlorhexidine clinell wipes to clean the site . following this , routine sampling of csf from an evd was not advocated unless there was a specific clinical indication for fear of introducing contamination into the sterile closed circuit . patients presenting with shunt infection or de novo ventriculitis requiring evd insertion had csf sampling daily . for all other cases , csf sampling was performed on the day prior to removal of the evd or as part of a septic screen should the child demonstrate clinical evidence of infection . all csf samples were sent to the on - site microbiology department and processed immediately . a cell count was made up of the neat csf and a gram stain performed on the spun deposit , for a differential white cell count and for the presence of organisms . primary culture of the spun deposit was performed on blood and chocolate agar incubated for 4048 h at 3537 c in 510 % co2 , macconkey agar incubated for 1824 h in air at 37 c , sabouraud agar incubated for 5 days in air at 3537 c and blood agar incubated anaerobically for 5 days . additional enrichment was performed by inoculation of brain - heart infusion for overnight enrichment at 3537 c followed by subculture on blood and chocolate incubated for 1824 h. evd catheter tips were cultured by rolling the terminal 5 cm across a blood agar plate and incubating at 3537 c for 1824 h. in this study , a ltevd infection was defined as a novel bacterial growth or detection in the csf and/or ltevd catheter that had arisen during the period of the ltevd being in situ and that resulted in a change in treatment ( antibiotic therapy and/or change of evd ) . when assessing suspected cases of ltevd infection , systemic factors , white cell count in the csf , bacterial growth on culture from csf / ltevd catheter samples and antibiotic treatment were all considered before coming to a conclusion as to whether a ltevd infection was present . ltevd csf infection was considered to have occurred if there was growth on primary culture , growth on subculture only and organisms were seen , growth in repeated samples on subculture when no organisms had been seen or where organisms were seen on repeated gram stain without growth . growth from the tip without growth in the csf was not automatically graded as a ltevd csf infection . each case was managed on an individual basis . the lead microbiology consultant for neurosurgery and the neurosurgeon took the final decision as to whether the ltevd had been infected during the management of the case and the following data were retrieved from the departmental operative database , operating theatre log and clinical record . the total number of ltevd procedures performed during the study period , the number of ltevd infections , the duration of insertion and the rate of inadvertent ltevd dislodgement were recorded . it was noted whether the removal of the ltevd was part of another surgical procedure ( such as insertion of ventriculoperitoneal ( vp ) shunt ) or whether the removal was performed in isolation . underlying diagnosis and patient age at the time of ltevd insertion was also recorded . patients who required ltevd insertion due to an existing infection were excluded from the data set when considering the final infection rate , due to possible treatment - induced discrepancies when testing for novel ltevd infection . when reporting the extra procedures needed for ltevd removal , ltevd insertions for the cohort with existing infection ( n = 48 ) were omitted , as these patients would have had a subsequent procedure to reinsert a shunt in theatre , irrespective of the type of evd inserted . a 2 2 fisher s exact test and student s t test were used for assessing a significant difference between the categorical data . a local guideline for insertion of ltevds was in place for the duration of this study . this comprised antibiotic prophylaxis ( flucloxacillin and amikacin ) at induction of anaesthesia and for a further period of 24 h , clipping of hair at least 2 cm away from the wound and preparation of the field with povidone iodine solution and/or chlorhexidine ( 0.5 % in 70 % alcohol ) . bactiseal antibiotic - impregnated ventricular and peritoneal catheters were connected to a miethke ventricular access device . subcutaneous tunnelling was performed from the cranial wound to the chest wall or abdomen , allowing the distal catheter to be passed . a purse - string suture was applied to the exit site and covered with a waterproof dressing . the peritoneal catheter was not cut , allowing a long length of catheter to extend beyond the exit site . post - operatively , surgical and nursing staff followed the external ventricular drain clinical guideline of great ormond street hospital . drain management involved hourly checks of the amount and colour of csf drained , exit site condition and the patients neurological condition . redness , inflammation , oozing of blood and csf leakage at the exit site were all documented . the post - operative dressing was changed at 24 h using 2 % chlorhexidine clinell wipes to clean the site . routine sampling of csf from an evd was not advocated unless there was a specific clinical indication for fear of introducing contamination into the sterile closed circuit . patients presenting with shunt infection or de novo ventriculitis requiring evd insertion had csf sampling daily . for all other cases , csf sampling was performed on the day prior to removal of the evd or as part of a septic screen should the child demonstrate clinical evidence of infection . all csf samples were sent to the on - site microbiology department and processed immediately . a cell count was made up of the neat csf and a gram stain performed on the spun deposit , for a differential white cell count and for the presence of organisms . primary culture of the spun deposit was performed on blood and chocolate agar incubated for 4048 h at 3537 c in 510 % co2 , macconkey agar incubated for 1824 h in air at 37 c , sabouraud agar incubated for 5 days in air at 3537 c and blood agar incubated anaerobically for 5 days . additional enrichment was performed by inoculation of brain - heart infusion for overnight enrichment at 3537 c followed by subculture on blood and chocolate incubated for 1824 h. evd catheter tips were cultured by rolling the terminal 5 cm across a blood agar plate and incubating at 3537 c for 1824 h. in this study , a ltevd infection was defined as a novel bacterial growth or detection in the csf and/or ltevd catheter that had arisen during the period of the ltevd being in situ and that resulted in a change in treatment ( antibiotic therapy and/or change of evd ) . when assessing suspected cases of ltevd infection , systemic factors , white cell count in the csf , bacterial growth on culture from csf / ltevd catheter samples and antibiotic treatment were all considered before coming to a conclusion as to whether a ltevd infection was present . ltevd csf infection was considered to have occurred if there was growth on primary culture , growth on subculture only and organisms were seen , growth in repeated samples on subculture when no organisms had been seen or where organisms were seen on repeated gram stain without growth . growth from the tip without growth in the csf was not automatically graded as a ltevd csf infection . each case was managed on an individual basis . the lead microbiology consultant for neurosurgery and the neurosurgeon took the final decision as to whether the ltevd had been infected during the management of the case and the following data were retrieved from the departmental operative database , operating theatre log and clinical record . the total number of ltevd procedures performed during the study period , the number of ltevd infections , the duration of insertion and the rate of inadvertent ltevd dislodgement were recorded . it was noted whether the removal of the ltevd was part of another surgical procedure ( such as insertion of ventriculoperitoneal ( vp ) shunt ) or whether the removal was performed in isolation . underlying diagnosis and patient age at the time of ltevd insertion was also recorded . patients who required ltevd insertion due to an existing infection were excluded from the data set when considering the final infection rate , due to possible treatment - induced discrepancies when testing for novel ltevd infection . when reporting the extra procedures needed for ltevd removal , ltevd insertions for the cohort with existing infection ( n = 48 ) were omitted , as these patients would have had a subsequent procedure to reinsert a shunt in theatre , irrespective of the type of evd inserted . a 2 2 fisher s exact test and student s t test were used for assessing a significant difference between the categorical data . one hundred seventy - seven patients had 181 ltevds placed between 2 january 2008 and 9 march 2012 . the mean age of the patients was 6.6 years with a range of 015.5 years . of the 181 procedures , 85 were inserted for intraventricular haemorrhage ( 47 % ) , 48 for infection ( 27 % ) , 13 for tumour - related hydrocephalus ( 7.2 % ) , 31 as a temporising measure ( 17 % ) ( where an evd was inserted as an emergency measure and a definitive csf diversion was performed subsequently ) and 4 for trauma ( 2.2 % ) . of the 133 patients with no existing infection , microbiology analysis identified five cases of novel ltevd infection , an infection rate of 3.8 % . no ltevd infections were identified in the cohort ( n = 48 ) that required a ltevd due to an existing infection . out of the total 181 procedures , five novel cases of ltevd infection were identified , an infection rate of 2.76 % . three other bacterial growths were recorded but were explained by sample contamination ( two cases ) and existing surgical site infection ( one case ) . as can be seen in table 1 , there was no obvious trend for infection in younger or older patients , with ltevds from a wide range of age groups being infected . when comparing the ages of the infected and non - infected groups , statistical analysis showed no evidence for age as a risk factor for ltevd infections ( p = 0.8967 ) . four of the five infected ltevds were inserted for longer than the median duration of insertion ; however , there was no significant difference in ltevd insertion duration between the infected ( n = 5 ) and non - infected ( n = 128 ) groups ( p = 0.1881 ) . four out of the five infected ltevd cases were caused by coagulase - negative staphylococci ( cons).table 1ltevd infections in this series : an overview describing the organism found in the csf , the age at ltevd insertion , the duration of insertion of the ltevd and the reason for insertiondata set numberorganism on cultureage at insertionduration of insertion ( days)reason for insertion145coagulase - negative staphylococcus5 years and 11 months7a temporising measure ( post endoscopic procedure)169gram - positive cocci9 months28intraventricular haemorrhage51coagulase - negative staphylococcus15 years and 0 month23intraventricular haemorrhage88coagulase - negative staphylococcus2 days21temporising measure ( spinal dysraphism)79coagulase - negative staphylococcus15 years and 4 months12intraventricular haemorrhage ltevd infections in this series : an overview describing the organism found in the csf , the age at ltevd insertion , the duration of insertion of the ltevd and the reason for insertion the mean duration of ltevd insertion was 11.2 days ( range 0 to 42 days and a median of 10 days , interquartile range of 19 days ) . four out of 181 ( 2.2 % ) of the ltevd procedures carried out during this time inadvertently dislodged , requiring an extra procedure to reinsert the evd . thirteen extra procedures were carried out in theatre , specifically to remove the evd , affecting 13/133 ( 9.8 % ) of our patients ( excluding the 48 ltevds inserted due to existing infection , as these would have an inevitable subsequent procedure to reinsert a non - infected shunt ) , where as the remainder of the removals were carried out during a subsequent procedure the patient required ; thus , no extra procedures were needed to remove the evd for these patients . the total number of extra operations required in this ltevd cohort was thus 17 ( 9.4 % ) . a major complication of evd insertion is csf infection , posing a significant risk to patients as well as placing an additional burden on hospital resources . a range of infection rates from short - tunnelled evds has been reported , from 3.4 to 32.2 % [ 28 , 1220 ] . the data for infection rates of short - tunnelled evds are summarised in fig . 1 . this study , utilising ltevd , reports a lower evd infection rate when compared with previously published literature.fig . 1a comparison of evd infection rates across previous series , using either a subcutaneous long - tunnelled evd or a short / non - tunnelled procedure a comparison of evd infection rates across previous series , using either a subcutaneous long - tunnelled evd or a short / non - tunnelled procedure numerous studies have attempted to identify factors contributing to evd - related infection . csf leakage particularly near the catheter exit site [ 21 , 22 ] , duration of evd insertion [ 3 , 12 ] , evd reinsertion , presence or absence of prophylactic antibiotics and csf sampling have all been suggested as significant contributing factors towards the onset of evd - related infection . other factors such as breaches of the closed system and surgical protocol violation and regular catheter change have also been investigated . comparing existing series is fraught with difficulty due to the varying age groups studied ( adult and paediatric ) , differing underlying aetiology and lack of standardised protocols for the insertion and management of evds . currently , therefore , there is little agreement about the relative significance of these contributing factors towards csf infection in evd patients . this exclusively paediatric study of long - tunnelled evd incorporating a predefined guideline for perioperative evd management has revealed an infection rate of 3.8 % , a rate substantially less than that previously reported . csf leakage , either at the cranial site or the evd exit site , is strongly correlated with infection risk . by increasing the distance from the burr hole to the exit site , the resistance to csf flow around the outside of the catheter will be greater , thus reducing csf leakage . clearly , the distance that bacteria have to migrate in order to colonise the csf compartment is also greater . omar and haspani found that tunnelling only as far as 5 cm from the burr hole was sufficient to reduce the infection rate from 62.9 to 11.5 % . for postero - parietal or parieto - occipital burr holes in children , the options for local tunnelling are limited . we avoid exit sites in the region of the neck as this is a mobile area with multiple skin creases . furthermore , wounds here are difficult to dress , are cosmetically unsatisfactory and may lie along the course of a subsequent shunt . thus , we advocate tunnelling to the anterior abdominal wall or , less commonly , the anterior chest wall ( well away from the nipple or breast bud ) , resulting in a tunnelling distance of at least 20 cm . the results presented in this paper demonstrate comparable results to the other two studies [ 27 , 28 ] that have investigated the effectiveness of ltevds . in the study by khanna et al . , a 0 % infection rate was achieved for the first 16 days after ltevd insertion and an overall infection rate of 4 % , in 100 adult and paediatric patients . it was concluded that ltevds had a low rate of infection and could remain in place for up to 40 days . a more recent study , using a non - antibiotic - impregnated ltevd , in 114 adult and paediatric patients requiring more than 7 days drainage , found the infection rate to be 6.8 % , which was deemed comparable with the infection rate of standard short - tunnelled evds . notably , ltevds were capable of remaining in place for long periods of time ( up to 60 days , with a mean duration of 20 days ) and no additional morbidity was associated with the ltevd procedure . both of these studies indicate promise of the ltevd technique though without conclusive evidence of their effectiveness in the paediatric population . our study demonstrated a ltevd infection rate of 3.8 % ( five out of 133 ) , which , as demonstrated by fig . 1 , compares favourably with previously published data on non - ltevd series . a review of evd infection , using data from 23 studies , comprising 5,733 evd procedures in both adults and children , reported an infection rate of 8.8 % . figure 1 compares the infection rates in 15 publications [ 2 , 3 , 8 , 9 , 11 , 19 , 20 , 23 , 2935 ] using short - tunnelled evds and 3 publications using ltevds [ 2729 ] with our series . a statistically significant difference ( p = < 0.05 ) was found in the evd - related infection rate between our study and 14 of the studies using the short - tunnelled procedure . furthermore , there was no significant difference in the infection rates when comparing our study to the other three published studies using a long subcutaneous tunnel , suggesting that tunnelling seems to be a major contributing factor when considering evd infection . the infection rate reported does not include the 48 cases that required a ltevd insertion for an existing infection as these patients may have been on active treatment for existing infection , which may affect the ability to detect a novel infection . infection is a well - recognised complication of evd insertion in patients with clean csf before insertion . however , there is also a rate of novel infection , with new organisms , in the group where the evd is placed as a part of the treatment for infection . there were no reported novel evd infections in this group with an existing infection ( n = 48 ) . therefore , the total infection rate , including this group that require a ltevd for infection , is 2.76 % ( five out of 181 ) . four out of 181 ( 2.2 % ) ltevds in this study were inadvertently dislodged and required a separate procedure to reinsert the evd . data on the rate of displacement of evd are sparse . in another study , using short - tunnelled evd , a dislodgement rate of two out of 51 ( 8.1 % ) was reported . this would imply that the combination of a reservoir and ltevd affords some protection against this complication . the average duration of insertion was 11.2 days , with one ltevd remaining in place for 42 days . our data and the data of the other ltevd studies [ 2729 ] suggest that ltevds can safely remain in place for long periods of time without the attendant risks of infection and dislodgement . it was noted that four of the five infected ltevds were inserted for longer than the median duration of insertion ; however , a statistical comparison found no significant difference in the ages of the two groups . duration of evd insertion has been shown to be a significant risk factor when using short - tunnelled evds [ 2 , 3 ] , further strengthening the argument for a ltevd protocol . additionally , one of the infected cases was inserted immediately after an endoscopic procedure , suggesting that this may have been a contributory factor to the ltevd infection , which would concur with the previous literature where intraoperative endoscope use was considered to be an additional risk factor when considering shunt infection rates in the paediatric population . the use of a ltevd incorporating a reservoir will inevitably result in an extra surgical procedure ( to remove the evd ) in some children . we would argue that the benefits of the long tunnel in terms of reducing infection rate and inadvertent dislodgement outweigh this inconvenience . the importance of using an antibiotic - impregnated catheter ( aic ) to lower infection rates in this cohort must not be underestimated . the bactiseal catheters used for this cohort contained 0.15 % clindamycin and 0.054 % rifampicin . several recent studies have demonstrated the effectiveness of using antibiotic - impregnated evd catheters in both the adult and paediatric population . a recent systematic review and meta - analysis identified several studies in both the adult and paediatric populations where antibiotic impregnation of the evd catheter was beneficial in reducing evd - related infections . a six - centre randomised controlled trial in the adult population has also shown a significant reduction in ltevd infections , from 9.4 to 1.3 % when using minocycline- and rifampicin - impregnated catheters . in a prospective versus historical cohort study of 91 paediatric patients , tamburrini et al . reported a significant reduction in evd infection rates , from 31.8 to 2.1 % when using aics in paediatric evds . in a further study , when aics were inserted in a centre already using prophylactic , systemic antibiotic treatment , evd infection rates were reduced from 23.5 to 4.3 % , further strengthening the argument for aic use in evds . when considering the antibiotics impregnated into the catheters , both minocycline / rifampin - impregnated ventricular catheter and clindamycin / rifampin - impregnated evd catheter ( used in this study ) however , not all of the data suggest that aics have a beneficial impact in evd insertion . a randomised controlled trial of 184 patients from another centre showed that aics did not have a significantly higher rate of csf infection ( 1 % ) compared to catheters without antibiotic impregnation ( 3 % ) , and there was no significant difference of clinical outcome at 6 months . a more recent randomised controlled study also demonstrated similar findings in 348 adult patients , where aics used in evds did not contribute to a reduction in infection rate ; however , this could be attributed to a low baseline rate of infection and so difficult to prove an aic - related reduction . the risk of a false negative csf culture when drawing csf from an aic must also be considered . using an in vitro model , an increased risk of false negative results of infected csf has been demonstrated . additionally , an increased rate of infection has been noted in paediatric vp shunt patients with prior evd insertion using an aic , the authors postulated that prior use of an aic increased the potential for resistant organisms . in summary , despite the possible risk of false negative csf sampling and antibiotic resistance , there is a body of both level i and level ii evidences , supporting the conclusion that aics do reduce evd infection rates . the role of aics used as a part of the treatment protocol for patients with pre - existing infection has been debated . however , for the cohort requiring ltevd insertion for pre - existing infection in this study ( n = 48 ) , the aic tubing was not employed as a specific treatment for the infection , for which appropriate intravenous and intraventricular antibiotics were administered before ltevd insertion . therefore , despite the licence for bactiseal aic catheters being for prevention of infection only , it was departmental practice to use aics for all evd insertions , whether infection was suspected at insertion or not . the relative contribution of aics , a long subcutaneous tunnel and a pre - defined protocol for perioperative management of the evd is difficult to elucidate in this study . there is evidence from another study that a comprehensive evd management protocol , similar to that implemented in this cohort , was effective in reducing evd - related infection rates . cited a reduction in infection rate from 1.06 to 0 % in evd procedures , over a 4-year period . the effectiveness of protocols in reducing shunt infection has also been well demonstrated in the north american shunt registry . this low infection rate and low dislodgement rate shown for evds in this study , using a ltevd , an aic and a perioperative management protocol , represents a potential for both clinical and economic benefits . fewer evd infections would reduce the patient s duration of hospital admission and reduce the risk from further surgery required to replace an infected evd . a lower dislodgement rate is both safer for the patient and cost - effective , as there is a reduced risk and cost brought about by extra theatre time . our study does have some limitations . the multi - study comparison ( fig . 1 ) does not take into account the different operating protocols and infection definitions across the centres at which the procedures were carried out . as discussed , evd - related infection is a multi - factorial problem and it would be difficult to disregard all the compounding factors apart from the tunnelling length and the use of aics that may influence the comparison with the other studies . this study was also conducted in the paediatric population , and comparing with adult studies may not offer a true comparison . further studies may wish to assess the cost - effectiveness of this procedure , specifically in relation to hospital stay and extra operating time . csf leakage particularly near the catheter exit site [ 21 , 22 ] , duration of evd insertion [ 3 , 12 ] , evd reinsertion , presence or absence of prophylactic antibiotics and csf sampling have all been suggested as significant contributing factors towards the onset of evd - related infection . other factors such as breaches of the closed system and surgical protocol violation and regular catheter change have also been investigated . comparing existing series is fraught with difficulty due to the varying age groups studied ( adult and paediatric ) , differing underlying aetiology and lack of standardised protocols for the insertion and management of evds . currently , therefore , there is little agreement about the relative significance of these contributing factors towards csf infection in evd patients . this exclusively paediatric study of long - tunnelled evd incorporating a predefined guideline for perioperative evd management has revealed an infection rate of 3.8 % , a rate substantially less than that previously reported . csf leakage , either at the cranial site or the evd exit site , is strongly correlated with infection risk . by increasing the distance from the burr hole to the exit site , the resistance to csf flow around the outside of the catheter will be greater , thus reducing csf leakage . clearly , the distance that bacteria have to migrate in order to colonise the csf compartment is also greater . omar and haspani found that tunnelling only as far as 5 cm from the burr hole was sufficient to reduce the infection rate from 62.9 to 11.5 % . for postero - parietal or parieto - occipital burr holes in children , the options for local tunnelling are limited . we avoid exit sites in the region of the neck as this is a mobile area with multiple skin creases . furthermore , wounds here are difficult to dress , are cosmetically unsatisfactory and may lie along the course of a subsequent shunt . thus , we advocate tunnelling to the anterior abdominal wall or , less commonly , the anterior chest wall ( well away from the nipple or breast bud ) , resulting in a tunnelling distance of at least 20 cm . the results presented in this paper demonstrate comparable results to the other two studies [ 27 , 28 ] that have investigated the effectiveness of ltevds . in the study by khanna et al . , a 0 % infection rate was achieved for the first 16 days after ltevd insertion and an overall infection rate of 4 % , in 100 adult and paediatric patients . it was concluded that ltevds had a low rate of infection and could remain in place for up to 40 days . a more recent study , using a non - antibiotic - impregnated ltevd , in 114 adult and paediatric patients requiring more than 7 days drainage , found the infection rate to be 6.8 % , which was deemed comparable with the infection rate of standard short - tunnelled evds . notably , ltevds were capable of remaining in place for long periods of time ( up to 60 days , with a mean duration of 20 days ) and no additional morbidity was associated with the ltevd procedure . both of these studies indicate promise of the ltevd technique though without conclusive evidence of their effectiveness in the paediatric population . our study demonstrated a ltevd infection rate of 3.8 % ( five out of 133 ) , which , as demonstrated by fig . 1 , compares favourably with previously published data on non - ltevd series . a review of evd infection , using data from 23 studies , comprising 5,733 evd procedures in both adults and children , reported an infection rate of 8.8 % . figure 1 compares the infection rates in 15 publications [ 2 , 3 , 8 , 9 , 11 , 19 , 20 , 23 , 2935 ] using short - tunnelled evds and 3 publications using ltevds [ 2729 ] with our series . a statistically significant difference ( p = < 0.05 ) was found in the evd - related infection rate between our study and 14 of the studies using the short - tunnelled procedure . furthermore , there was no significant difference in the infection rates when comparing our study to the other three published studies using a long subcutaneous tunnel , suggesting that tunnelling seems to be a major contributing factor when considering evd infection . the infection rate reported does not include the 48 cases that required a ltevd insertion for an existing infection as these patients may have been on active treatment for existing infection , which may affect the ability to detect a novel infection . infection is a well - recognised complication of evd insertion in patients with clean csf before insertion . however , there is also a rate of novel infection , with new organisms , in the group where the evd is placed as a part of the treatment for infection . there were no reported novel evd infections in this group with an existing infection ( n = 48 ) . therefore , the total infection rate , including this group that require a ltevd for infection , is 2.76 % ( five out of 181 ) . four out of 181 ( 2.2 % ) ltevds in this study were inadvertently dislodged and required a separate procedure to reinsert the evd . data on the rate of displacement of evd are sparse . in another study , using short - tunnelled evd , a dislodgement rate of two out of 51 ( 8.1 % ) was reported . this would imply that the combination of a reservoir and ltevd affords some protection against this complication . the average duration of insertion was 11.2 days , with one ltevd remaining in place for 42 days . our data and the data of the other ltevd studies [ 2729 ] suggest that ltevds can safely remain in place for long periods of time without the attendant risks of infection and dislodgement . it was noted that four of the five infected ltevds were inserted for longer than the median duration of insertion ; however , a statistical comparison found no significant difference in the ages of the two groups . duration of evd insertion has been shown to be a significant risk factor when using short - tunnelled evds [ 2 , 3 ] , further strengthening the argument for a ltevd protocol . additionally , one of the infected cases was inserted immediately after an endoscopic procedure , suggesting that this may have been a contributory factor to the ltevd infection , which would concur with the previous literature where intraoperative endoscope use was considered to be an additional risk factor when considering shunt infection rates in the paediatric population . the use of a ltevd incorporating a reservoir will inevitably result in an extra surgical procedure ( to remove the evd ) in some children . we would argue that the benefits of the long tunnel in terms of reducing infection rate and inadvertent dislodgement outweigh this inconvenience . the importance of using an antibiotic - impregnated catheter ( aic ) to lower infection rates in this cohort must not be underestimated . the bactiseal catheters used for this cohort contained 0.15 % clindamycin and 0.054 % rifampicin . several recent studies have demonstrated the effectiveness of using antibiotic - impregnated evd catheters in both the adult and paediatric population . a recent systematic review and meta - analysis identified several studies in both the adult and paediatric populations where antibiotic impregnation of the evd catheter was beneficial in reducing evd - related infections . a six - centre randomised controlled trial in the adult population has also shown a significant reduction in ltevd infections , from 9.4 to 1.3 % when using minocycline- and rifampicin - impregnated catheters . in a prospective versus historical cohort study of 91 paediatric patients , tamburrini et al . reported a significant reduction in evd infection rates , from 31.8 to 2.1 % when using aics in paediatric evds . in a further study , when aics were inserted in a centre already using prophylactic , systemic antibiotic treatment , evd infection rates were reduced from 23.5 to 4.3 % , further strengthening the argument for aic use in evds . when considering the antibiotics impregnated into the catheters , both minocycline / rifampin - impregnated ventricular catheter and clindamycin / rifampin - impregnated evd catheter ( used in this study ) have been shown to be equally effective at reducing evd - related infections . however , not all of the data suggest that aics have a beneficial impact in evd insertion . a randomised controlled trial of 184 patients from another centre showed that aics did not have a significantly higher rate of csf infection ( 1 % ) compared to catheters without antibiotic impregnation ( 3 % ) , and there was no significant difference of clinical outcome at 6 months . a more recent randomised controlled study also demonstrated similar findings in 348 adult patients , where aics used in evds did not contribute to a reduction in infection rate ; however , this could be attributed to a low baseline rate of infection and so difficult to prove an aic - related reduction . the risk of a false negative csf culture when drawing csf from an aic must also be considered . using an in vitro model , an increased risk of false negative results of infected csf has been demonstrated . additionally , an increased rate of infection has been noted in paediatric vp shunt patients with prior evd insertion using an aic , the authors postulated that prior use of an aic increased the potential for resistant organisms . in summary , despite the possible risk of false negative csf sampling and antibiotic resistance , there is a body of both level i and level ii evidences , supporting the conclusion that aics do reduce evd infection rates . the role of aics used as a part of the treatment protocol for patients with pre - existing infection has been debated . however , for the cohort requiring ltevd insertion for pre - existing infection in this study ( n = 48 ) , the aic tubing was not employed as a specific treatment for the infection , for which appropriate intravenous and intraventricular antibiotics were administered before ltevd insertion . therefore , despite the licence for bactiseal aic catheters being for prevention of infection only , it was departmental practice to use aics for all evd insertions , whether infection was suspected at insertion or not . the relative contribution of aics , a long subcutaneous tunnel and a pre - defined protocol for perioperative management of the evd is difficult to elucidate in this study . there is evidence from another study that a comprehensive evd management protocol , similar to that implemented in this cohort , was effective in reducing evd - related infection rates . cited a reduction in infection rate from 1.06 to 0 % in evd procedures , over a 4-year period . the effectiveness of protocols in reducing shunt infection has also been well demonstrated in the north american shunt registry . this low infection rate and low dislodgement rate shown for evds in this study , using a ltevd , an aic and a perioperative management protocol , represents a potential for both clinical and economic benefits . fewer evd infections would reduce the patient s duration of hospital admission and reduce the risk from further surgery required to replace an infected evd . a lower dislodgement rate is both safer for the patient and cost - effective , as there is a reduced risk and cost brought about by extra theatre time . our study does have some limitations . the multi - study comparison ( fig . 1 ) does not take into account the different operating protocols and infection definitions across the centres at which the procedures were carried out . as discussed , evd - related infection is a multi - factorial problem and it would be difficult to disregard all the compounding factors apart from the tunnelling length and the use of aics that may influence the comparison with the other studies . this study was also conducted in the paediatric population , and comparing with adult studies may not offer a true comparison . further studies may wish to assess the cost - effectiveness of this procedure , specifically in relation to hospital stay and extra operating time . long - tunnelled evds , using an antibiotic - impregnated catheter and managed according to an agreed perioperative management protocol , carry a lower infection rate and dislodgement rate than the more widely used short - tunnelled procedure . we believe that the reduced morbidity and costs associated with such a policy outweigh the disadvantage of requiring an additional procedure for evd removal .

purposethe aim of this study is to report the efficacy of long subcutaneous tunnelling of external ventricular drains in reducing rates of infection and catheter displacement in a paediatric population.methodsin children requiring external ventricular drainage , a long - tunnelled drain was placed and managed according to a locally agreed guideline . end points were novel csf infection incurred during the time of drainage and re - operation to re - site displaced catheters . data were compared to other published series.resultsone hundred eighty - one long - tunnelled external ventricular drains ( ltevds ) were inserted . the mean age was 6.6 years ( range 015.5 years ) . reasons for insertion included intraventricular haemorrhage ( 47 % ) , infection ( 27 % ) , tumour - related hydrocephalus ( 7.2 % ) , as a temporising measure ( 17 % ) and trauma ( 2.2 % ) . the overall new infection rate for ltevd was 2.76 % . if the 48 cases where ltevds were inserted to treat an existing infection are excluded , the infection rate was 3.8 % ( 5/133 ) . the mean duration of insertion was 10 days ( range 042 days ) . four ltevds ( 2.2 % ) were inadvertently dislodged , requiring reinsertion . thirteen patients required removal of evd alone.there was a significant difference ( p < 0.05 ) when comparing our infection rate to 14 publications of infection rates in short - tunnelled evds ; however , there was no difference when comparing our data to three publications using ltevds.conclusionthe use of an antibiotic - impregnated ltevd , managed according to a predefined guideline , is associated with significantly reduced infection and displacement rates when compared with contemporary series . it is suggested that this reduction is of both clinical and economic benefits .

Introduction Methods Protocol Data collection Results Discussion EVD infections: the risk factors The evidence for tunnelling in reducing EVD infection Further strengthening the LTEVD argument: experience from this single-centre study Antibiotic-impregnated catheters: their importance in reducing EVD infections Implications and limitations Conclusion Conflict of interest

insertion of external ventricular drains ( evds ) is one of the most common neurosurgical procedures performed in neurosurgery today , with over 20,000 evds inserted annually in the usa alone . the most widely practised surgical technique for insertion of evds involves the insertion of a ventricular catheter into the ventricle with tunnelling of the distal end a short distance away from the incision , a short - tunnelled evd . the complication rate from short - tunnelled evds is high and includes infection , csf leak , blockage , misplacement of the ventricular catheter at the time of surgery and the inadvertent migration of the ventricular catheter following surgery . we therefore hypothesised that insertion of a long - tunnelled evd ( ltevd ) would reduce the rate of novel infection and , since ltevds require the incorporation of a reservoir , would also reduce the rate of inadvertent migration of the catheter out of the ventricle . a local guideline for insertion of ltevds was in place for the duration of this study . additional enrichment was performed by inoculation of brain - heart infusion for overnight enrichment at 3537 c followed by subculture on blood and chocolate incubated for 1824 h. evd catheter tips were cultured by rolling the terminal 5 cm across a blood agar plate and incubating at 3537 c for 1824 h. in this study , a ltevd infection was defined as a novel bacterial growth or detection in the csf and/or ltevd catheter that had arisen during the period of the ltevd being in situ and that resulted in a change in treatment ( antibiotic therapy and/or change of evd ) . the total number of ltevd procedures performed during the study period , the number of ltevd infections , the duration of insertion and the rate of inadvertent ltevd dislodgement were recorded . when reporting the extra procedures needed for ltevd removal , ltevd insertions for the cohort with existing infection ( n = 48 ) were omitted , as these patients would have had a subsequent procedure to reinsert a shunt in theatre , irrespective of the type of evd inserted . a 2 2 fisher s exact test and student s t test were used for assessing a significant difference between the categorical data . a local guideline for insertion of ltevds was in place for the duration of this study . additional enrichment was performed by inoculation of brain - heart infusion for overnight enrichment at 3537 c followed by subculture on blood and chocolate incubated for 1824 h. evd catheter tips were cultured by rolling the terminal 5 cm across a blood agar plate and incubating at 3537 c for 1824 h. in this study , a ltevd infection was defined as a novel bacterial growth or detection in the csf and/or ltevd catheter that had arisen during the period of the ltevd being in situ and that resulted in a change in treatment ( antibiotic therapy and/or change of evd ) . the total number of ltevd procedures performed during the study period , the number of ltevd infections , the duration of insertion and the rate of inadvertent ltevd dislodgement were recorded . patients who required ltevd insertion due to an existing infection were excluded from the data set when considering the final infection rate , due to possible treatment - induced discrepancies when testing for novel ltevd infection . when reporting the extra procedures needed for ltevd removal , ltevd insertions for the cohort with existing infection ( n = 48 ) were omitted , as these patients would have had a subsequent procedure to reinsert a shunt in theatre , irrespective of the type of evd inserted . a 2 2 fisher s exact test and student s t test were used for assessing a significant difference between the categorical data . the mean age of the patients was 6.6 years with a range of 015.5 years . of the 181 procedures , 85 were inserted for intraventricular haemorrhage ( 47 % ) , 48 for infection ( 27 % ) , 13 for tumour - related hydrocephalus ( 7.2 % ) , 31 as a temporising measure ( 17 % ) ( where an evd was inserted as an emergency measure and a definitive csf diversion was performed subsequently ) and 4 for trauma ( 2.2 % ) . of the 133 patients with no existing infection , microbiology analysis identified five cases of novel ltevd infection , an infection rate of 3.8 % . as can be seen in table 1 , there was no obvious trend for infection in younger or older patients , with ltevds from a wide range of age groups being infected . when comparing the ages of the infected and non - infected groups , statistical analysis showed no evidence for age as a risk factor for ltevd infections ( p = 0.8967 ) . four of the five infected ltevds were inserted for longer than the median duration of insertion ; however , there was no significant difference in ltevd insertion duration between the infected ( n = 5 ) and non - infected ( n = 128 ) groups ( p = 0.1881 ) . four out of the five infected ltevd cases were caused by coagulase - negative staphylococci ( cons).table 1ltevd infections in this series : an overview describing the organism found in the csf , the age at ltevd insertion , the duration of insertion of the ltevd and the reason for insertiondata set numberorganism on cultureage at insertionduration of insertion ( days)reason for insertion145coagulase - negative staphylococcus5 years and 11 months7a temporising measure ( post endoscopic procedure)169gram - positive cocci9 months28intraventricular haemorrhage51coagulase - negative staphylococcus15 years and 0 month23intraventricular haemorrhage88coagulase - negative staphylococcus2 days21temporising measure ( spinal dysraphism)79coagulase - negative staphylococcus15 years and 4 months12intraventricular haemorrhage ltevd infections in this series : an overview describing the organism found in the csf , the age at ltevd insertion , the duration of insertion of the ltevd and the reason for insertion the mean duration of ltevd insertion was 11.2 days ( range 0 to 42 days and a median of 10 days , interquartile range of 19 days ) . four out of 181 ( 2.2 % ) of the ltevd procedures carried out during this time inadvertently dislodged , requiring an extra procedure to reinsert the evd . thirteen extra procedures were carried out in theatre , specifically to remove the evd , affecting 13/133 ( 9.8 % ) of our patients ( excluding the 48 ltevds inserted due to existing infection , as these would have an inevitable subsequent procedure to reinsert a non - infected shunt ) , where as the remainder of the removals were carried out during a subsequent procedure the patient required ; thus , no extra procedures were needed to remove the evd for these patients . a range of infection rates from short - tunnelled evds has been reported , from 3.4 to 32.2 % [ 28 , 1220 ] . the data for infection rates of short - tunnelled evds are summarised in fig . this study , utilising ltevd , reports a lower evd infection rate when compared with previously published literature.fig . 1a comparison of evd infection rates across previous series , using either a subcutaneous long - tunnelled evd or a short / non - tunnelled procedure a comparison of evd infection rates across previous series , using either a subcutaneous long - tunnelled evd or a short / non - tunnelled procedure numerous studies have attempted to identify factors contributing to evd - related infection . csf leakage particularly near the catheter exit site [ 21 , 22 ] , duration of evd insertion [ 3 , 12 ] , evd reinsertion , presence or absence of prophylactic antibiotics and csf sampling have all been suggested as significant contributing factors towards the onset of evd - related infection . this exclusively paediatric study of long - tunnelled evd incorporating a predefined guideline for perioperative evd management has revealed an infection rate of 3.8 % , a rate substantially less than that previously reported . a more recent study , using a non - antibiotic - impregnated ltevd , in 114 adult and paediatric patients requiring more than 7 days drainage , found the infection rate to be 6.8 % , which was deemed comparable with the infection rate of standard short - tunnelled evds . notably , ltevds were capable of remaining in place for long periods of time ( up to 60 days , with a mean duration of 20 days ) and no additional morbidity was associated with the ltevd procedure . our study demonstrated a ltevd infection rate of 3.8 % ( five out of 133 ) , which , as demonstrated by fig . figure 1 compares the infection rates in 15 publications [ 2 , 3 , 8 , 9 , 11 , 19 , 20 , 23 , 2935 ] using short - tunnelled evds and 3 publications using ltevds [ 2729 ] with our series . a statistically significant difference ( p = < 0.05 ) was found in the evd - related infection rate between our study and 14 of the studies using the short - tunnelled procedure . furthermore , there was no significant difference in the infection rates when comparing our study to the other three published studies using a long subcutaneous tunnel , suggesting that tunnelling seems to be a major contributing factor when considering evd infection . the infection rate reported does not include the 48 cases that required a ltevd insertion for an existing infection as these patients may have been on active treatment for existing infection , which may affect the ability to detect a novel infection . therefore , the total infection rate , including this group that require a ltevd for infection , is 2.76 % ( five out of 181 ) . four out of 181 ( 2.2 % ) ltevds in this study were inadvertently dislodged and required a separate procedure to reinsert the evd . in another study , using short - tunnelled evd , a dislodgement rate of two out of 51 ( 8.1 % ) was reported . our data and the data of the other ltevd studies [ 2729 ] suggest that ltevds can safely remain in place for long periods of time without the attendant risks of infection and dislodgement . it was noted that four of the five infected ltevds were inserted for longer than the median duration of insertion ; however , a statistical comparison found no significant difference in the ages of the two groups . duration of evd insertion has been shown to be a significant risk factor when using short - tunnelled evds [ 2 , 3 ] , further strengthening the argument for a ltevd protocol . additionally , one of the infected cases was inserted immediately after an endoscopic procedure , suggesting that this may have been a contributory factor to the ltevd infection , which would concur with the previous literature where intraoperative endoscope use was considered to be an additional risk factor when considering shunt infection rates in the paediatric population . the importance of using an antibiotic - impregnated catheter ( aic ) to lower infection rates in this cohort must not be underestimated . in a further study , when aics were inserted in a centre already using prophylactic , systemic antibiotic treatment , evd infection rates were reduced from 23.5 to 4.3 % , further strengthening the argument for aic use in evds . when considering the antibiotics impregnated into the catheters , both minocycline / rifampin - impregnated ventricular catheter and clindamycin / rifampin - impregnated evd catheter ( used in this study ) however , not all of the data suggest that aics have a beneficial impact in evd insertion . a randomised controlled trial of 184 patients from another centre showed that aics did not have a significantly higher rate of csf infection ( 1 % ) compared to catheters without antibiotic impregnation ( 3 % ) , and there was no significant difference of clinical outcome at 6 months . a more recent randomised controlled study also demonstrated similar findings in 348 adult patients , where aics used in evds did not contribute to a reduction in infection rate ; however , this could be attributed to a low baseline rate of infection and so difficult to prove an aic - related reduction . additionally , an increased rate of infection has been noted in paediatric vp shunt patients with prior evd insertion using an aic , the authors postulated that prior use of an aic increased the potential for resistant organisms . however , for the cohort requiring ltevd insertion for pre - existing infection in this study ( n = 48 ) , the aic tubing was not employed as a specific treatment for the infection , for which appropriate intravenous and intraventricular antibiotics were administered before ltevd insertion . the relative contribution of aics , a long subcutaneous tunnel and a pre - defined protocol for perioperative management of the evd is difficult to elucidate in this study . this low infection rate and low dislodgement rate shown for evds in this study , using a ltevd , an aic and a perioperative management protocol , represents a potential for both clinical and economic benefits . this exclusively paediatric study of long - tunnelled evd incorporating a predefined guideline for perioperative evd management has revealed an infection rate of 3.8 % , a rate substantially less than that previously reported . a more recent study , using a non - antibiotic - impregnated ltevd , in 114 adult and paediatric patients requiring more than 7 days drainage , found the infection rate to be 6.8 % , which was deemed comparable with the infection rate of standard short - tunnelled evds . notably , ltevds were capable of remaining in place for long periods of time ( up to 60 days , with a mean duration of 20 days ) and no additional morbidity was associated with the ltevd procedure . our study demonstrated a ltevd infection rate of 3.8 % ( five out of 133 ) , which , as demonstrated by fig . figure 1 compares the infection rates in 15 publications [ 2 , 3 , 8 , 9 , 11 , 19 , 20 , 23 , 2935 ] using short - tunnelled evds and 3 publications using ltevds [ 2729 ] with our series . a statistically significant difference ( p = < 0.05 ) was found in the evd - related infection rate between our study and 14 of the studies using the short - tunnelled procedure . furthermore , there was no significant difference in the infection rates when comparing our study to the other three published studies using a long subcutaneous tunnel , suggesting that tunnelling seems to be a major contributing factor when considering evd infection . the infection rate reported does not include the 48 cases that required a ltevd insertion for an existing infection as these patients may have been on active treatment for existing infection , which may affect the ability to detect a novel infection . however , there is also a rate of novel infection , with new organisms , in the group where the evd is placed as a part of the treatment for infection . there were no reported novel evd infections in this group with an existing infection ( n = 48 ) . therefore , the total infection rate , including this group that require a ltevd for infection , is 2.76 % ( five out of 181 ) . four out of 181 ( 2.2 % ) ltevds in this study were inadvertently dislodged and required a separate procedure to reinsert the evd . in another study , using short - tunnelled evd , a dislodgement rate of two out of 51 ( 8.1 % ) was reported . the average duration of insertion was 11.2 days , with one ltevd remaining in place for 42 days . our data and the data of the other ltevd studies [ 2729 ] suggest that ltevds can safely remain in place for long periods of time without the attendant risks of infection and dislodgement . it was noted that four of the five infected ltevds were inserted for longer than the median duration of insertion ; however , a statistical comparison found no significant difference in the ages of the two groups . duration of evd insertion has been shown to be a significant risk factor when using short - tunnelled evds [ 2 , 3 ] , further strengthening the argument for a ltevd protocol . additionally , one of the infected cases was inserted immediately after an endoscopic procedure , suggesting that this may have been a contributory factor to the ltevd infection , which would concur with the previous literature where intraoperative endoscope use was considered to be an additional risk factor when considering shunt infection rates in the paediatric population . the importance of using an antibiotic - impregnated catheter ( aic ) to lower infection rates in this cohort must not be underestimated . a randomised controlled trial of 184 patients from another centre showed that aics did not have a significantly higher rate of csf infection ( 1 % ) compared to catheters without antibiotic impregnation ( 3 % ) , and there was no significant difference of clinical outcome at 6 months . a more recent randomised controlled study also demonstrated similar findings in 348 adult patients , where aics used in evds did not contribute to a reduction in infection rate ; however , this could be attributed to a low baseline rate of infection and so difficult to prove an aic - related reduction . additionally , an increased rate of infection has been noted in paediatric vp shunt patients with prior evd insertion using an aic , the authors postulated that prior use of an aic increased the potential for resistant organisms . however , for the cohort requiring ltevd insertion for pre - existing infection in this study ( n = 48 ) , the aic tubing was not employed as a specific treatment for the infection , for which appropriate intravenous and intraventricular antibiotics were administered before ltevd insertion . the relative contribution of aics , a long subcutaneous tunnel and a pre - defined protocol for perioperative management of the evd is difficult to elucidate in this study . there is evidence from another study that a comprehensive evd management protocol , similar to that implemented in this cohort , was effective in reducing evd - related infection rates . this low infection rate and low dislodgement rate shown for evds in this study , using a ltevd , an aic and a perioperative management protocol , represents a potential for both clinical and economic benefits . long - tunnelled evds , using an antibiotic - impregnated catheter and managed according to an agreed perioperative management protocol , carry a lower infection rate and dislodgement rate than the more widely used short - tunnelled procedure .

improving glycemic control can influence the prognosis for patients with type 2 diabetes as it reduces the risk of developing microvascular complications ( nephropathy , neuropathy and retinopathy ) . recent guidelines from the national institute of clinical excellence ( nice ) recommend the initial use of diet and exercise and , when these fail to maintain glycemic control , metformin should be prescribed . monotherapy with any treatment , however , is often unable to sustain target hba1c levels of 6.57.5% in the majority of patients . sulphonylureas have been frequently used in combination with metformin , but are not always appropriate choices as these may cause weight gain and increase the risk of hypoglycaemia . the development of newer insulin secretagogues , such as nateglinide , provides physicians with an alternative to sulphonylureas when selecting the optimal combination of oral agents for an individual patient . nateglinide ( 120 mg three times per day ) is advantageous over other agents in that it helps to control postprandial glucose ( ppg ) levels , along with glycosylated hemoglobin , and also can be used in combination with metformin ( 500 mg three times per day ) . the use of combination therapy subsequent to the failure of monotherapy helps some patients to achieve the recommend levels of glycemic control . however , use of any combination is clearly also associated with an increased cost compared with metformin as monotherapy . the purpose of this study was to estimate the potential long - term health and economic impact of adding nateglinide to metformin in order to improve glycemic control and thereby reduce complication rates . together with the clinical data on the therapeutic efficacy of combination therapy , these economic analyses facilitate assessment of the long - term cost - effectiveness from the perspective of the health care system , of using this combination to achieve improved glycemic control . this model was developed to simulate the lifetime risk of developing diabetes - related complications rates ( microvascular and macrovascular ) in a cohort of patients diagnosed with type 2 diabetes ( figure 1 ) . in this updated version of the model , both the level of hba1c ( glycosylated haemoglobin ) and two - hour postprandial glucose ( ppg ) each year of remaining life is simulated for all the patients in the cohort and during each cycle , the patient is exposed to the risks of developing each type of complication . these risks are determined from the degree of glycemic control , as well as other known risk factors , such as duration of diabetes . schematic representation of model ( reprinted with permission from can j diabetes . 2003 ; 27(1 ) : 3341 ) . the microvascular complications ( nephropathy , retinopathy , and neuropathy ) have several stages through which each patient can progress . the most severe stages for the microvascular complications are end stage renal disease , blindness or amputations . the stages of a complication are assumed irreversible only progression to more severe stages is possible . complications such as hypoglycaemia and foot ulcer were assumed to resolve in the course of each cycle of one year . for the purpose of this model , macrovascular complications ( stroke and myocardial infarction ) were considered as finite events , rather than progressive conditions . each simulated patient had clinical characteristics that were determined by the input distributions specified . using a monte carlo technique , each patient in the cohort the assignment of cholesterol level , smoking status , body mass index and systolic blood pressure was then determined using the distributions and associations observed amongst patients with type 2 diabetes [ 10 - 12 ] . for thirty annual cycles , the model checks each patient who has survived to that point , and updates the age , duration of disease and hba1c level . over each cycle , the estimated risks of developing a new complication or progressing to the next stage of an established one are assigned to each simulated patient in the cohort . during a pre - model period of seven years , the patients were allowed to accumulate complications but costs from managing these complications are not considered in the comparisons . previous analyses using the model have been evaluated by peer review [ 6 - 9 ] . source data and other independently obtained results were used as comparisons to determine predictive validity . model results for relative risk over 10 years for all - cause mortality and for microvascular disease and retinopathy at 12 years were consistent with ukpds patients in intensive and conventional treatment groups . the risk of death in this updated model was linked to both ppg and hba1c levels . weibull functions were derived from the diabetes epidemiology : collaborative analysis of diagnostic criteria in europe ( decode ) study and estimates were based on the patients ' age , gender , systolic blood pressure , total cholesterol , body mass index , smoking status , and ppg level . as in the original model , the risk of death was also assessed from the age- and gender - dependent mortality for patients diagnosed with type 2 diabetes , with an adjustment if nephropathy develops . the higher of these three death risk estimates in each model cycle was applied . the estimates for microvascular complications ( nephropathy , retinopathy , and neuropathy ) were determined from the available epidemiological studies [ 19 - 21 ] and the risk gradients observed in the diabetes control and complications trial ( dcct ) were assumed to apply to type 2 diabetes , an accepted assumption [ 23 - 25 ] confirmed by the ukpds . the risks of each microvascular complication are estimated by adjusting each according to the patient 's hba1c level at a specific point in time ( risk = 1 - e , where = bhr , and hr is the hba1c value relative to a standard and is a complication - specific coefficient ) . the base hazard for a complication depends on factors such as duration of diabetes , race and for the retinopathy module , for example , also the probability of detection and treatment . evidence has recently been published that indicates ppg is an independent predictor of the occurrence of macrovascular complications , as well as of mortality . in this updated model , the risk of stroke or myocardial infarction was estimated using weibull functions derived from the decode study . the risk equations derived from the decode study include established risk factors for macrovascular disease such as age , gender , systolic blood pressure , total cholesterol , body mass index , smoking status , as well as ppg level . for each complication , the direct medical costs were estimated for the immediate impact of the event ( costs arising in the year the event occurs ) and the subsequent impact of the complication ( costs accrued in years subsequent to the year of the event ) . clarke et al combined resource use data collected from the ukpds with cost estimates for these services , and published regression equations for estimating the cost of major complications . the annual hospital in - patient costs , and non - hospital costs ( general practioners , nurses , podiatrists , opticians , dieticians , hospital outpatient clinics ) were estimated using these regression equations for the event year and subsequent years . as the inpatient costs were estimated for myocardial infarction , stroke , blindness , or an amputation . the inpatient costs of less severe stages of these complications were not included in these estimates the cost estimates are quite conservative . all complication costs are expressed in 1999 great britain pounds ( 1 gbp = $ 1.7 usd = 1.4 euros ) . it should be noted that the cost of end stage renal disease was estimated based on data from 1996 . we elected not to inflate this cost , however , as the applicability of general inflation rates to something as specialized as the management of end stage renal disease is fraught with inaccuracy and this was the most expensive complication ( 21,456 per year ) . the daily cost for metformin ( 1500 mg per day ) was 0.07 , and 0.87 for the combination of nateglinide ( 360 mg / day = 0.80 ) with metformin ( 1500 mg per day ) . the distributions of hba1c and ppg at the beginning of the model period , as well as the effects of each treatment regimen were obtained from a clinical trial assessing the efficacy of combining nateglinide ( 360 mg / day ) with metformin ( 1500 mg per day ) compared with metformin alone ( table 1 ) . the mean hba1c at baseline was 8.4% , at the trial end point the hba1c was reduced with both metformin and for the combination ( -0.8% , and -1.5% respectively ) , as was the ppg level ( -0.9 , and -2.3 respectively ) . clinical characteristics of simulated cohort after processing each cohort of 10,000 patients over thirty years , the model provides estimates of the mean survival time , the frequency of each type of complication , and the mean accumulated complication and treatment costs per patient . survival time is also weighted by the quality of life ; the utility assigned depending on the complications present . the utilities assigned were as follows ; amputation 0.50 , stroke 0.62 , blindness 0.71 and myocardial infarction 0.73 , end stage renal disease 0.59 . the cost per life year gained ( lyg ) and cost per quality adjusted life year ( qaly ) was determined . sensitivity analyses were conducted on model parameters and uncertainty in the base case estimates was examined using the bootstrap technique with 250 model replications , and 1000 re - samples from the results of these simulations . this model was developed to simulate the lifetime risk of developing diabetes - related complications rates ( microvascular and macrovascular ) in a cohort of patients diagnosed with type 2 diabetes ( figure 1 ) . in this updated version of the model , both the level of hba1c ( glycosylated haemoglobin ) and two - hour postprandial glucose ( ppg ) each year of remaining life is simulated for all the patients in the cohort and during each cycle , the patient is exposed to the risks of developing each type of complication . these risks are determined from the degree of glycemic control , as well as other known risk factors , such as duration of diabetes . schematic representation of model ( reprinted with permission from can j diabetes . 2003 ; 27(1 ) : 3341 ) . the microvascular complications ( nephropathy , retinopathy , and neuropathy ) have several stages through which each patient can progress . the most severe stages for the microvascular complications are end stage renal disease , blindness or amputations . the stages of a complication are assumed irreversible only progression to more severe stages is possible . complications such as hypoglycaemia and foot ulcer were assumed to resolve in the course of each cycle of one year . for the purpose of this model , macrovascular complications ( stroke and myocardial infarction ) were considered as finite events , rather than progressive conditions . each simulated patient had clinical characteristics that were determined by the input distributions specified . using a monte carlo technique , each patient in the cohort the assignment of cholesterol level , smoking status , body mass index and systolic blood pressure was then determined using the distributions and associations observed amongst patients with type 2 diabetes [ 10 - 12 ] . for thirty annual cycles , the model checks each patient who has survived to that point , and updates the age , duration of disease and hba1c level . over each cycle , the estimated risks of developing a new complication or progressing to the next stage of an established one are assigned to each simulated patient in the cohort . during a pre - model period of seven years , the patients were allowed to accumulate complications but costs from managing these complications are not considered in the comparisons . previous analyses using the model have been evaluated by peer review [ 6 - 9 ] . source data and other independently obtained results were used as comparisons to determine predictive validity . model results for relative risk over 10 years for all - cause mortality and for microvascular disease and retinopathy at 12 years were consistent with ukpds patients in intensive and conventional treatment groups . the risk of death in this updated model was linked to both ppg and hba1c levels . weibull functions were derived from the diabetes epidemiology : collaborative analysis of diagnostic criteria in europe ( decode ) study and estimates were based on the patients ' age , gender , systolic blood pressure , total cholesterol , body mass index , smoking status , and ppg level . as in the original model , the risk of death was also assessed from the age- and gender - dependent mortality for patients diagnosed with type 2 diabetes , with an adjustment if nephropathy develops . the estimates for microvascular complications ( nephropathy , retinopathy , and neuropathy ) were determined from the available epidemiological studies [ 19 - 21 ] and the risk gradients observed in the diabetes control and complications trial ( dcct ) were assumed to apply to type 2 diabetes , an accepted assumption [ 23 - 25 ] confirmed by the ukpds . the risks of each microvascular complication are estimated by adjusting each according to the patient 's hba1c level at a specific point in time ( risk = 1 - e , where = bhr , and hr is the hba1c value relative to a standard and is a complication - specific coefficient ) . the base hazard for a complication depends on factors such as duration of diabetes , race and for the retinopathy module , for example , also the probability of detection and treatment . evidence has recently been published that indicates ppg is an independent predictor of the occurrence of macrovascular complications , as well as of mortality . in this updated model , the risk of stroke or myocardial infarction was estimated using weibull functions derived from the decode study . the risk equations derived from the decode study include established risk factors for macrovascular disease such as age , gender , systolic blood pressure , total cholesterol , body mass index , smoking status , as well as ppg level . for each complication , the direct medical costs were estimated for the immediate impact of the event ( costs arising in the year the event occurs ) and the subsequent impact of the complication ( costs accrued in years subsequent to the year of the event ) . clarke et al combined resource use data collected from the ukpds with cost estimates for these services , and published regression equations for estimating the cost of major complications . the annual hospital in - patient costs , and non - hospital costs ( general practioners , nurses , podiatrists , opticians , dieticians , hospital outpatient clinics ) were estimated using these regression equations for the event year and subsequent years . as the inpatient costs were estimated for myocardial infarction , stroke , blindness , or an amputation . the inpatient costs of less severe stages of these complications were not included in these estimates the cost estimates are quite conservative . all complication costs are expressed in 1999 great britain pounds ( 1 gbp = $ 1.7 usd = 1.4 euros ) . it should be noted that the cost of end stage renal disease was estimated based on data from 1996 . we elected not to inflate this cost , however , as the applicability of general inflation rates to something as specialized as the management of end stage renal disease is fraught with inaccuracy and this was the most expensive complication ( 21,456 per year ) . the daily cost for metformin ( 1500 mg per day ) was 0.07 , and 0.87 for the combination of nateglinide ( 360 mg / day = 0.80 ) with metformin ( 1500 mg per day ) . the distributions of hba1c and ppg at the beginning of the model period , as well as the effects of each treatment regimen were obtained from a clinical trial assessing the efficacy of combining nateglinide ( 360 mg / day ) with metformin ( 1500 mg per day ) compared with metformin alone ( table 1 ) . the mean hba1c at baseline was 8.4% , at the trial end point the hba1c was reduced with both metformin and for the combination ( -0.8% , and -1.5% respectively ) , as was the ppg level ( -0.9 , and -2.3 respectively ) . clinical characteristics of simulated cohort after processing each cohort of 10,000 patients over thirty years , the model provides estimates of the mean survival time , the frequency of each type of complication , and the mean accumulated complication and treatment costs per patient . survival time is also weighted by the quality of life ; the utility assigned depending on the complications present . the utilities assigned were as follows ; amputation 0.50 , stroke 0.62 , blindness 0.71 and myocardial infarction 0.73 , end stage renal disease 0.59 . the cost per life year gained ( lyg ) and cost per quality adjusted life year ( qaly ) was determined . sensitivity analyses were conducted on model parameters and uncertainty in the base case estimates was examined using the bootstrap technique with 250 model replications , and 1000 re - samples from the results of these simulations . our analyses simulated a cohort of patients treated with metformin and estimated the mean survival time to be 13.5 years . over their lifetime , retinopathy was the most common affecting over a quarter of the patients , as well as foot ulcers and microalbuminuria ( table 2 ) . the model predicted mean lifetime discounted costs per patient of about five thousand pounds ( table 3 ) . macrovascular disease was common ( table 2 ) and accounted for about 40% of the lifetime costs due to complications , with myocardial infarction being the slightly larger component of the macrovascular costs ( 63% ) . frequency of microvascular and macrovascular complications by treatment health benefits and costs for metformin and the combination of metformin with nateglinide lyg = life year gained qaly = quality adjusted life year the improvement in glycemic control , in terms of both the hba1c and the ppg , expected with the combination nateglinide with metformin is estimated to increase survival on average 0.39 years per patient ( 0.32 discounted years ) or 0.46 ( 0.37 discounted ) qaly ( table 3 ) . moreover , complications were expected to occur less frequently , or at least progress more slowly ( table 2 ) . combination therapy is expected to reduce the frequency of complications and prolong survival , but also increase the average costs by an average of 2,066 per patient . to determine the impact of the nateglinide - metformin combination on the cost of managing complications , the difference in mean cost between metformin alone and the combination group was determined ( table 3 ) . thus , savings of 464 were estimated regarding the lifetime cost of managing complications . these arise mainly from a reduction in the costs of treating end stage renal disease ( 72% ) and neuropathy ( 19% ) . the increase in the treatment costs due to combination therapy are therefore predicted to be partially offset by this reduction in the cost of managing complications , leaving an increment of 2,066 in the lifetime costs per patient ( table 3 ) . this translates into a cost - effectiveness ratio of 6,772 ( 95%ci : 6,134 to 7,464 ) per additional discounted year of life , and 5,609 per discounted qaly . the model inputs were varied to reflect different scenarios and table 4 shows the impact on the estimates . if a population with higher glycemic levels at baseline is modeled , a larger proportion of the cohort develops severe complications on metformin alone . varying the discount rate had a major effect on the cost - effectiveness results . varying the efficacy of the combination of nateglinide and metformin on ppg values had a minor effect , a 50% reduction in efficacy led to a 3% increase in macrovascular disease related costs . varying the impact of the combination of nateglinide and metformin treatment on hba1c values had a larger impact on the total cost predicted . decreasing the efficacy by 10% , or 25% led to total cost increases of 3% , and 9% , respectively . the improvement in glycemic control , in terms of both the hba1c and the ppg , expected with the combination nateglinide with metformin is estimated to increase survival on average 0.39 years per patient ( 0.32 discounted years ) or 0.46 ( 0.37 discounted ) qaly ( table 3 ) . moreover , complications were expected to occur less frequently , or at least progress more slowly ( table 2 ) . combination therapy is expected to reduce the frequency of complications and prolong survival , but also increase the average costs by an average of 2,066 per patient . to determine the impact of the nateglinide - metformin combination on the cost of managing complications , the difference in mean cost between metformin alone and the combination group was determined ( table 3 ) . thus , savings of 464 were estimated regarding the lifetime cost of managing complications . these arise mainly from a reduction in the costs of treating end stage renal disease ( 72% ) and neuropathy ( 19% ) . the increase in the treatment costs due to combination therapy are therefore predicted to be partially offset by this reduction in the cost of managing complications , leaving an increment of 2,066 in the lifetime costs per patient ( table 3 ) . 6,134 to 7,464 ) per additional discounted year of life , and 5,609 per discounted qaly . the model inputs were varied to reflect different scenarios and table 4 shows the impact on the estimates . if a population with higher glycemic levels at baseline is modeled , a larger proportion of the cohort develops severe complications on metformin alone . varying the discount rate had a major effect on the cost - effectiveness results . varying the efficacy of the combination of nateglinide and metformin on ppg values had a minor effect , a 50% reduction in efficacy led to a 3% increase in macrovascular disease related costs . varying the impact of the combination of nateglinide and metformin treatment on hba1c values had a larger impact on the total cost predicted . decreasing the efficacy by 10% , or 25% led to total cost increases of 3% , and 9% , respectively . improving glycemic control using combination therapy will inevitably increase drug treatment costs when compared with monotherapy . however , the reduction in hba1c and ppg levels when treating patients with type 2 diabetes with a combination of nateglinide and metformin has the potential to translate into reduced complication rates . long term therefore , combination treatment is likely to result in substantial offsets in overall costs . thus , the additional glycemic control is achieved at a rate of 6,772 per year of additional life , an estimate generally considered cost - effective . diabetes - related complications have been shown in several uk studies to require expensive medical interventions , frequently provided in a hospital inpatient setting [ 36 - 39 ] . the ukpds demonstrated that keeping glucose levels near normal decreased the incidence of microvascular complications over ten years . in addition , cost - effectiveness analyses based on the ukpds results indicate the costs of managing complications would be expected to be reduced , and , specifically , intensive blood glucose control with metformin is predicted to result in lower complications costs amongst overweight patients . the dcct results showed improved glycemic control can lower microvascular complication rates in patients with type 1 diabetes , and one key assumption of this model is that these rates also apply to type 2 diabetes . this model predicts comparable results to those of the ukpds patients in the intensive and conventional treatment groups in terms of relative risk over ten years for microvascular disease or retinopathy at 12 years . the economic implications of combination therapy depend to some extent on the characteristics of the cohort analyzed . for example , the sensitivity analyses illustrate that greater savings are predicted for patients diagnosed when they are young , with longer duration of disease and poorer glycemic control initially . macrovascular disease is predicted to be the major component of the costs accounting for over one third of the costs accrued over a lifetime from managing diabetes related complications . this is of particular importance as these complications tend to arise earlier in the course of the disease than those that are microvascular in nature , and are the leading cause of death . thus , from both the clinical and economic perspectives , it is important that in addition to glycemic control , any risk factors for cardiovascular disease that are known to be modifiable are managed such as smoking cessation , reducing obesity , high blood pressure and hypercholesterolaemia . the equations developed for predicting the risk of stroke and of myocardial infarction included the ppg level . these predictions are based on the results of the decode study that investigated the prevalence of macrovascular disease and mortality in europe . thus , the assumption in the model that reducing ppg levels will reduce the risk of macrovascular disease remains to be proven conclusively . the long - term predictions were based on the efficacy of combining nateglinide with metformin demonstrated in clinical trials . even though these analyses were based on the efficacy observed in a randomized , controlled trial , it was necessary to make some assumptions about long - term glycemic control . given the lack of specific data on the combination over longer timeframes , it was assumed that after the initial improvement in glycemic control , the hba1c would begin to drift upward as it did with metformin and other hypo glycemic agents employed in the ukpds . this is a conservative assumption as it is quite possible that with the combination there will be a slower , or at least delayed , upward drift . the cost inputs for these economic analyses were limited to only the most severe stages of the complications . this was done in order to accord with the estimates ' source , the ukpds . the costs also did not include the less severe stages of the complications ( such as gross proteinuria , foot ulcers or photocoagulation ) . similarly , the macrovascular costs do not include the management of milder conditions such as angina or transient ischaemic attacks . in conclusion , prescribing the combination of nateglinide and metformin for patients who are not maintaining good glycemic control on monotherapy alone should be cost - effective , as the combination is expected to reduce the rates of diabetes - related complications at an acceptable additional cost . caro research of which jaime caro is a shareholder , received a grant from novartis pharma ag , ( united kingdom ) , which provided funding for portions of the study .

backgroundto reduce the likelihood of complications in persons with type 2 diabetes , it is critical to control hyperglycaemia . monotherapy with metformin or insulin secretagogues may fail to sustain control after an initial reduction in glycemic levels . thus , combining metformin with other agents is frequently necessary . these analyses model the potential long - term economic and health impact of using combination therapy to improve glycemic control.methodsan existing model that simulates the long - term course of type 2 diabetes in relation to glycosylated haemoglobin ( hba1c ) and post - prandial glucose ( ppg ) was used to compare the combination of nateglinide with metformin to monotherapy with metformin . complication rates were estimated for major diabetes - related complications ( macrovascular and microvascular ) based on existing epidemiologic studies and clinical trial data . utilities and costs were estimated using data collected in the united kingdom prospective diabetes study ( ukpds ) . survival , life years gained ( lyg ) , quality - adjusted life years ( qaly ) , complication rates and associated costs were estimated . costs were discounted at 6% and benefits at 1.5% per year.resultscombination therapy was predicted to reduce complication rates and associated costs compared with metformin . survival increased by 0.39 ( 0.32 discounted ) and qaly by 0.46 years ( 0.37 discounted ) implying costs of 6,772 per discounted lyg and 5,609 per discounted qaly . sensitivity analyses showed the results to be consistent over broad ranges.conclusionalthough drug treatment costs are increased by combination therapy , this cost is expected to be partially offset by a reduction in the costs of treating long - term diabetes complications .

Background Methods Model framework Risk estimates Costs Analyses Results Base case Sensitivity analyses Discussion Conclusion Competing interests Authors' contributions

improving glycemic control can influence the prognosis for patients with type 2 diabetes as it reduces the risk of developing microvascular complications ( nephropathy , neuropathy and retinopathy ) . recent guidelines from the national institute of clinical excellence ( nice ) recommend the initial use of diet and exercise and , when these fail to maintain glycemic control , metformin should be prescribed . monotherapy with any treatment , however , is often unable to sustain target hba1c levels of 6.57.5% in the majority of patients . the development of newer insulin secretagogues , such as nateglinide , provides physicians with an alternative to sulphonylureas when selecting the optimal combination of oral agents for an individual patient . nateglinide ( 120 mg three times per day ) is advantageous over other agents in that it helps to control postprandial glucose ( ppg ) levels , along with glycosylated hemoglobin , and also can be used in combination with metformin ( 500 mg three times per day ) . the use of combination therapy subsequent to the failure of monotherapy helps some patients to achieve the recommend levels of glycemic control . however , use of any combination is clearly also associated with an increased cost compared with metformin as monotherapy . the purpose of this study was to estimate the potential long - term health and economic impact of adding nateglinide to metformin in order to improve glycemic control and thereby reduce complication rates . together with the clinical data on the therapeutic efficacy of combination therapy , these economic analyses facilitate assessment of the long - term cost - effectiveness from the perspective of the health care system , of using this combination to achieve improved glycemic control . this model was developed to simulate the lifetime risk of developing diabetes - related complications rates ( microvascular and macrovascular ) in a cohort of patients diagnosed with type 2 diabetes ( figure 1 ) . in this updated version of the model , both the level of hba1c ( glycosylated haemoglobin ) and two - hour postprandial glucose ( ppg ) each year of remaining life is simulated for all the patients in the cohort and during each cycle , the patient is exposed to the risks of developing each type of complication . these risks are determined from the degree of glycemic control , as well as other known risk factors , such as duration of diabetes . complications such as hypoglycaemia and foot ulcer were assumed to resolve in the course of each cycle of one year . for the purpose of this model , macrovascular complications ( stroke and myocardial infarction ) were considered as finite events , rather than progressive conditions . using a monte carlo technique , each patient in the cohort the assignment of cholesterol level , smoking status , body mass index and systolic blood pressure was then determined using the distributions and associations observed amongst patients with type 2 diabetes [ 10 - 12 ] . over each cycle , the estimated risks of developing a new complication or progressing to the next stage of an established one are assigned to each simulated patient in the cohort . during a pre - model period of seven years , the patients were allowed to accumulate complications but costs from managing these complications are not considered in the comparisons . as in the original model , the risk of death was also assessed from the age- and gender - dependent mortality for patients diagnosed with type 2 diabetes , with an adjustment if nephropathy develops . the estimates for microvascular complications ( nephropathy , retinopathy , and neuropathy ) were determined from the available epidemiological studies [ 19 - 21 ] and the risk gradients observed in the diabetes control and complications trial ( dcct ) were assumed to apply to type 2 diabetes , an accepted assumption [ 23 - 25 ] confirmed by the ukpds . the base hazard for a complication depends on factors such as duration of diabetes , race and for the retinopathy module , for example , also the probability of detection and treatment . in this updated model , the risk of stroke or myocardial infarction was estimated using weibull functions derived from the decode study . for each complication , the direct medical costs were estimated for the immediate impact of the event ( costs arising in the year the event occurs ) and the subsequent impact of the complication ( costs accrued in years subsequent to the year of the event ) . the annual hospital in - patient costs , and non - hospital costs ( general practioners , nurses , podiatrists , opticians , dieticians , hospital outpatient clinics ) were estimated using these regression equations for the event year and subsequent years . as the inpatient costs were estimated for myocardial infarction , stroke , blindness , or an amputation . the inpatient costs of less severe stages of these complications were not included in these estimates the cost estimates are quite conservative . all complication costs are expressed in 1999 great britain pounds ( 1 gbp = $ 1.7 usd = 1.4 euros ) . it should be noted that the cost of end stage renal disease was estimated based on data from 1996 . we elected not to inflate this cost , however , as the applicability of general inflation rates to something as specialized as the management of end stage renal disease is fraught with inaccuracy and this was the most expensive complication ( 21,456 per year ) . the daily cost for metformin ( 1500 mg per day ) was 0.07 , and 0.87 for the combination of nateglinide ( 360 mg / day = 0.80 ) with metformin ( 1500 mg per day ) . the distributions of hba1c and ppg at the beginning of the model period , as well as the effects of each treatment regimen were obtained from a clinical trial assessing the efficacy of combining nateglinide ( 360 mg / day ) with metformin ( 1500 mg per day ) compared with metformin alone ( table 1 ) . the mean hba1c at baseline was 8.4% , at the trial end point the hba1c was reduced with both metformin and for the combination ( -0.8% , and -1.5% respectively ) , as was the ppg level ( -0.9 , and -2.3 respectively ) . clinical characteristics of simulated cohort after processing each cohort of 10,000 patients over thirty years , the model provides estimates of the mean survival time , the frequency of each type of complication , and the mean accumulated complication and treatment costs per patient . the cost per life year gained ( lyg ) and cost per quality adjusted life year ( qaly ) was determined . sensitivity analyses were conducted on model parameters and uncertainty in the base case estimates was examined using the bootstrap technique with 250 model replications , and 1000 re - samples from the results of these simulations . this model was developed to simulate the lifetime risk of developing diabetes - related complications rates ( microvascular and macrovascular ) in a cohort of patients diagnosed with type 2 diabetes ( figure 1 ) . in this updated version of the model , both the level of hba1c ( glycosylated haemoglobin ) and two - hour postprandial glucose ( ppg ) each year of remaining life is simulated for all the patients in the cohort and during each cycle , the patient is exposed to the risks of developing each type of complication . these risks are determined from the degree of glycemic control , as well as other known risk factors , such as duration of diabetes . the microvascular complications ( nephropathy , retinopathy , and neuropathy ) have several stages through which each patient can progress . complications such as hypoglycaemia and foot ulcer were assumed to resolve in the course of each cycle of one year . for the purpose of this model , macrovascular complications ( stroke and myocardial infarction ) were considered as finite events , rather than progressive conditions . using a monte carlo technique , each patient in the cohort the assignment of cholesterol level , smoking status , body mass index and systolic blood pressure was then determined using the distributions and associations observed amongst patients with type 2 diabetes [ 10 - 12 ] . over each cycle , the estimated risks of developing a new complication or progressing to the next stage of an established one are assigned to each simulated patient in the cohort . weibull functions were derived from the diabetes epidemiology : collaborative analysis of diagnostic criteria in europe ( decode ) study and estimates were based on the patients ' age , gender , systolic blood pressure , total cholesterol , body mass index , smoking status , and ppg level . as in the original model , the risk of death was also assessed from the age- and gender - dependent mortality for patients diagnosed with type 2 diabetes , with an adjustment if nephropathy develops . the estimates for microvascular complications ( nephropathy , retinopathy , and neuropathy ) were determined from the available epidemiological studies [ 19 - 21 ] and the risk gradients observed in the diabetes control and complications trial ( dcct ) were assumed to apply to type 2 diabetes , an accepted assumption [ 23 - 25 ] confirmed by the ukpds . the base hazard for a complication depends on factors such as duration of diabetes , race and for the retinopathy module , for example , also the probability of detection and treatment . in this updated model , the risk of stroke or myocardial infarction was estimated using weibull functions derived from the decode study . for each complication , the direct medical costs were estimated for the immediate impact of the event ( costs arising in the year the event occurs ) and the subsequent impact of the complication ( costs accrued in years subsequent to the year of the event ) . the annual hospital in - patient costs , and non - hospital costs ( general practioners , nurses , podiatrists , opticians , dieticians , hospital outpatient clinics ) were estimated using these regression equations for the event year and subsequent years . as the inpatient costs were estimated for myocardial infarction , stroke , blindness , or an amputation . the inpatient costs of less severe stages of these complications were not included in these estimates the cost estimates are quite conservative . all complication costs are expressed in 1999 great britain pounds ( 1 gbp = $ 1.7 usd = 1.4 euros ) . we elected not to inflate this cost , however , as the applicability of general inflation rates to something as specialized as the management of end stage renal disease is fraught with inaccuracy and this was the most expensive complication ( 21,456 per year ) . the daily cost for metformin ( 1500 mg per day ) was 0.07 , and 0.87 for the combination of nateglinide ( 360 mg / day = 0.80 ) with metformin ( 1500 mg per day ) . the distributions of hba1c and ppg at the beginning of the model period , as well as the effects of each treatment regimen were obtained from a clinical trial assessing the efficacy of combining nateglinide ( 360 mg / day ) with metformin ( 1500 mg per day ) compared with metformin alone ( table 1 ) . the mean hba1c at baseline was 8.4% , at the trial end point the hba1c was reduced with both metformin and for the combination ( -0.8% , and -1.5% respectively ) , as was the ppg level ( -0.9 , and -2.3 respectively ) . clinical characteristics of simulated cohort after processing each cohort of 10,000 patients over thirty years , the model provides estimates of the mean survival time , the frequency of each type of complication , and the mean accumulated complication and treatment costs per patient . the cost per life year gained ( lyg ) and cost per quality adjusted life year ( qaly ) was determined . sensitivity analyses were conducted on model parameters and uncertainty in the base case estimates was examined using the bootstrap technique with 250 model replications , and 1000 re - samples from the results of these simulations . our analyses simulated a cohort of patients treated with metformin and estimated the mean survival time to be 13.5 years . macrovascular disease was common ( table 2 ) and accounted for about 40% of the lifetime costs due to complications , with myocardial infarction being the slightly larger component of the macrovascular costs ( 63% ) . frequency of microvascular and macrovascular complications by treatment health benefits and costs for metformin and the combination of metformin with nateglinide lyg = life year gained qaly = quality adjusted life year the improvement in glycemic control , in terms of both the hba1c and the ppg , expected with the combination nateglinide with metformin is estimated to increase survival on average 0.39 years per patient ( 0.32 discounted years ) or 0.46 ( 0.37 discounted ) qaly ( table 3 ) . moreover , complications were expected to occur less frequently , or at least progress more slowly ( table 2 ) . combination therapy is expected to reduce the frequency of complications and prolong survival , but also increase the average costs by an average of 2,066 per patient . to determine the impact of the nateglinide - metformin combination on the cost of managing complications , the difference in mean cost between metformin alone and the combination group was determined ( table 3 ) . thus , savings of 464 were estimated regarding the lifetime cost of managing complications . these arise mainly from a reduction in the costs of treating end stage renal disease ( 72% ) and neuropathy ( 19% ) . the increase in the treatment costs due to combination therapy are therefore predicted to be partially offset by this reduction in the cost of managing complications , leaving an increment of 2,066 in the lifetime costs per patient ( table 3 ) . this translates into a cost - effectiveness ratio of 6,772 ( 95%ci : 6,134 to 7,464 ) per additional discounted year of life , and 5,609 per discounted qaly . if a population with higher glycemic levels at baseline is modeled , a larger proportion of the cohort develops severe complications on metformin alone . varying the efficacy of the combination of nateglinide and metformin on ppg values had a minor effect , a 50% reduction in efficacy led to a 3% increase in macrovascular disease related costs . varying the impact of the combination of nateglinide and metformin treatment on hba1c values had a larger impact on the total cost predicted . the improvement in glycemic control , in terms of both the hba1c and the ppg , expected with the combination nateglinide with metformin is estimated to increase survival on average 0.39 years per patient ( 0.32 discounted years ) or 0.46 ( 0.37 discounted ) qaly ( table 3 ) . combination therapy is expected to reduce the frequency of complications and prolong survival , but also increase the average costs by an average of 2,066 per patient . to determine the impact of the nateglinide - metformin combination on the cost of managing complications , the difference in mean cost between metformin alone and the combination group was determined ( table 3 ) . thus , savings of 464 were estimated regarding the lifetime cost of managing complications . these arise mainly from a reduction in the costs of treating end stage renal disease ( 72% ) and neuropathy ( 19% ) . the increase in the treatment costs due to combination therapy are therefore predicted to be partially offset by this reduction in the cost of managing complications , leaving an increment of 2,066 in the lifetime costs per patient ( table 3 ) . 6,134 to 7,464 ) per additional discounted year of life , and 5,609 per discounted qaly . if a population with higher glycemic levels at baseline is modeled , a larger proportion of the cohort develops severe complications on metformin alone . varying the efficacy of the combination of nateglinide and metformin on ppg values had a minor effect , a 50% reduction in efficacy led to a 3% increase in macrovascular disease related costs . varying the impact of the combination of nateglinide and metformin treatment on hba1c values had a larger impact on the total cost predicted . improving glycemic control using combination therapy will inevitably increase drug treatment costs when compared with monotherapy . however , the reduction in hba1c and ppg levels when treating patients with type 2 diabetes with a combination of nateglinide and metformin has the potential to translate into reduced complication rates . thus , the additional glycemic control is achieved at a rate of 6,772 per year of additional life , an estimate generally considered cost - effective . diabetes - related complications have been shown in several uk studies to require expensive medical interventions , frequently provided in a hospital inpatient setting [ 36 - 39 ] . in addition , cost - effectiveness analyses based on the ukpds results indicate the costs of managing complications would be expected to be reduced , and , specifically , intensive blood glucose control with metformin is predicted to result in lower complications costs amongst overweight patients . the dcct results showed improved glycemic control can lower microvascular complication rates in patients with type 1 diabetes , and one key assumption of this model is that these rates also apply to type 2 diabetes . this model predicts comparable results to those of the ukpds patients in the intensive and conventional treatment groups in terms of relative risk over ten years for microvascular disease or retinopathy at 12 years . for example , the sensitivity analyses illustrate that greater savings are predicted for patients diagnosed when they are young , with longer duration of disease and poorer glycemic control initially . macrovascular disease is predicted to be the major component of the costs accounting for over one third of the costs accrued over a lifetime from managing diabetes related complications . this is of particular importance as these complications tend to arise earlier in the course of the disease than those that are microvascular in nature , and are the leading cause of death . thus , from both the clinical and economic perspectives , it is important that in addition to glycemic control , any risk factors for cardiovascular disease that are known to be modifiable are managed such as smoking cessation , reducing obesity , high blood pressure and hypercholesterolaemia . these predictions are based on the results of the decode study that investigated the prevalence of macrovascular disease and mortality in europe . thus , the assumption in the model that reducing ppg levels will reduce the risk of macrovascular disease remains to be proven conclusively . the long - term predictions were based on the efficacy of combining nateglinide with metformin demonstrated in clinical trials . even though these analyses were based on the efficacy observed in a randomized , controlled trial , it was necessary to make some assumptions about long - term glycemic control . given the lack of specific data on the combination over longer timeframes , it was assumed that after the initial improvement in glycemic control , the hba1c would begin to drift upward as it did with metformin and other hypo glycemic agents employed in the ukpds . this is a conservative assumption as it is quite possible that with the combination there will be a slower , or at least delayed , upward drift . the costs also did not include the less severe stages of the complications ( such as gross proteinuria , foot ulcers or photocoagulation ) . in conclusion , prescribing the combination of nateglinide and metformin for patients who are not maintaining good glycemic control on monotherapy alone should be cost - effective , as the combination is expected to reduce the rates of diabetes - related complications at an acceptable additional cost . caro research of which jaime caro is a shareholder , received a grant from novartis pharma ag , ( united kingdom ) , which provided funding for portions of the study .

for efficient solar energy conversion lightharvesting materials that can tap into large portions of the electromagnetic spectrum , and , in particular , can utilize red and near infrared light , are essential.1 to illustrate the benefits of such panchromic materials , it has been suggested that extending the photosynthetically useful spectral range from 400700 nm to 400750 nm would increase the usable photon flux by 19 % .1b , 2 porphyrin polymers are increasingly investigated for this purpose because of their attractive chargetransport properties and intense absorptions.3 applications of these versatile materials as semiconductors,3a as stimuliresponsive materials4 and as catalysts5 are also actively pursued . a variety of transformations have been successfully applied to the polymerization of diverse porphyrin monomers , such as metalcatalyzed crosscouplings , chemical oxidations , and oxidative and reductive electropolymerizations.3a , 3b , 3d , 6 the lightharvesting and chargetransport properties of the polymers are governed by the monomers and the nature of the formed connection . all these parameters have been investigated to some extent , with the exception of the building blocks these have almost exclusively been porphyrins , with nonideal photophysical properties as a consequence , that is , negligible absorption outside of the blue . black porphyrins and phthalocyanines have recently been obtained,7 but are not readily polymerized due to the lack of suitable functionalities . conjugated or fused porphyrin oligomers with redshifted absorptions are known , however , their syntheses are demanding.8 chlorins share the carbon framework with porphyrins , with one of the pyrrolic double bonds removed . consequently , chlorins have characteristic intense red absorptions in addition to the soret band , which suggests that their polymers have potential as lightharvesting materials for the underutilized low energypart of the solar spectrum . the uv / vis absorption spectra of chlorins are sensitive to the type and position of the peripheral substituents . a striking example of spectral tuning is seen in chlorophylls ( figure 1 ) . vinyl and formyl groups positioned along the spectroscopic axes ( i.e. , the 2,3 , 7,8 and 12,13positions , figure 1 , top ) move the qyband from abs=652 nm ( in meoh)9 in chl b to abs=707 nm ( in meoh)10 in what is the most redshifted naturallyoccurring chlorophyll , chl f. several other auxochromes , such as alkynes , ketones , cyclic esters and amides , as well as extended conjugated fragments have been extensively investigated for the past 10 years.11 in addition to a deepened understanding of tetrapyrrole photophysics , these studies have provided intensely absorbing chromophores for photodynamic therapy and fluorescence imaging.12 a limitation of these groups is that while some offer possibilities for functionalization ( e.g. , by traditional carbonyl chemistry ) , most conceivable transformations are detrimental to their abilities as auxochromes . comparison of chlorins and porphyrins , numbering , and spectroscopic axes ( top ) , chlorophylls a , b , d and f ( middle ) , and established naturallyoccurring and synthetic auxochromes , and the auxochromes investigated here ( bottom ) . chlorins extracted from natural sources ( e.g. , chlorophylls ) and those prepared from porphyrins by reduction13 or dihydroxylation14 are prone to decomposition . the chlorin redox state can be lockedin by placing geminal dialkyl groups in the pyrroline ring.15 gemdialkyl groups can be installed by , for example , claisen rearrangement starting from 1hydroxyethyl porphyrins , which in turn are available from vinyl or acetylporphyrins.16 a nichlorin carrying electropolymerizable pyrrole groups was prepared this way.17 polymerization on an electrode surface yielded immobilized coenzyme mimics . electropolymerization did not alter the chlorin properties , presumably due to the relatively long macrocycleanchoring group distance ( > 8 atoms).17a gemdialkylstabilized chlorins are also accessible by total synthesis.15b , 18 of the currently existing methods , the one developed by lindsey is amenable to the introduction of a variety of peripheral substituents , for example , bioconjugatable,12b surface attachment19 and solubilizing groups,20 auxochromes,11e , 11i , 21 metal chelates22 and even other tetrapyrroles.23 we hypothesized that fivemembered aromatic heterocycles would be sufficiently small to adopt coplanar conformations with the macrocycle , thus extending conjugation and redshifting the chlorin absorption and emission ( figure 2 ) . the advantage of using these substituents is their ease of installation and robustness under a range of conditions . the results presented here establish furans and thiophenes as powerful auxochromes for hydroporphyrins ( chlorins ) , and bisthienylchlorins as viable monomers to access redabsorbing polymers . the chlorins were prepared using the lindsey method ( schemes 1 and 2 ) . this is a twostep onepot protocol consisting of an initial acidcatalyzed condensation of a dihydrodipyrrin ( western half , e.g. , 1 ) and a 1bromo9formyl dipyrromethane ( eastern half , e.g. , 2s ) , followed by znmediated , intramolecular oxidative cyclization under highdilution conditions.11h , 18e the znchelates formed in the macrocyclization can be difficult to purify and are poor substrates for subsequent transformations ; thus they were demetallated in situ . the furan and thiophenebased heterocycles were introduced premacrocyclization into the 10position and the 3position in chl313 . postmacrocyclization installation via suzuki coupling between the appropriate bromochlorin and the heterocycle boronic acid derivative was also possible . the bromochlorins themselves were available either from brominated precursors ( e.g. , 1br ) , or through chlorin bromination under acidic conditions , which is known to be selective for the most electron rich and not sterically hindered 15position.24 the successful synthesis of these heterocyclefunctionalized chlorins shows that chlorins with thiophene or furan pendent groups undergo halogenation in the tetrapyrrole , and that electronrich heterocycles are compatible with the oxidizing macrocylization conditions . the mild chlorin synthesis conditions enable the introduction of two nonidentical heterocyclic substituents into the final product by installing one substituent before the macrocyclization , and the second after . suzuki coupling between boronic acid 3s and 13n , followed by removal of the ts protecting group yielded the thienylfunctionalized western half 13 . macrocyclization with brominated eastern half 2br , followed by demetallation with tfa in ch2cl2 afforded the brominated , thienylfunctionalized chlorin chl313 . this species was readily coupled with boronic acid 3 under standard suzuki conditions , affording chl313 in good yield after column chromatography on silica gel . the uv / vis absorption and fluorescence emission spectra of the chlorins were recorded in ch2cl2 ( table 1 and figure 1 ) . the absorption and emission properties of chl3 and chl3 were found to be essentially solvent independent ( table s1 ) . the largest redshifts in the soret and qyband absorptions of chl3 were =8 and 4 nm , respectively , upon going from meoh to toluene . the situation was similar for chl3 , and the absorption spectrum shifted to the red by =8 nm ( soret ) and 5 nm ( q y ) upon replacing acetonitrile with toluene . changes in the fluorescence excitation and emission maxima were even smaller ( table s1 ) . the molar extinction coefficients ( ) could not be accurately determined due to the limited amount of material available.22a therefore , absorption spectra were normalized to enable direct comparison of substituent effects on the b / qband ratio.11b a 2thienyl group at the 3 or 13positions redshifts the absorption maximum by 10 nm compared to the unsubstituted free base chlorin ( max=634 nm in toluene25 ) , a 5formyl2thienyl group in the same positions results in a 20 nmred shift . the effect of 10substitution is small ( 6 nm redshift for thiophene ) , comparable to the effect of a mesophgroup.26 these differences are in line with the fact that the 3 and the 13positions lie along one of the spectroscopic axes of the chlorins ( figure 1 ) , thus , their substitutions have a larger effect on the photophysical properties than a 10substitution . the simultaneous 3thienyl , 13formylthienyl substitution ( chl313 ) affords dramatically redshifted absorption ( to 668 nm ) , and a fluorescence emission with a maximum at 680 nm . the effect of the heterocycles is comparable to those of vinyl , formyl or acetyl auxochromes at the same positions.11a for example , 3alkynyl or 3vinylgroups shift the qyabsorption of znchl from 602 to 627 nm or 620 nm , respectively , while 13acetylation results in max=632 nm.11a thus these established auxochromes can be replaced by , for example , thiophene , which is also a convenient synthetic handle for further functionalizations , such as modifications at their positions or oxidative radicalpolymerization ( vide infra ) . the fluorescence spectrum in all cases consists of a qyband and a lowintensity satellite ( figure 3 ) . the order of emission bands generally follows the same order as the qy absorption bands . this order is reversed for chl3 and chl3 , and similarly for chl13 and chl13 . a formylfuryl substituent results in a larger redshift in the absorption spectrum than a formylthienyl group in the same position . this is explained by the extension of the conjugation over the formylfuryl group ( figure s3 ) , and the resulting decrease in the homo lumo gap , as shown by dft calculations ( table s2 ) . the calculated dihedral angles are smaller for the furanappended chlorins than for the thienylchlorins ( table s2 ) , which is in line with extended conjugation for the former , but not the latter . one explanation for this may be that the twisted thienylchlorins relax into a more coplanar conformation after excitation . an xray crystallographic analysis of chl313 showed a dihedral angle of 22.8(11) ( 24.2(5) ) between the least squares plane ( l.s.pl . ) of the thienyl and the adjacent pyrrole ring , showing that such quite planar conformations are accessible for 3 , and presumably 13thienylchlorins . in contrast , the same substituent in the mesoposition ( in chl103 ) shows significantly larger torsion of the chlorin and the thiopehene l.s.pl . normalized absorption ( top ) and fluorescence emission ( bottom ) spectra of selected heterocyclebearing chlorins in ch2cl2 at room temperature . ellipsoids at 50 % probability level . for clarity only one of the disordered thienyl units in chl313 it is interesting to note that imahori and coworkers have observed an increased stokes shift for polymesofurylporphyrin but not for polymesothienylporphyrin.3b this was explained with the larger steric bulk of the thienyl group , which in the mesoposition precludes coplanarization with the macrocycle . we propose that in the less crowded position the small furan can be coplanar , and the thiophene can become significantly coplanar in the excited state . freezing thfsolutions of chl3 and chl3 at 77 k afforded excitation maxima at 651 nm for both species ( table s3 ) . the difference between the excitation and emission maxima diminished to 2 nm ( 47 cm , from 115 cm ) for chl3 and to 6 nm ( 93 cm , from 279 cm ) for chl3 . these observations are consistent with freezing resulting in similar conformations for the two chlorins ; hence the similar excitation maxima . the small difference between ex and em could be due to the lack of conformational changes for chl3 in the solid state ; this effect is less pronounced for chl3 . the fluorescence quantum yields were found to be typical of previously reported free base chlorins , with values ranging from 0.11 to 0.34 . the photophysical properties of chl313 are particularly appealing , combining intense redshifted absorption and emission with the highest quantum yield in this series . therefore , we note that already the attachment of a single formylthienyl group to either the 3 or the 13position affords emission above 660 nm . the redox properties of the chlorins were studied by cyclic voltammetry ( cv ) in ch2cl2 with nbu4pf6 as supporting electrolyte . all chlorin derivatives show one reversible ( 1.66 to 1.70 v ) and one quasireversible reduction ( 2.06 to 2.20 v , figure 5 ) . the first quasireversible oxidation ( 0.37 to 0.44 v ) is typically chlorin based,11a while the second oxidation is irreversible ( 0.830.86 v ) , and is assigned to thienyl oxidation.27 depending on the bisthienylchlorin substitution pattern , we have observed different increased increments of current upon repeated oxidative scans , indicative of deposition of polymeric species on the glassy carbon ( gc ) working electrode . [ a ] conditions : measured with [ analyte]=for 1 mm in ch2cl2 with 0.1 m nbu4pf6 on glassy celectrode ; n=100 mv s. potentials are given versus fc . [ b ] reversible peak , the reported value is e 1/2=(e pa+e pc)/2 . cyclic voltammograms of the bisthienylchlorin monomers ( left ) , the electropolymerizations of the monomers , and the characterization of the polymer films ( right ) . monomers were recorded in dry , deareated ch2cl2 using a threeelectrode setup with a gc working electrode . the potentials are referenced internally to the fc couple . generally , a scan rate of 100 mv s was used ; semireversible redox events were investigated at higher scan rates ( 500 mv s , dotted lines ) . polymerization was performed on fluorine doped tin oxide glass ( fto ) which was used as the working electrode in a threeelectrode setup . the initial scan is highlighted in red , with subsequent scans going from black to light gray . after polymer deposition the films are thoroughly washed with ch2cl2 and placed in pure electrolyte solution . the electrochemical response of the films was investigated at 100 mv s using different potential windows ( red dotted line ) . with these data in hand we attempted a controlled electropolymerization of bisthienylchlorins chl310 , chl313 , chl315 and chl1015 ( figure s4 ) . we have carried out the polymerization on fluorine doped tin oxide ( fto ) conductive glass substrates by repeated cycling between oxidative ( ca . 1.00 v ) and reductive potentials ( 0.5 to 0.2 v ) , which gives rise to homogenous polymer films in all cases ( vide infra ) . the polymerization of chl1015 and chl310 proceeded slowly showing only moderate current increases after 45 and 80 cycles , respectively . by contrast , chlorins chl315 and chl313 showed rapid polymer deposition concomitant with a substantial current increase . the cyclic voltammograms of p chl313 and p chl315 displayed persistent oxidative peaks at + 0.04 v and 0.01 v , respectively , upon cycling to negative potentials . this could be attributed to formation of metallated tin chlorin.28 although p chl310 shows an ambipolar conduction behavior we observe a rapid decomposition of the film with applied reductive potentials as observed by a significant current decrease within the first five scans ( film s1 vs. film s5 , figure 5 ) . this contrasts the behavior observed for p chl1015 , which exhibits good bipolar conduction behavior between 0.56 and + 0.37 v. the polymer films on the fto substrate had broad absorptions with bands around 410420 nm and 650680 nm ( figure s4 ) with the exception of p chl310 ( which was probably not observed due to low concentrations , that is , thin films ) . the films were essentially nonemissive because of selfquenching due to the short intrachlorin distances ( figures s6 , s7 ) . scanning electron microscopy ( sem ) analysis of the films revealed a uniform film formation on the fto substrate during the electropolymerization indicative of a controlled radical polymerization ( figure s9 ) . in order to circumvent the selfquenching observed in the electropolymerized systems , an organicsoluble chlorin polymer ( p chl313hex ) was prepared by treating a 1:20 mixture of chl313 and 3hexylthiophene with fecl3 . h nmr analysis of the resulting copolymer ( figure s1 ) showed unique broadened signals assigned to the macrocycle ( 4 % incorporation ; 2 ppm , 4.6 ppm , > 10 ppm ) , along with typical resonance associated with regioirregular hexylthiophene ( e.g. , 23 ppm ) . the polymer was analyzed by gel permeation chromatography , which yielded a weight average molecular weight ( m w ) of 5299 g mol , a number average molecular weight ( m n ) of 2020 g mol and a polydispersity index ( pdi ) of 2.62 ( figure s2 ) . the relatively large pdi is typical of noncontrolled radical polymerization ; we have not attempted to optimize this procedure yet . a hexylthiophene 20mer containing a single bisthienylchlorin , which corresponds to an 4 % incorporation is expected to have a m w of 3865 g mol . p chl313hex had slightly broadened soret and qbands ( figure s5 ) , and was much more fluorescent than the films ( max=681 nm in ch2cl2 , figures s6s8 ) , which is consistent with the larger spacing between the chromophores . the chlorins were prepared using the lindsey method ( schemes 1 and 2 ) . this is a twostep onepot protocol consisting of an initial acidcatalyzed condensation of a dihydrodipyrrin ( western half , e.g. , 1 ) and a 1bromo9formyl dipyrromethane ( eastern half , e.g. , 2s ) , followed by znmediated , intramolecular oxidative cyclization under highdilution conditions.11h , 18e the znchelates formed in the macrocyclization can be difficult to purify and are poor substrates for subsequent transformations ; thus they were demetallated in situ . the furan and thiophenebased heterocycles were introduced premacrocyclization into the 10position and the 3position in chl313 . postmacrocyclization installation via suzuki coupling between the appropriate bromochlorin and the heterocycle boronic acid derivative was also possible . the bromochlorins themselves were available either from brominated precursors ( e.g. , 1br ) , or through chlorin bromination under acidic conditions , which is known to be selective for the most electron rich and not sterically hindered 15position.24 the successful synthesis of these heterocyclefunctionalized chlorins shows that chlorins with thiophene or furan pendent groups undergo halogenation in the tetrapyrrole , and that electronrich heterocycles are compatible with the oxidizing macrocylization conditions . the mild chlorin synthesis conditions enable the introduction of two nonidentical heterocyclic substituents into the final product by installing one substituent before the macrocyclization , and the second after . suzuki coupling between boronic acid 3s and 13n , followed by removal of the ts protecting group yielded the thienylfunctionalized western half 13 . macrocyclization with brominated eastern half 2br , followed by demetallation with tfa in ch2cl2 afforded the brominated , thienylfunctionalized chlorin chl313 . this species was readily coupled with boronic acid 3 under standard suzuki conditions , affording chl313 in good yield after column chromatography on silica gel . the uv / vis absorption and fluorescence emission spectra of the chlorins were recorded in ch2cl2 ( table 1 and figure 1 ) . the absorption and emission properties of chl3 and chl3 were found to be essentially solvent independent ( table s1 ) . the largest redshifts in the soret and qyband absorptions of chl3 were =8 and 4 nm , respectively , upon going from meoh to toluene . the situation was similar for chl3 , and the absorption spectrum shifted to the red by =8 nm ( soret ) and 5 nm ( q y ) upon replacing acetonitrile with toluene . changes in the fluorescence excitation and emission maxima were even smaller ( table s1 ) . the molar extinction coefficients ( ) could not be accurately determined due to the limited amount of material available.22a therefore , absorption spectra were normalized to enable direct comparison of substituent effects on the b / qband ratio.11b a 2thienyl group at the 3 or 13positions redshifts the absorption maximum by 10 nm compared to the unsubstituted free base chlorin ( max=634 nm in toluene25 ) , a 5formyl2thienyl group in the same positions results in a 20 nmred shift . the effect of 10substitution is small ( 6 nm redshift for thiophene ) , comparable to the effect of a mesophgroup.26 these differences are in line with the fact that the 3 and the 13positions lie along one of the spectroscopic axes of the chlorins ( figure 1 ) , thus , their substitutions have a larger effect on the photophysical properties than a 10substitution . the simultaneous 3thienyl , 13formylthienyl substitution ( chl313 ) affords dramatically redshifted absorption ( to 668 nm ) , and a fluorescence emission with a maximum at 680 nm . the effect of the heterocycles is comparable to those of vinyl , formyl or acetyl auxochromes at the same positions.11a for example , 3alkynyl or 3vinylgroups shift the qyabsorption of znchl from 602 to 627 nm or 620 nm , respectively , while 13acetylation results in max=632 nm.11a thus these established auxochromes can be replaced by , for example , thiophene , which is also a convenient synthetic handle for further functionalizations , such as modifications at their positions or oxidative radicalpolymerization ( vide infra ) . the fluorescence spectrum in all cases consists of a qyband and a lowintensity satellite ( figure 3 ) . the order of emission bands generally follows the same order as the qy absorption bands . this order is reversed for chl3 and chl3 , and similarly for chl13 and chl13 . a formylfuryl substituent results in a larger redshift in the absorption spectrum than a formylthienyl group in the same position . this is explained by the extension of the conjugation over the formylfuryl group ( figure s3 ) , and the resulting decrease in the homo the calculated dihedral angles are smaller for the furanappended chlorins than for the thienylchlorins ( table s2 ) , which is in line with extended conjugation for the former , but not the latter . one explanation for this may be that the twisted thienylchlorins relax into a more coplanar conformation after excitation . an xray crystallographic analysis of chl313 showed a dihedral angle of 22.8(11) ( 24.2(5) ) between the least squares plane ( l.s.pl . ) of the thienyl and the adjacent pyrrole ring , showing that such quite planar conformations are accessible for 3 , and presumably 13thienylchlorins . in contrast , the same substituent in the mesoposition ( in chl103 ) shows significantly larger torsion of the chlorin and the thiopehene l.s.pl . normalized absorption ( top ) and fluorescence emission ( bottom ) spectra of selected heterocyclebearing chlorins in ch2cl2 at room temperature . ellipsoids at 50 % probability level . for clarity only one of the disordered thienyl units in chl313 is shown . solution and refinement parameters it is interesting to note that imahori and coworkers have observed an increased stokes shift for polymesofurylporphyrin but not for polymesothienylporphyrin.3b this was explained with the larger steric bulk of the thienyl group , which in the mesoposition precludes coplanarization with the macrocycle . we propose that in the less crowded position the small furan can be coplanar , and the thiophene can become significantly coplanar in the excited state . freezing thfsolutions of chl3 and chl3 at 77 k afforded excitation maxima at 651 nm for both species ( table s3 ) . the difference between the excitation and emission maxima diminished to 2 nm ( 47 cm , from 115 cm ) for chl3 and to 6 nm ( 93 cm , from 279 cm ) for chl3 . these observations are consistent with freezing resulting in similar conformations for the two chlorins ; hence the similar excitation maxima . the small difference between ex and em could be due to the lack of conformational changes for chl3 in the solid state ; this effect is less pronounced for chl3 . the fluorescence quantum yields were found to be typical of previously reported free base chlorins , with values ranging from 0.11 to 0.34 . the photophysical properties of chl313 are particularly appealing , combining intense redshifted absorption and emission with the highest quantum yield in this series . therefore , we note that already the attachment of a single formylthienyl group to either the 3 or the 13position affords emission above 660 nm . the redox properties of the chlorins were studied by cyclic voltammetry ( cv ) in ch2cl2 with nbu4pf6 as supporting electrolyte . all chlorin derivatives show one reversible ( 1.66 to 1.70 v ) and one quasireversible reduction ( 2.06 to 2.20 v , figure 5 ) . the first quasireversible oxidation ( 0.37 to 0.44 v ) is typically chlorin based,11a while the second oxidation is irreversible ( 0.830.86 v ) , and is assigned to thienyl oxidation.27 depending on the bisthienylchlorin substitution pattern , we have observed different increased increments of current upon repeated oxidative scans , indicative of deposition of polymeric species on the glassy carbon ( gc ) working electrode . [ a ] conditions : measured with [ analyte]=for 1 mm in ch2cl2 with 0.1 m nbu4pf6 on glassy celectrode ; n=100 mv s. potentials are given versus fc . [ b ] reversible peak , the reported value is e 1/2=(e pa+e pc)/2 . cyclic voltammograms of the bisthienylchlorin monomers ( left ) , the electropolymerizations of the monomers , and the characterization of the polymer films ( right ) . monomers were recorded in dry , deareated ch2cl2 using a threeelectrode setup with a gc working electrode . the potentials are referenced internally to the fc couple . generally , a scan rate of 100 mv s was used ; semireversible redox events were investigated at higher scan rates ( 500 mv s , dotted lines ) . polymerization was performed on fluorine doped tin oxide glass ( fto ) which was used as the working electrode in a threeelectrode setup . the initial scan is highlighted in red , with subsequent scans going from black to light gray . after polymer deposition the films are thoroughly washed with ch2cl2 and placed in pure electrolyte solution . the electrochemical response of the films was investigated at 100 mv s using different potential windows ( red dotted line ) . with these data in hand we attempted a controlled electropolymerization of bisthienylchlorins chl310 , chl313 , chl315 and chl1015 ( figure s4 ) . we have carried out the polymerization on fluorine doped tin oxide ( fto ) conductive glass substrates by repeated cycling between oxidative ( ca . 1.00 v ) and reductive potentials ( 0.5 to 0.2 v ) , which gives rise to homogenous polymer films in all cases ( vide infra ) . the polymerization of chl1015 and chl310 proceeded slowly showing only moderate current increases after 45 and 80 cycles , respectively . by contrast , chlorins chl315 and chl313 showed rapid polymer deposition concomitant with a substantial current increase . the cyclic voltammograms of p chl313 and p chl315 displayed persistent oxidative peaks at + 0.04 v and 0.01 v , respectively , upon cycling to negative potentials . this could be attributed to formation of metallated tin chlorin.28 although p chl310 shows an ambipolar conduction behavior we observe a rapid decomposition of the film with applied reductive potentials as observed by a significant current decrease within the first five scans ( film s1 vs. film s5 , figure 5 ) . this contrasts the behavior observed for p chl1015 , which exhibits good bipolar conduction behavior between 0.56 and + 0.37 v. the polymer films on the fto substrate had broad absorptions with bands around 410420 nm and 650680 nm ( figure s4 ) with the exception of p chl310 ( which was probably not observed due to low concentrations , that is , thin films ) . the films were essentially nonemissive because of selfquenching due to the short intrachlorin distances ( figures s6 , s7 ) . scanning electron microscopy ( sem ) analysis of the films revealed a uniform film formation on the fto substrate during the electropolymerization indicative of a controlled radical polymerization ( figure s9 ) . in order to circumvent the selfquenching observed in the electropolymerized systems , an organicsoluble chlorin polymer ( p chl313hex ) was prepared by treating a 1:20 mixture of chl313 and 3hexylthiophene with fecl3 . h nmr analysis of the resulting copolymer ( figure s1 ) showed unique broadened signals assigned to the macrocycle ( 4 % incorporation ; 2 ppm , 4.6 ppm , > 10 ppm ) , along with typical resonance associated with regioirregular hexylthiophene ( e.g. , 23 ppm ) . the polymer was analyzed by gel permeation chromatography , which yielded a weight average molecular weight ( m w ) of 5299 g mol , a number average molecular weight ( m n ) of 2020 g mol and a polydispersity index ( pdi ) of 2.62 ( figure s2 ) . the relatively large pdi is typical of noncontrolled radical polymerization ; we have not attempted to optimize this procedure yet . a hexylthiophene 20mer containing a single bisthienylchlorin , which corresponds to an 4 % incorporation is expected to have a m w of 3865 g mol . p chl313hex had slightly broadened soret and qbands ( figure s5 ) , and was much more fluorescent than the films ( max=681 nm in ch2cl2 , figures s6s8 ) , which is consistent with the larger spacing between the chromophores . chlorins functionalized with furans and thiophenes in the peripheral positions were prepared by de novo synthesis from substituted dihydrodipyrrins or dipyrromethanes , or by suzuki coupling between furanyl / thienyl boronic acids and bromochlorins . the reported chlorins have redshifted absorption and emission spectra compared to the parent macrocycles . in the case of furanyl and formylfuranyl substitution , the redshift could be ascribed to the extension of the conjugation . an interesting increase in stokes shift was noted for thiophene derivatives compared to those of furansubstituted chlorins , which was tentatively attributed to the adoption of a coplanar conformation of the thienyl substituent and the chlorin in the excited state . the thienylchlorin regioisomers differed greatly in polymerization efficiency and the electrochemical properties of the resulting films , which underscores the impact of monomer structure . both the films and the soluble polymer had absorptions extending beyond 700 nm , and substantial absorption over large portions of the visible spectrum . additionally , the soluble polymer retained the chlorin monomer 's intense red emission . taken together , our results showcase the utility of small heterocycles as chlorin auxochromes that are analogous to the wellestablished vinyl , formyl and acetyl groups with the benefit of undergoing straightforward chemical and electrochemical polymerization . these new materials are expected to be useful for panchromic light harvesting in artificial photosynthesis , and as red emitters for sensing and imaging applications . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

abstractthe de novo syntheses of chemically stable chlorins with fivemembered heterocyclic ( furane , thiophene , formylfurane and formylthiophene ) substituents in selected meso and positions are reported . heterocycle incorporation in the 3 and 13positions shifted the chlorin absorption and emission to the red ( up to em=680 nm ) , thus these readily incorporated substituents function analogously to auxochromes present in chlorophylls , for example , formyl and vinyl groups . photophysical , theoretical and xray crystallographic experiments revealed small but significant differences between the behavior of the furan and the thiophenebased auxochromes . four regioisomeric bisthienylchlorins ( 3,10 ; 3,13 , 3,15 and 10,15 ) were oxidatively electropolymerized ; the chlorin monomer geometry had a profound impact on the polymerization efficiency and the electrochemical properties of the resulting material . chemical copolymerization of 3,13bisthienylchlorin with 3hexylthiophene yielded an organicsoluble redemitting polymer .

Introduction Results and Discussion Synthesis Photophysical characterization Electrochemistry Polymerization Conclusion Supporting information

a variety of transformations have been successfully applied to the polymerization of diverse porphyrin monomers , such as metalcatalyzed crosscouplings , chemical oxidations , and oxidative and reductive electropolymerizations.3a , 3b , 3d , 6 the lightharvesting and chargetransport properties of the polymers are governed by the monomers and the nature of the formed connection . consequently , chlorins have characteristic intense red absorptions in addition to the soret band , which suggests that their polymers have potential as lightharvesting materials for the underutilized low energypart of the solar spectrum . the uv / vis absorption spectra of chlorins are sensitive to the type and position of the peripheral substituents . comparison of chlorins and porphyrins , numbering , and spectroscopic axes ( top ) , chlorophylls a , b , d and f ( middle ) , and established naturallyoccurring and synthetic auxochromes , and the auxochromes investigated here ( bottom ) . the chlorin redox state can be lockedin by placing geminal dialkyl groups in the pyrroline ring.15 gemdialkyl groups can be installed by , for example , claisen rearrangement starting from 1hydroxyethyl porphyrins , which in turn are available from vinyl or acetylporphyrins.16 a nichlorin carrying electropolymerizable pyrrole groups was prepared this way.17 polymerization on an electrode surface yielded immobilized coenzyme mimics . electropolymerization did not alter the chlorin properties , presumably due to the relatively long macrocycleanchoring group distance ( > 8 atoms).17a gemdialkylstabilized chlorins are also accessible by total synthesis.15b , 18 of the currently existing methods , the one developed by lindsey is amenable to the introduction of a variety of peripheral substituents , for example , bioconjugatable,12b surface attachment19 and solubilizing groups,20 auxochromes,11e , 11i , 21 metal chelates22 and even other tetrapyrroles.23 we hypothesized that fivemembered aromatic heterocycles would be sufficiently small to adopt coplanar conformations with the macrocycle , thus extending conjugation and redshifting the chlorin absorption and emission ( figure 2 ) . , 2s ) , followed by znmediated , intramolecular oxidative cyclization under highdilution conditions.11h , 18e the znchelates formed in the macrocyclization can be difficult to purify and are poor substrates for subsequent transformations ; thus they were demetallated in situ . the furan and thiophenebased heterocycles were introduced premacrocyclization into the 10position and the 3position in chl313 . postmacrocyclization installation via suzuki coupling between the appropriate bromochlorin and the heterocycle boronic acid derivative was also possible . , 1br ) , or through chlorin bromination under acidic conditions , which is known to be selective for the most electron rich and not sterically hindered 15position.24 the successful synthesis of these heterocyclefunctionalized chlorins shows that chlorins with thiophene or furan pendent groups undergo halogenation in the tetrapyrrole , and that electronrich heterocycles are compatible with the oxidizing macrocylization conditions . the uv / vis absorption and fluorescence emission spectra of the chlorins were recorded in ch2cl2 ( table 1 and figure 1 ) . the absorption and emission properties of chl3 and chl3 were found to be essentially solvent independent ( table s1 ) . the situation was similar for chl3 , and the absorption spectrum shifted to the red by =8 nm ( soret ) and 5 nm ( q y ) upon replacing acetonitrile with toluene . changes in the fluorescence excitation and emission maxima were even smaller ( table s1 ) . the molar extinction coefficients ( ) could not be accurately determined due to the limited amount of material available.22a therefore , absorption spectra were normalized to enable direct comparison of substituent effects on the b / qband ratio.11b a 2thienyl group at the 3 or 13positions redshifts the absorption maximum by 10 nm compared to the unsubstituted free base chlorin ( max=634 nm in toluene25 ) , a 5formyl2thienyl group in the same positions results in a 20 nmred shift . the effect of 10substitution is small ( 6 nm redshift for thiophene ) , comparable to the effect of a mesophgroup.26 these differences are in line with the fact that the 3 and the 13positions lie along one of the spectroscopic axes of the chlorins ( figure 1 ) , thus , their substitutions have a larger effect on the photophysical properties than a 10substitution . the simultaneous 3thienyl , 13formylthienyl substitution ( chl313 ) affords dramatically redshifted absorption ( to 668 nm ) , and a fluorescence emission with a maximum at 680 nm . the effect of the heterocycles is comparable to those of vinyl , formyl or acetyl auxochromes at the same positions.11a for example , 3alkynyl or 3vinylgroups shift the qyabsorption of znchl from 602 to 627 nm or 620 nm , respectively , while 13acetylation results in max=632 nm.11a thus these established auxochromes can be replaced by , for example , thiophene , which is also a convenient synthetic handle for further functionalizations , such as modifications at their positions or oxidative radicalpolymerization ( vide infra ) . this is explained by the extension of the conjugation over the formylfuryl group ( figure s3 ) , and the resulting decrease in the homo lumo gap , as shown by dft calculations ( table s2 ) . an xray crystallographic analysis of chl313 showed a dihedral angle of 22.8(11) ( 24.2(5) ) between the least squares plane ( l.s.pl . ) of the thienyl and the adjacent pyrrole ring , showing that such quite planar conformations are accessible for 3 , and presumably 13thienylchlorins . in contrast , the same substituent in the mesoposition ( in chl103 ) shows significantly larger torsion of the chlorin and the thiopehene l.s.pl . for clarity only one of the disordered thienyl units in chl313 it is interesting to note that imahori and coworkers have observed an increased stokes shift for polymesofurylporphyrin but not for polymesothienylporphyrin.3b this was explained with the larger steric bulk of the thienyl group , which in the mesoposition precludes coplanarization with the macrocycle . we propose that in the less crowded position the small furan can be coplanar , and the thiophene can become significantly coplanar in the excited state . the difference between the excitation and emission maxima diminished to 2 nm ( 47 cm , from 115 cm ) for chl3 and to 6 nm ( 93 cm , from 279 cm ) for chl3 . the small difference between ex and em could be due to the lack of conformational changes for chl3 in the solid state ; this effect is less pronounced for chl3 . the photophysical properties of chl313 are particularly appealing , combining intense redshifted absorption and emission with the highest quantum yield in this series . the redox properties of the chlorins were studied by cyclic voltammetry ( cv ) in ch2cl2 with nbu4pf6 as supporting electrolyte . the first quasireversible oxidation ( 0.37 to 0.44 v ) is typically chlorin based,11a while the second oxidation is irreversible ( 0.830.86 v ) , and is assigned to thienyl oxidation.27 depending on the bisthienylchlorin substitution pattern , we have observed different increased increments of current upon repeated oxidative scans , indicative of deposition of polymeric species on the glassy carbon ( gc ) working electrode . cyclic voltammograms of the bisthienylchlorin monomers ( left ) , the electropolymerizations of the monomers , and the characterization of the polymer films ( right ) . the electrochemical response of the films was investigated at 100 mv s using different potential windows ( red dotted line ) . this contrasts the behavior observed for p chl1015 , which exhibits good bipolar conduction behavior between 0.56 and + 0.37 v. the polymer films on the fto substrate had broad absorptions with bands around 410420 nm and 650680 nm ( figure s4 ) with the exception of p chl310 ( which was probably not observed due to low concentrations , that is , thin films ) . scanning electron microscopy ( sem ) analysis of the films revealed a uniform film formation on the fto substrate during the electropolymerization indicative of a controlled radical polymerization ( figure s9 ) . in order to circumvent the selfquenching observed in the electropolymerized systems , an organicsoluble chlorin polymer ( p chl313hex ) was prepared by treating a 1:20 mixture of chl313 and 3hexylthiophene with fecl3 . h nmr analysis of the resulting copolymer ( figure s1 ) showed unique broadened signals assigned to the macrocycle ( 4 % incorporation ; 2 ppm , 4.6 ppm , > 10 ppm ) , along with typical resonance associated with regioirregular hexylthiophene ( e.g. p chl313hex had slightly broadened soret and qbands ( figure s5 ) , and was much more fluorescent than the films ( max=681 nm in ch2cl2 , figures s6s8 ) , which is consistent with the larger spacing between the chromophores . the furan and thiophenebased heterocycles were introduced premacrocyclization into the 10position and the 3position in chl313 . postmacrocyclization installation via suzuki coupling between the appropriate bromochlorin and the heterocycle boronic acid derivative was also possible . , 1br ) , or through chlorin bromination under acidic conditions , which is known to be selective for the most electron rich and not sterically hindered 15position.24 the successful synthesis of these heterocyclefunctionalized chlorins shows that chlorins with thiophene or furan pendent groups undergo halogenation in the tetrapyrrole , and that electronrich heterocycles are compatible with the oxidizing macrocylization conditions . the uv / vis absorption and fluorescence emission spectra of the chlorins were recorded in ch2cl2 ( table 1 and figure 1 ) . the absorption and emission properties of chl3 and chl3 were found to be essentially solvent independent ( table s1 ) . the situation was similar for chl3 , and the absorption spectrum shifted to the red by =8 nm ( soret ) and 5 nm ( q y ) upon replacing acetonitrile with toluene . the molar extinction coefficients ( ) could not be accurately determined due to the limited amount of material available.22a therefore , absorption spectra were normalized to enable direct comparison of substituent effects on the b / qband ratio.11b a 2thienyl group at the 3 or 13positions redshifts the absorption maximum by 10 nm compared to the unsubstituted free base chlorin ( max=634 nm in toluene25 ) , a 5formyl2thienyl group in the same positions results in a 20 nmred shift . the effect of 10substitution is small ( 6 nm redshift for thiophene ) , comparable to the effect of a mesophgroup.26 these differences are in line with the fact that the 3 and the 13positions lie along one of the spectroscopic axes of the chlorins ( figure 1 ) , thus , their substitutions have a larger effect on the photophysical properties than a 10substitution . the simultaneous 3thienyl , 13formylthienyl substitution ( chl313 ) affords dramatically redshifted absorption ( to 668 nm ) , and a fluorescence emission with a maximum at 680 nm . the effect of the heterocycles is comparable to those of vinyl , formyl or acetyl auxochromes at the same positions.11a for example , 3alkynyl or 3vinylgroups shift the qyabsorption of znchl from 602 to 627 nm or 620 nm , respectively , while 13acetylation results in max=632 nm.11a thus these established auxochromes can be replaced by , for example , thiophene , which is also a convenient synthetic handle for further functionalizations , such as modifications at their positions or oxidative radicalpolymerization ( vide infra ) . this is explained by the extension of the conjugation over the formylfuryl group ( figure s3 ) , and the resulting decrease in the homo the calculated dihedral angles are smaller for the furanappended chlorins than for the thienylchlorins ( table s2 ) , which is in line with extended conjugation for the former , but not the latter . an xray crystallographic analysis of chl313 showed a dihedral angle of 22.8(11) ( 24.2(5) ) between the least squares plane ( l.s.pl . ) of the thienyl and the adjacent pyrrole ring , showing that such quite planar conformations are accessible for 3 , and presumably 13thienylchlorins . in contrast , the same substituent in the mesoposition ( in chl103 ) shows significantly larger torsion of the chlorin and the thiopehene l.s.pl . solution and refinement parameters it is interesting to note that imahori and coworkers have observed an increased stokes shift for polymesofurylporphyrin but not for polymesothienylporphyrin.3b this was explained with the larger steric bulk of the thienyl group , which in the mesoposition precludes coplanarization with the macrocycle . we propose that in the less crowded position the small furan can be coplanar , and the thiophene can become significantly coplanar in the excited state . the difference between the excitation and emission maxima diminished to 2 nm ( 47 cm , from 115 cm ) for chl3 and to 6 nm ( 93 cm , from 279 cm ) for chl3 . the small difference between ex and em could be due to the lack of conformational changes for chl3 in the solid state ; this effect is less pronounced for chl3 . the photophysical properties of chl313 are particularly appealing , combining intense redshifted absorption and emission with the highest quantum yield in this series . the redox properties of the chlorins were studied by cyclic voltammetry ( cv ) in ch2cl2 with nbu4pf6 as supporting electrolyte . the first quasireversible oxidation ( 0.37 to 0.44 v ) is typically chlorin based,11a while the second oxidation is irreversible ( 0.830.86 v ) , and is assigned to thienyl oxidation.27 depending on the bisthienylchlorin substitution pattern , we have observed different increased increments of current upon repeated oxidative scans , indicative of deposition of polymeric species on the glassy carbon ( gc ) working electrode . cyclic voltammograms of the bisthienylchlorin monomers ( left ) , the electropolymerizations of the monomers , and the characterization of the polymer films ( right ) . the electrochemical response of the films was investigated at 100 mv s using different potential windows ( red dotted line ) . this contrasts the behavior observed for p chl1015 , which exhibits good bipolar conduction behavior between 0.56 and + 0.37 v. the polymer films on the fto substrate had broad absorptions with bands around 410420 nm and 650680 nm ( figure s4 ) with the exception of p chl310 ( which was probably not observed due to low concentrations , that is , thin films ) . scanning electron microscopy ( sem ) analysis of the films revealed a uniform film formation on the fto substrate during the electropolymerization indicative of a controlled radical polymerization ( figure s9 ) . in order to circumvent the selfquenching observed in the electropolymerized systems , an organicsoluble chlorin polymer ( p chl313hex ) was prepared by treating a 1:20 mixture of chl313 and 3hexylthiophene with fecl3 . h nmr analysis of the resulting copolymer ( figure s1 ) showed unique broadened signals assigned to the macrocycle ( 4 % incorporation ; 2 ppm , 4.6 ppm , > 10 ppm ) , along with typical resonance associated with regioirregular hexylthiophene ( e.g. p chl313hex had slightly broadened soret and qbands ( figure s5 ) , and was much more fluorescent than the films ( max=681 nm in ch2cl2 , figures s6s8 ) , which is consistent with the larger spacing between the chromophores . chlorins functionalized with furans and thiophenes in the peripheral positions were prepared by de novo synthesis from substituted dihydrodipyrrins or dipyrromethanes , or by suzuki coupling between furanyl / thienyl boronic acids and bromochlorins . the reported chlorins have redshifted absorption and emission spectra compared to the parent macrocycles . in the case of furanyl and formylfuranyl substitution , the redshift could be ascribed to the extension of the conjugation . an interesting increase in stokes shift was noted for thiophene derivatives compared to those of furansubstituted chlorins , which was tentatively attributed to the adoption of a coplanar conformation of the thienyl substituent and the chlorin in the excited state . the thienylchlorin regioisomers differed greatly in polymerization efficiency and the electrochemical properties of the resulting films , which underscores the impact of monomer structure . both the films and the soluble polymer had absorptions extending beyond 700 nm , and substantial absorption over large portions of the visible spectrum . additionally , the soluble polymer retained the chlorin monomer 's intense red emission . taken together , our results showcase the utility of small heterocycles as chlorin auxochromes that are analogous to the wellestablished vinyl , formyl and acetyl groups with the benefit of undergoing straightforward chemical and electrochemical polymerization .

the online version of this article ( doi:10.1007/s13402 - 011 - 0045 - 5 ) contains supplementary material , which is available to authorized users . the choice of therapy for breast cancer is based on clinico - pathological features such as estrogen receptor ( er ) and progesterone receptor ( pr ) status , her2 ( human epidermal growth factor receptor 2 ) status , tumor size and grade . expression of er is predictive for endocrine therapy response and an important prognostic factor for breast cancer . the gene coding for this receptor , esr1 ( estrogen receptor 1 ) , is located on chromosome 6q25 . er regulates the transcription of many genes ( approximately 5% of the genome ) by binding to estrogen responsive elements ( ere ) thereby leading to e.g. cell proliferation . estrogen and er are involved in sexual development and reproductive function , but in pathological situations elevated levels of er are seen . ductal carcinoma in situ of the breast generally expressess er , and more than two - thirds of invasive breast cancers are er positive . several endocrine therapies targeting er have successfully been developed , leading to a substantial decrease in tumor growth in about 3050% of er expressing patients . tamoxifen is a selective estrogen receptor modulator ( serm ) and is the standard endocrine therapy for er - positive breast cancers . it competes with estrogen for binding to er and thereby inhibits gene transcription activation and thus cell growth . other endocrine therapies that are increasingly used in breast cancer treatment are aromatase inhibitors ( ais ) , which block the synthesis of estrogen and fulvestrant , an estrogen - receptor destabilizator and downregulator ( serd ) . overexpression of er can be caused by esr1 amplification and esr1 amplification by fluorescence in situ hybridization ( fish ) was originally reported to be a relatively frequent event in breast cancer , associated with a significantly longer survival for patients treated with adjuvant tamoxifen monotherapy . amplification of esr1 has subsequently been studied by several other groups that have questioned the frequency of er amplification in breast cancer patients . er gene amplification detection using fish provided a frequency of 20.6% and 22.6% , whereas acgh , cish and qpcr provided a frequency of 010% [ 1013 ] . differences in amplification frequencies were assigned to missing the amplification due to the small amplicon ( 600 kb ) and the lower levels of amplification , to tumor heterogeneity and to contamination by normal cells . the latter two would lead to an underestimation of the amplification frequency detected by non - morphological techniques . multiplex ligation - dependent probe amplification ( mlpa ) is a molecular technique to detect gene copy number changes , that , for her2 , has been shown to correlate well with immunohistochemistry ( ihc ) , fluorescence in situ hybridization ( fish ) and chromogenic in situ hybridization ( cish ) . mlpa is a pcr - based method for gene copy number quantification in dna extracted from frozen or paraffin embedded tissue . mlpa kits contain up to 45 probes which can be simultaneously detected in one pcr reaction [ 15 , 16 ] . this study aimed to analyze the frequency of esr1 amplification by mlpa in a large group of breast cancer patients and to compare esr1 gains and amplifications detected by mlpa with fish and clinicopathologic features . this study will show that esr1 amplification detected by mlpa is rare in breast cancer , and seems to be associated with high er expression , high age , high grade and high proliferation . tissue samples of invasive breast cancer patients were collected between november 2004 and september 2009 at the department of pathology of the university medical centre in utrecht ( umcu ) , the netherlands . this study randomly selected 135 tissue samples from this consecutive series . according to the dutch federation of medical scientific societies ( the federa ) , use of redundant tissue for research purposes does not require informed consent , if the patients are offered the possibilities to refuse this ( opting out system ) and material has been used anonymously or coded . in the umc utrecht , all patients are informed that research may happen with their redundant tissue , and are offered to opt out . no material of patients that have opted out has been used in the present study , and all materials were used anonymously . the research protocol for this study grading was performed according to the nottingham modification of bloom - richardson system , and mitoses were counted according to a strict protocol as before to arrive at the mitotic activity index ( mai ) . all tissue samples were analyzed with mlpa to determine esr1 and her2 gene copy number alternations . furthermore , her2 , er and pr protein expression were assessed by immunohistochemistry , and histological type , tumor size , histological grade and age at diagnosis were determined for all patients . tumors showing esr1 gain or amplification by mlpa were re - analyzed by fish , as were 14 tumors with normal mlpa esr1 results . for all fish - analyzed tumors in this study , her2 cish data were already available from previous studies [ 14 , 19 ] . ihc for her2 was performed using the hercep test ( dako , glostrup , denmark ) according to the manufacturers instructions on 4 m thick sections from the neutral buffered formaldehyde fixed tissue blocks . ihc membrane staining was semi - quantitatively scored as negative ( 0 ) , weakly positive ( 1 + ) , equivocal ( 2 + ) and strongly positive ( 3 + ) according to the dako fda - approved scoring system . interpretation of staining was done by 2 experienced breast pathologists . as control a small tissue array containing a 0 , 1 + , 2 + and 3 + breast tumor samples was taken along on the same slide as the tumor to be analyzed . immunohistochemical staining for er ( 1d5 , 1:80 , dako ) and pr ( pgr636 , 1:200 , dako ) was performed using a bond - max automated staining machine ( vision biosystems , newcastle , uk ) with the bond polymer refine detection kit ( vision biosystems , cat . invasive tumor areas as identified on serial h&e sections were harvested from one or two whole 4 m thick paraffin sections ( corresponding to approximately 1 square cm of tumor tissue ) with a scalpel . dna was isolated from these tissue fragments by 1 hr incubation in proteinase k ( 10 mg / ml ; roche , almere , the netherlands ) and lysis buffer ( 50 mm tris - hcl buffer ph 8.0 with 0.5% tween 20 ) at 56c followed by boiling for 10 min . this dna solution ( 50100 l ) was , after centrifugation , used in the mlpa analysis according the manufacturers instructions , using the p004-b1 kit ( mrc holland , amsterdam , the netherlands ) . this kit contains , amongst others , two probes for esr1 and three for her2 . all tests were performed in duplicate in an abi 9700 pcr machine ( applied biosystems , foster city , ca , usa ) . gene copy numbers were analyzed using genescan ( applied biosystems ) and coffalyser ( version 9.4 ) software ( mrc - holland ) . for genes with more than one probe present in the kit , the mean of all the probe peaks of this gene in duplicate was calculated . if this mean value was below 0.7 the respective gene was defined as lost , a value between 0.71.3 was defined as normal , 1.32.0 as gain , and values > 2.0 as amplified , as previously established [ 20 , 21 ] . fish was performed using the zytolightspec esr1/cen 6 dual color probe kit ( zytovision , germany , z-2070 - 20 ) according to the manufacturer s instructions . briefly , slides were deparaffinised and incubated for 15 min in heat pretreatment solution citric at 98c . slides were incubated in a pepsin solution for 10 min at 37 c , washed in wash buffer ssc , dehydrated and air dried . subsequently , 10 l of zytolightspec esr1/cen 6 dual color probe was applied to the slides followed by denaturation at 75c for 10 min and incubation overnight at 37c in a thermobrite statspin system ( abbott molecular ) . after hybridization , coverslips were removed in wash buffer a at 37c for 2 min , followed by a wash in the same wash buffer for 2 5 min at 37c , dehydration and dapi / antifade solution for 15 min in the dark . evaluation of the sample was carried out using a leica dm5500 b fluorescence microscope ( leica microsystems , germany ) and leica af6000 software . the esr1 probe was labelled with zygreen ( green , excitation at 503 nm and emission at 528 nm ) and the cep6 alpha - satellite probe with zyorange ( orange , excitation at 547 nm and emission at 572 nm ) . interpretation of the results was based on the counting of at least 30 tumor cells in at least two different areas by one blinded observer , and we used two different approaches : the esr1 absolute copy number and the esr1/cep6 ratio . a sample was defined as gained if the absolute esr1 copy number was > 2 or the esr1/cep6 ratio was > 1.0 , and as amplified if the absolute esr1 copy number was > 10 or if the esr1/cep6 ratio was > 2.2 . tissue samples of invasive breast cancer patients were collected between november 2004 and september 2009 at the department of pathology of the university medical centre in utrecht ( umcu ) , the netherlands . this study randomly selected 135 tissue samples from this consecutive series . according to the dutch federation of medical scientific societies ( the federa ) , use of redundant tissue for research purposes does not require informed consent , if the patients are offered the possibilities to refuse this ( opting out system ) and material has been used anonymously or coded . in the umc utrecht , all patients are informed that research may happen with their redundant tissue , and are offered to opt out . no material of patients that have opted out has been used in the present study , and all materials were used anonymously . the research protocol for this study grading was performed according to the nottingham modification of bloom - richardson system , and mitoses were counted according to a strict protocol as before to arrive at the mitotic activity index ( mai ) . all tissue samples were analyzed with mlpa to determine esr1 and her2 gene copy number alternations . furthermore , her2 , er and pr protein expression were assessed by immunohistochemistry , and histological type , tumor size , histological grade and age at diagnosis were determined for all patients . tumors showing esr1 gain or amplification by mlpa were re - analyzed by fish , as were 14 tumors with normal mlpa esr1 results . for all fish - analyzed tumors in this study , her2 cish data were already available from previous studies [ 14 , 19 ] . ihc for her2 was performed using the hercep test ( dako , glostrup , denmark ) according to the manufacturers instructions on 4 m thick sections from the neutral buffered formaldehyde fixed tissue blocks . ihc membrane staining was semi - quantitatively scored as negative ( 0 ) , weakly positive ( 1 + ) , equivocal ( 2 + ) and strongly positive ( 3 + ) according to the dako fda - approved scoring system . interpretation of staining was done by 2 experienced breast pathologists . as control a small tissue array containing a 0 , 1 + , 2 + and 3 + breast tumor samples was taken along on the same slide as the tumor to be analyzed . immunohistochemical staining for er ( 1d5 , 1:80 , dako ) and pr ( pgr636 , 1:200 , dako ) was performed using a bond - max automated staining machine ( vision biosystems , newcastle , uk ) with the bond polymer refine detection kit ( vision biosystems , cat . invasive tumor areas as identified on serial h&e sections were harvested from one or two whole 4 m thick paraffin sections ( corresponding to approximately 1 square cm of tumor tissue ) with a scalpel . dna was isolated from these tissue fragments by 1 hr incubation in proteinase k ( 10 mg / ml ; roche , almere , the netherlands ) and lysis buffer ( 50 mm tris - hcl buffer ph 8.0 with 0.5% tween 20 ) at 56c followed by boiling for 10 min . this dna solution ( 50100 l ) was , after centrifugation , used in the mlpa analysis according the manufacturers instructions , using the p004-b1 kit ( mrc holland , amsterdam , the netherlands ) . this kit contains , amongst others , two probes for esr1 and three for her2 . all tests were performed in duplicate in an abi 9700 pcr machine ( applied biosystems , foster city , ca , usa ) . gene copy numbers were analyzed using genescan ( applied biosystems ) and coffalyser ( version 9.4 ) software ( mrc - holland ) . for genes with more than one probe present in the kit , the mean of all the probe peaks of this gene in duplicate was calculated . if this mean value was below 0.7 the respective gene was defined as lost , a value between 0.71.3 was defined as normal , 1.32.0 as gain , and values > 2.0 as amplified , as previously established [ 20 , 21 ] . fish was performed using the zytolightspec esr1/cen 6 dual color probe kit ( zytovision , germany , z-2070 - 20 ) according to the manufacturer s instructions . briefly , slides were deparaffinised and incubated for 15 min in heat pretreatment solution citric at 98c . slides were incubated in a pepsin solution for 10 min at 37 c , washed in wash buffer ssc , dehydrated and air dried . subsequently , 10 l of zytolightspec esr1/cen 6 dual color probe was applied to the slides followed by denaturation at 75c for 10 min and incubation overnight at 37c in a thermobrite statspin system ( abbott molecular ) . after hybridization , coverslips were removed in wash buffer a at 37c for 2 min , followed by a wash in the same wash buffer for 2 5 min at 37c , dehydration and dapi / antifade solution for 15 min in the dark . evaluation of the sample was carried out using a leica dm5500 b fluorescence microscope ( leica microsystems , germany ) and leica af6000 software . the esr1 probe was labelled with zygreen ( green , excitation at 503 nm and emission at 528 nm ) and the cep6 alpha - satellite probe with zyorange ( orange , excitation at 547 nm and emission at 572 nm ) . interpretation of the results was based on the counting of at least 30 tumor cells in at least two different areas by one blinded observer , and we used two different approaches : the esr1 absolute copy number and the esr1/cep6 ratio . a sample was defined as gained if the absolute esr1 copy number was > 2 or the esr1/cep6 ratio was > 1.0 , and as amplified if the absolute esr1 copy number was > 10 or if the esr1/cep6 ratio was > 2.2 . of the 135 tumors analyzed by mlpa , 2% showed esr1 amplification ( 3/135 ) and 6% showed esr1 gain ( 8/135 ) . fifteen percent of the patients showed her2 amplification ( 20/135 ) by mlpa , and 5% more showed her2 gain ( 7/135 ) . none of the 3 true esr1 amplifications were associated with her2 amplification , but 4/8 esr1 gains were associated with her2 gain ( 2/4 ) or her2 amplification ( 2/4 ) . there were no losses of esr1 by mlpa in this study , although 10 tumors had copy number ratios in the lower range between 0.7 and 0.8 . supplementary table 1 shows the association between esr1 copy number status by mlpa and esr1 status by fish ( separated into the esr1/cep6 ratio and the esr1 absolute copy number ) . figure 1 and tables 1 and 2 show that all three amplifications by mlpa were also amplified by fish using both fish interpretation methods . table 1 shows that four of the 8 mlpa - detected esr1 gains were also fish amplified , 2/8 were fish gained and 2/8 were not amplified by fish using the ratio interpretation . of all 25 fish - analyzed samples , 13/25 ( 52% ) showed an increased ( > 2 ) cep6 copy number . 1esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in redtable 1concordance between esr1 mlpa ratio and fish esr1/cep6 ratiofish esr1/cep6 ratiototalnagamlpana101314g2248a0033total1231025amplification ( a ) , gain ( g ) , no amplification ( na)table 2concordance between esr1 mlpa ratio and fish absolute esr1 copy numberfish esr1 absolute copytotalnagamlpana67114g1438a0033total711725amplification ( a ) , gain ( g ) , no amplification ( na ) esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in red concordance between esr1 mlpa ratio and fish esr1/cep6 ratio amplification ( a ) , gain ( g ) , no amplification ( na ) concordance between esr1 mlpa ratio and fish absolute esr1 copy number amplification ( a ) , gain ( g ) , no amplification ( na ) using the absolute copy number interpretation as shown in table 2 , 3/8 mlpa - detected gains were also fish amplified , 4/8 were fish gained and 1/8 mlpa gains was not amplified by fish . we also analyzed 14 mlpa non - amplified breast tumors by fish of which 10/14 were normal , 1/14 was gained and 3/14 were amplified by fish using the ratio interpretation . using the absolute copy number interpretation , 6/14 mlpa non - amplified tumors showed no amplification by fish , 7/14 were gained and 1/14 was amplified by fish . this study found two esr1 losses by fish which were both associated with lower mlpa copy number ratios ( 0.72 and 0.73 ) . table 1 shows that all three mlpa - detected esr1 amplifications were associated with 100% er positivity . of the eight esr1 gains by mlpa , 5 had very high er expression ( 90100% ) and 3 were er negative . two of the 3 tumors with esr1 amplifications were grade 3 and 1 was grade 1 . all 8 tumors with esr1 gains were non - low grade ( 5/8 grade 3 , 3/8 grade 2 ) and , in total , 24/25 grade 1 tumors had normal esr1 gene dosage . of the three esr1 amplifications and eight esr1 gains , 2/3 and 6/8 had high mai ( > 10 ) , respectively . all but one ( lobular ) tumors with esr1 amplifications or gains were of the ductal type . all patients with esr1 gain / amplification by mlpa were older than 50 years . two of the three mlpa - detected amplifications were pt1 , the other was pt2 . six of the 8 esr1 gains were pt1 ( < 2 cm ) , whereas 2/8 were pt2 . the lymph node status of the 3 patients with mlpa - detected amplifications was pn0 in 2/3 , and pn1 in 1/3 . table 1 shows 10 esr1 fish amplifications and 3 gains using the esr1/cep6 ratio for interpretation . all 10 fish amplifications had high er expression ( 90100% ) but 2/3 gains were completely er negative . none of the esr1 fish amplified tumors were her2 cish amplified , but all 3 fish - detected gains were her2 cish amplified . nine of the 10 fish amplifications were non - low grade ( 7/9 grade 3 , 2/9 grade 2 ) . all three gains were non - low grade ( 1/3 grade 3 , 2/3 grade 2 ) , had a high mai , were pn0 , pt1 and older than 50 . using the absolute esr1 copy number for interpretation , all 7 fish amplifications had high er expression ( 90100% ) but only 5/11 fish gains were er positive . none of the esr1 fish amplified tumors were her2 cish amplified , but 3/11 fish - detected gains were her2 cish amplified . five of the seven amplifications were non - low grade ( 4/5 grade 3 and 1/5 grade 2 ) , and all but two of the amplifications were associated with high mai . of the 7 amplifications , all but one of the 11 fish - detected gains were non - low grade ( 7/10 grade 3 and 3/10 grade 2 ) and 9/11 were associated with high mai . of the 11 detected esr1 gains , 5/11 were pn0 , 4 were pn1 and 2 were pn2 . seven of these 11 gains were associated with pt1 , 3/11 with pt2 and 1/11 was pt3 . five of the 11 patients with fish - detected esr1 gains were younger than 50 . of the 135 tumors analyzed by mlpa , 2% showed esr1 amplification ( 3/135 ) and 6% showed esr1 gain ( 8/135 ) . fifteen percent of the patients showed her2 amplification ( 20/135 ) by mlpa , and 5% more showed her2 gain ( 7/135 ) . none of the 3 true esr1 amplifications were associated with her2 amplification , but 4/8 esr1 gains were associated with her2 gain ( 2/4 ) or her2 amplification ( 2/4 ) . there were no losses of esr1 by mlpa in this study , although 10 tumors had copy number ratios in the lower range between 0.7 and 0.8 . supplementary table 1 shows the association between esr1 copy number status by mlpa and esr1 status by fish ( separated into the esr1/cep6 ratio and the esr1 absolute copy number ) . figure 1 and tables 1 and 2 show that all three amplifications by mlpa were also amplified by fish using both fish interpretation methods . table 1 shows that four of the 8 mlpa - detected esr1 gains were also fish amplified , 2/8 were fish gained and 2/8 were not amplified by fish using the ratio interpretation . of all 25 fish - analyzed samples , 13/25 ( 52% ) showed an increased ( > 2 ) cep6 copy number . 1esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in redtable 1concordance between esr1 mlpa ratio and fish esr1/cep6 ratiofish esr1/cep6 ratiototalnagamlpana101314g2248a0033total1231025amplification ( a ) , gain ( g ) , no amplification ( na)table 2concordance between esr1 mlpa ratio and fish absolute esr1 copy numberfish esr1 absolute copytotalnagamlpana67114g1438a0033total711725amplification ( a ) , gain ( g ) , no amplification ( na ) esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in red concordance between esr1 mlpa ratio and fish esr1/cep6 ratio amplification ( a ) , gain ( g ) , no amplification ( na ) concordance between esr1 mlpa ratio and fish absolute esr1 copy number amplification ( a ) , gain ( g ) , no amplification ( na ) using the absolute copy number interpretation as shown in table 2 , 3/8 mlpa - detected gains were also fish amplified , 4/8 were fish gained and 1/8 mlpa gains was not amplified by fish . we also analyzed 14 mlpa non - amplified breast tumors by fish of which 10/14 were normal , 1/14 was gained and 3/14 were amplified by fish using the ratio interpretation . using the absolute copy number interpretation , 6/14 mlpa non - amplified tumors showed no amplification by fish , 7/14 were gained and 1/14 was amplified by fish . this study found two esr1 losses by fish which were both associated with lower mlpa copy number ratios ( 0.72 and 0.73 ) . supplementary table 1 shows that all three mlpa - detected esr1 amplifications were associated with 100% er positivity . of the eight esr1 gains by mlpa , 5 had very high er expression ( 90100% ) and 3 were er negative . two of the 3 tumors with esr1 amplifications were grade 3 and 1 was grade 1 . all 8 tumors with esr1 gains were non - low grade ( 5/8 grade 3 , 3/8 grade 2 ) and , in total , 24/25 grade 1 tumors had normal esr1 gene dosage . of the three esr1 amplifications and eight esr1 gains , 2/3 and 6/8 had high mai ( > 10 ) , respectively . all but one ( lobular ) tumors with esr1 amplifications or gains were of the ductal type . all patients with esr1 gain / amplification by mlpa were older than 50 years . two of the three mlpa - detected amplifications were pt1 , the other was pt2 . six of the 8 esr1 gains were pt1 ( < 2 cm ) , whereas 2/8 were pt2 . the lymph node status of the 3 patients with mlpa - detected amplifications was pn0 in 2/3 , and pn1 in 1/3 . for the mlpa - detected gains , 5/8 were pn0 and 3/8 pn1 . table 1 shows 10 esr1 fish amplifications and 3 gains using the esr1/cep6 ratio for interpretation . all 10 fish amplifications had high er expression ( 90100% ) but 2/3 gains were completely er negative . none of the esr1 fish amplified tumors were her2 cish amplified , but all 3 fish - detected gains were her2 cish amplified . nine of the 10 fish amplifications were non - low grade ( 7/9 grade 3 , 2/9 grade 2 ) . all three gains were non - low grade ( 1/3 grade 3 , 2/3 grade 2 ) , had a high mai , were pn0 , pt1 and older than 50 . using the absolute esr1 copy number for interpretation , all 7 fish amplifications had high er expression ( 90100% ) but only 5/11 fish gains were er positive . none of the esr1 fish amplified tumors were her2 cish amplified , but 3/11 fish - detected gains were her2 cish amplified . five of the seven amplifications were non - low grade ( 4/5 grade 3 and 1/5 grade 2 ) , and all but two of the amplifications were associated with high mai . of the 7 amplifications , 4/7 were pn0 and 3/7 pn1 , 3/7 were pt1 and 4/7 pt2 . all but one of the 11 fish - detected gains were non - low grade ( 7/10 grade 3 and 3/10 grade 2 ) and 9/11 were associated with high mai . of the 11 detected esr1 gains , 5/11 were pn0 , 4 were pn1 and 2 were pn2 . seven of these 11 gains were associated with pt1 , 3/11 with pt2 and 1/11 was pt3 . five of the 11 patients with fish - detected esr1 gains were younger than 50 . the aim of this study was to analyze the frequency of esr1 amplification by mlpa , to confirm these amplifications by fish , and to associate these amplifications with clinico - pathological features . esr1 amplification by mlpa was rare ( 2% ) and additionally 6% of the patients showed esr1 gain . all mlpa - detected esr1 amplifications and nearly all esr1 gains were also fish amplified and gained , but not all fish amplifications / gains were mlpa amplified / gained , with a 60% overall concordance ( 15/25 ) between mlpa and fish using the ratio interpretation method and a 52% concordance ( 13/25 ) between mlpa and fish using the absolute esr1 copy number . although the esr1/cep17 fish ratio showed a slightly better correlation with mlpa than the absolute fish esr1 copy number , there was overall not a very good concordance between mlpa and fish . fish seemed to detect more amplifications than mlpa ( in this selected group 40% ( 10/25 ) plus 12% ( 3/25 ) gains ) . our percentage of esr1 amplification by mlpa was much lower than the 20.6% found by holst et al . using bac fish on tissue microarrays , but was consistent with the lower range ( 010% ) of esr1 amplification reported by other groups using a broad range of techniques such as array comparative genomic hybridization ( acgh ) , cish , fish and qpcr [ 813 , 22 ] . several reasons have been postulated for the difference in amplification frequencies reported by these techniques including heterogeneity or contamination by normal dna ( acgh , qpcr , mlpa in this study although we microdissected the tumor area ) , the small esr1 amplicon size ( although her2 also has a small amplicon size : 500700 kb ) , low levels of amplification ( only 15% of its amplifications were > 10 copies , 41% 56 copies ) , large cish probes ( 360 kb ) , and an automated / manual scoring system . according to holst et al . given the low amount of esr1 gains and amplifications in this study , it was not deemed appropriate to perform statistical analysis . therefore , for the association with clinicopathological parameters , frequencies were used rather than statistical p - values . esr1 gain / amplification was not associated with low grade as reported by holst et al . , but in contrary , seemed to be associated with higher grade ( 2 or 3 ) and high mai . this could however explain in part the observation of ejlertsen et al . that patients with esr1 amplification treated with tamoxifen had a shorter time to recurrence and the fact that lyng et al . found esr1 amplification more frequently in patients with recurrence ( 6/7 amplified patients showed recurrence in contrast to 22/46 patients without esr1 amplification ) upon tamoxifen treatment . we did not find an obvious relation between esr1 gain / amplification and tumor size ( pt status ) or lymph node status ( pn status ) , but esr1 gains / amplifications seemed to be associated with higher age . , all esr1 amplifications detected by mlpa or fish were 90100% er positive . for esr1 gains in conclusion , this study used mlpa to detect esr1 amplification in a group of 135 patients and detected only 2% amplification and 6% more gains . all esr1 amplifications by mlpa or fish had high er expression by ihc , but for esr1 gains this was not obvious . surprisingly , esr1 gain / amplification was not preferably associated with low - grade tumors as reported previously , but in contrast seemed to be associated with high grade and high mai . esr1 amplification by mlpa was associated with higher age , but not with nodal status or pt status . all mlpa - detected esr1 amplifications and nearly all esr1 gains were also fish amplified and gained , but not all fish amplifications / gains were mlpa amplified / gained , leading to an overall concordance of only 60% between both techniques . this confirms previous studies that showed differences in the amplification frequencies detected by different techniques . esr1 status by multiplex ligation - dependent probe amplification ( mlpa ) and fluorescence in situ hybridisation ( fish , according to the ratio esr1/cep6 and the absolute esr1 copy number ) for 25 breast cancer patients .

backgroundexpression of estrogen receptor alpha ( er ) is predictive for endocrine therapy response and an important prognostic factor in breast cancer . overexpression of er can be caused by estrogen receptor 1 ( esr1 ) gene amplification and was originally reported to be a frequent event associated with a significantly longer survival for er - positive women treated with adjuvant tamoxifen monotherapy , which was however questioned by subsequent studies.methodsthis study aimed to reanalyze the frequency of esr1 amplification by multiplex ligation - dependent probe amplification ( mlpa ) and fluorescence in situ hybridisation ( fish ) , and to assess clinicopathologic correlations . mlpa was performed in a group of 135 breast cancer patients , and gains / amplifications were subjected to fish.resultstrue esr1 amplification by mlpa was rare ( 2% ) and only 6% more patients showed a modest gain of esr1 . all mlpa - detected esr1 amplifications and nearly all esr1 gains were also fish amplified and gained , but not all fish amplifications / gains were mlpa amplified / gained , leading to an overall concordance of only 60% between both techniques . all 3 mlpa and fish esr1 amplified cases had high er expression , but there was no obvious correlation between esr1 gain and er status by ihc . esr1 gains / amplifications were not associated with her2 gain / amplification , but seemed to be associated with older age . surprisingly , esr1 gain / amplification was not associated with low grade as reported previously , but correlated with high grade and high proliferation . furthermore , esr1 gain / amplification by mlpa was not associated with nodal status or tumor size ( pt status).conclusionsesr1 amplification as detected by mlpa is rare in breast cancer , and seems to be associated with high er expression , high age , high grade and high proliferation . this study confirms previous studies that showed differences in the esr1 amplification frequencies detected by different techniques.electronic supplementary materialthe online version of this article ( doi:10.1007/s13402 - 011 - 0045 - 5 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Materials and methods Patient material Immunohistochemistry Multiplex ligation-dependent probe amplification (MLPA) FISH Results MLPA FISH Association with clinicopathological features Discussion Conclusion Electronic supplementary material

the online version of this article ( doi:10.1007/s13402 - 011 - 0045 - 5 ) contains supplementary material , which is available to authorized users . the choice of therapy for breast cancer is based on clinico - pathological features such as estrogen receptor ( er ) and progesterone receptor ( pr ) status , her2 ( human epidermal growth factor receptor 2 ) status , tumor size and grade . expression of er is predictive for endocrine therapy response and an important prognostic factor for breast cancer . the gene coding for this receptor , esr1 ( estrogen receptor 1 ) , is located on chromosome 6q25 . estrogen and er are involved in sexual development and reproductive function , but in pathological situations elevated levels of er are seen . ductal carcinoma in situ of the breast generally expressess er , and more than two - thirds of invasive breast cancers are er positive . several endocrine therapies targeting er have successfully been developed , leading to a substantial decrease in tumor growth in about 3050% of er expressing patients . tamoxifen is a selective estrogen receptor modulator ( serm ) and is the standard endocrine therapy for er - positive breast cancers . other endocrine therapies that are increasingly used in breast cancer treatment are aromatase inhibitors ( ais ) , which block the synthesis of estrogen and fulvestrant , an estrogen - receptor destabilizator and downregulator ( serd ) . overexpression of er can be caused by esr1 amplification and esr1 amplification by fluorescence in situ hybridization ( fish ) was originally reported to be a relatively frequent event in breast cancer , associated with a significantly longer survival for patients treated with adjuvant tamoxifen monotherapy . amplification of esr1 has subsequently been studied by several other groups that have questioned the frequency of er amplification in breast cancer patients . differences in amplification frequencies were assigned to missing the amplification due to the small amplicon ( 600 kb ) and the lower levels of amplification , to tumor heterogeneity and to contamination by normal cells . multiplex ligation - dependent probe amplification ( mlpa ) is a molecular technique to detect gene copy number changes , that , for her2 , has been shown to correlate well with immunohistochemistry ( ihc ) , fluorescence in situ hybridization ( fish ) and chromogenic in situ hybridization ( cish ) . this study aimed to analyze the frequency of esr1 amplification by mlpa in a large group of breast cancer patients and to compare esr1 gains and amplifications detected by mlpa with fish and clinicopathologic features . this study will show that esr1 amplification detected by mlpa is rare in breast cancer , and seems to be associated with high er expression , high age , high grade and high proliferation . tissue samples of invasive breast cancer patients were collected between november 2004 and september 2009 at the department of pathology of the university medical centre in utrecht ( umcu ) , the netherlands . the research protocol for this study grading was performed according to the nottingham modification of bloom - richardson system , and mitoses were counted according to a strict protocol as before to arrive at the mitotic activity index ( mai ) . furthermore , her2 , er and pr protein expression were assessed by immunohistochemistry , and histological type , tumor size , histological grade and age at diagnosis were determined for all patients . tumors showing esr1 gain or amplification by mlpa were re - analyzed by fish , as were 14 tumors with normal mlpa esr1 results . for all fish - analyzed tumors in this study , her2 cish data were already available from previous studies [ 14 , 19 ] . immunohistochemical staining for er ( 1d5 , 1:80 , dako ) and pr ( pgr636 , 1:200 , dako ) was performed using a bond - max automated staining machine ( vision biosystems , newcastle , uk ) with the bond polymer refine detection kit ( vision biosystems , cat . tissue samples of invasive breast cancer patients were collected between november 2004 and september 2009 at the department of pathology of the university medical centre in utrecht ( umcu ) , the netherlands . the research protocol for this study grading was performed according to the nottingham modification of bloom - richardson system , and mitoses were counted according to a strict protocol as before to arrive at the mitotic activity index ( mai ) . furthermore , her2 , er and pr protein expression were assessed by immunohistochemistry , and histological type , tumor size , histological grade and age at diagnosis were determined for all patients . tumors showing esr1 gain or amplification by mlpa were re - analyzed by fish , as were 14 tumors with normal mlpa esr1 results . for all fish - analyzed tumors in this study , her2 cish data were already available from previous studies [ 14 , 19 ] . ihc membrane staining was semi - quantitatively scored as negative ( 0 ) , weakly positive ( 1 + ) , equivocal ( 2 + ) and strongly positive ( 3 + ) according to the dako fda - approved scoring system . immunohistochemical staining for er ( 1d5 , 1:80 , dako ) and pr ( pgr636 , 1:200 , dako ) was performed using a bond - max automated staining machine ( vision biosystems , newcastle , uk ) with the bond polymer refine detection kit ( vision biosystems , cat . of the 135 tumors analyzed by mlpa , 2% showed esr1 amplification ( 3/135 ) and 6% showed esr1 gain ( 8/135 ) . fifteen percent of the patients showed her2 amplification ( 20/135 ) by mlpa , and 5% more showed her2 gain ( 7/135 ) . none of the 3 true esr1 amplifications were associated with her2 amplification , but 4/8 esr1 gains were associated with her2 gain ( 2/4 ) or her2 amplification ( 2/4 ) . there were no losses of esr1 by mlpa in this study , although 10 tumors had copy number ratios in the lower range between 0.7 and 0.8 . supplementary table 1 shows the association between esr1 copy number status by mlpa and esr1 status by fish ( separated into the esr1/cep6 ratio and the esr1 absolute copy number ) . table 1 shows that four of the 8 mlpa - detected esr1 gains were also fish amplified , 2/8 were fish gained and 2/8 were not amplified by fish using the ratio interpretation . 1esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in redtable 1concordance between esr1 mlpa ratio and fish esr1/cep6 ratiofish esr1/cep6 ratiototalnagamlpana101314g2248a0033total1231025amplification ( a ) , gain ( g ) , no amplification ( na)table 2concordance between esr1 mlpa ratio and fish absolute esr1 copy numberfish esr1 absolute copytotalnagamlpana67114g1438a0033total711725amplification ( a ) , gain ( g ) , no amplification ( na ) esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in red concordance between esr1 mlpa ratio and fish esr1/cep6 ratio amplification ( a ) , gain ( g ) , no amplification ( na ) concordance between esr1 mlpa ratio and fish absolute esr1 copy number amplification ( a ) , gain ( g ) , no amplification ( na ) using the absolute copy number interpretation as shown in table 2 , 3/8 mlpa - detected gains were also fish amplified , 4/8 were fish gained and 1/8 mlpa gains was not amplified by fish . table 1 shows that all three mlpa - detected esr1 amplifications were associated with 100% er positivity . of the eight esr1 gains by mlpa , 5 had very high er expression ( 90100% ) and 3 were er negative . all 8 tumors with esr1 gains were non - low grade ( 5/8 grade 3 , 3/8 grade 2 ) and , in total , 24/25 grade 1 tumors had normal esr1 gene dosage . of the three esr1 amplifications and eight esr1 gains , 2/3 and 6/8 had high mai ( > 10 ) , respectively . all patients with esr1 gain / amplification by mlpa were older than 50 years . two of the three mlpa - detected amplifications were pt1 , the other was pt2 . six of the 8 esr1 gains were pt1 ( < 2 cm ) , whereas 2/8 were pt2 . the lymph node status of the 3 patients with mlpa - detected amplifications was pn0 in 2/3 , and pn1 in 1/3 . all 10 fish amplifications had high er expression ( 90100% ) but 2/3 gains were completely er negative . none of the esr1 fish amplified tumors were her2 cish amplified , but all 3 fish - detected gains were her2 cish amplified . nine of the 10 fish amplifications were non - low grade ( 7/9 grade 3 , 2/9 grade 2 ) . all three gains were non - low grade ( 1/3 grade 3 , 2/3 grade 2 ) , had a high mai , were pn0 , pt1 and older than 50 . using the absolute esr1 copy number for interpretation , all 7 fish amplifications had high er expression ( 90100% ) but only 5/11 fish gains were er positive . none of the esr1 fish amplified tumors were her2 cish amplified , but 3/11 fish - detected gains were her2 cish amplified . five of the seven amplifications were non - low grade ( 4/5 grade 3 and 1/5 grade 2 ) , and all but two of the amplifications were associated with high mai . of the 7 amplifications , all but one of the 11 fish - detected gains were non - low grade ( 7/10 grade 3 and 3/10 grade 2 ) and 9/11 were associated with high mai . five of the 11 patients with fish - detected esr1 gains were younger than 50 . of the 135 tumors analyzed by mlpa , 2% showed esr1 amplification ( 3/135 ) and 6% showed esr1 gain ( 8/135 ) . fifteen percent of the patients showed her2 amplification ( 20/135 ) by mlpa , and 5% more showed her2 gain ( 7/135 ) . none of the 3 true esr1 amplifications were associated with her2 amplification , but 4/8 esr1 gains were associated with her2 gain ( 2/4 ) or her2 amplification ( 2/4 ) . there were no losses of esr1 by mlpa in this study , although 10 tumors had copy number ratios in the lower range between 0.7 and 0.8 . supplementary table 1 shows the association between esr1 copy number status by mlpa and esr1 status by fish ( separated into the esr1/cep6 ratio and the esr1 absolute copy number ) . table 1 shows that four of the 8 mlpa - detected esr1 gains were also fish amplified , 2/8 were fish gained and 2/8 were not amplified by fish using the ratio interpretation . 1esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in redtable 1concordance between esr1 mlpa ratio and fish esr1/cep6 ratiofish esr1/cep6 ratiototalnagamlpana101314g2248a0033total1231025amplification ( a ) , gain ( g ) , no amplification ( na)table 2concordance between esr1 mlpa ratio and fish absolute esr1 copy numberfish esr1 absolute copytotalnagamlpana67114g1438a0033total711725amplification ( a ) , gain ( g ) , no amplification ( na ) esr1 ( green ) amplification by fish in 3 multiplex ligation - dependent probe amplification ( mlpa)-amplified patients . cep6 is indicated in red concordance between esr1 mlpa ratio and fish esr1/cep6 ratio amplification ( a ) , gain ( g ) , no amplification ( na ) concordance between esr1 mlpa ratio and fish absolute esr1 copy number amplification ( a ) , gain ( g ) , no amplification ( na ) using the absolute copy number interpretation as shown in table 2 , 3/8 mlpa - detected gains were also fish amplified , 4/8 were fish gained and 1/8 mlpa gains was not amplified by fish . supplementary table 1 shows that all three mlpa - detected esr1 amplifications were associated with 100% er positivity . of the eight esr1 gains by mlpa , 5 had very high er expression ( 90100% ) and 3 were er negative . all 8 tumors with esr1 gains were non - low grade ( 5/8 grade 3 , 3/8 grade 2 ) and , in total , 24/25 grade 1 tumors had normal esr1 gene dosage . of the three esr1 amplifications and eight esr1 gains , 2/3 and 6/8 had high mai ( > 10 ) , respectively . all patients with esr1 gain / amplification by mlpa were older than 50 years . two of the three mlpa - detected amplifications were pt1 , the other was pt2 . six of the 8 esr1 gains were pt1 ( < 2 cm ) , whereas 2/8 were pt2 . the lymph node status of the 3 patients with mlpa - detected amplifications was pn0 in 2/3 , and pn1 in 1/3 . all 10 fish amplifications had high er expression ( 90100% ) but 2/3 gains were completely er negative . none of the esr1 fish amplified tumors were her2 cish amplified , but all 3 fish - detected gains were her2 cish amplified . nine of the 10 fish amplifications were non - low grade ( 7/9 grade 3 , 2/9 grade 2 ) . all three gains were non - low grade ( 1/3 grade 3 , 2/3 grade 2 ) , had a high mai , were pn0 , pt1 and older than 50 . using the absolute esr1 copy number for interpretation , all 7 fish amplifications had high er expression ( 90100% ) but only 5/11 fish gains were er positive . none of the esr1 fish amplified tumors were her2 cish amplified , but 3/11 fish - detected gains were her2 cish amplified . five of the seven amplifications were non - low grade ( 4/5 grade 3 and 1/5 grade 2 ) , and all but two of the amplifications were associated with high mai . all but one of the 11 fish - detected gains were non - low grade ( 7/10 grade 3 and 3/10 grade 2 ) and 9/11 were associated with high mai . five of the 11 patients with fish - detected esr1 gains were younger than 50 . the aim of this study was to analyze the frequency of esr1 amplification by mlpa , to confirm these amplifications by fish , and to associate these amplifications with clinico - pathological features . esr1 amplification by mlpa was rare ( 2% ) and additionally 6% of the patients showed esr1 gain . all mlpa - detected esr1 amplifications and nearly all esr1 gains were also fish amplified and gained , but not all fish amplifications / gains were mlpa amplified / gained , with a 60% overall concordance ( 15/25 ) between mlpa and fish using the ratio interpretation method and a 52% concordance ( 13/25 ) between mlpa and fish using the absolute esr1 copy number . although the esr1/cep17 fish ratio showed a slightly better correlation with mlpa than the absolute fish esr1 copy number , there was overall not a very good concordance between mlpa and fish . our percentage of esr1 amplification by mlpa was much lower than the 20.6% found by holst et al . using bac fish on tissue microarrays , but was consistent with the lower range ( 010% ) of esr1 amplification reported by other groups using a broad range of techniques such as array comparative genomic hybridization ( acgh ) , cish , fish and qpcr [ 813 , 22 ] . several reasons have been postulated for the difference in amplification frequencies reported by these techniques including heterogeneity or contamination by normal dna ( acgh , qpcr , mlpa in this study although we microdissected the tumor area ) , the small esr1 amplicon size ( although her2 also has a small amplicon size : 500700 kb ) , low levels of amplification ( only 15% of its amplifications were > 10 copies , 41% 56 copies ) , large cish probes ( 360 kb ) , and an automated / manual scoring system . given the low amount of esr1 gains and amplifications in this study , it was not deemed appropriate to perform statistical analysis . esr1 gain / amplification was not associated with low grade as reported by holst et al . , but in contrary , seemed to be associated with higher grade ( 2 or 3 ) and high mai . we did not find an obvious relation between esr1 gain / amplification and tumor size ( pt status ) or lymph node status ( pn status ) , but esr1 gains / amplifications seemed to be associated with higher age . , all esr1 amplifications detected by mlpa or fish were 90100% er positive . for esr1 gains in conclusion , this study used mlpa to detect esr1 amplification in a group of 135 patients and detected only 2% amplification and 6% more gains . all esr1 amplifications by mlpa or fish had high er expression by ihc , but for esr1 gains this was not obvious . surprisingly , esr1 gain / amplification was not preferably associated with low - grade tumors as reported previously , but in contrast seemed to be associated with high grade and high mai . esr1 amplification by mlpa was associated with higher age , but not with nodal status or pt status . all mlpa - detected esr1 amplifications and nearly all esr1 gains were also fish amplified and gained , but not all fish amplifications / gains were mlpa amplified / gained , leading to an overall concordance of only 60% between both techniques . this confirms previous studies that showed differences in the amplification frequencies detected by different techniques . esr1 status by multiplex ligation - dependent probe amplification ( mlpa ) and fluorescence in situ hybridisation ( fish , according to the ratio esr1/cep6 and the absolute esr1 copy number ) for 25 breast cancer patients .