The social and legal acceptability of third-party reproduction varies around the world. In Australia, gamete and embryo donation and surrogacy are permitted within the regulatory framework set out by federal and state governments. The aim of this paper is to describe the social context and regulatory framework for third-party reproduction in Australia. This is a review of current laws and regulations related to third-party reproduction in Australia. Although subtle between-state differences exist, third-party reproduction is by and large a socially acceptable and legally permissible way to form a family throughout Australia. The overarching principles that govern the practice of third-party reproduction are altruism; the right of donorconceived people to be informed of their biological origins; and the provision of comprehensive counselling about the social, psychological, physical, ethical, financial and legal implications of third-party reproduction to those considering donating or receiving gametes or embryos and entering surrogacy arrangements. These principles ensure that donors are not motivated by financial gain, donor offspring can identify and meet with the person or persons who donated gametes or embryos, and prospective donors and recipients are aware of and have carefully considered the potential consequences of third-party reproduction.Australian state laws and federal guidelines prohibit commercial and anonymous third-party reproduction; mandate counselling of all parties involved in gamete and embryo donation and surrogacy arrangements; and require clinics to keep records with identifying and non-identifying information about the donor/s to allow donor-conceived offspring to trace their biological origins.  Assisted reproductive technologies (ART) offerthose who are affected by infertility an opportunityto have children. Most ART procedures involveheterosexual couples using their own oocytes andsperm to form embryos in the hope of having achild who is genetically linked to both partners.Couples where either the male or the female partneris affected by conditions that preclude the useof their own gametes may consider using donatedgametes in ART procedures.in vitro fertilisation(IVF) if the female partner also has a fertilityproblem. In more recent years, single womenand lesbian couples have been able to use DI andIVF with donor sperm to have children in somecountries. Donor oocytes may be used by coupleswhere ovarian failure or declining ovarian functionis the cause of infertility, poor oocyte qualityhas been identified in previous ART cycles, or thewoman is a carrier of a severe genetic condition, where the woman in couples with untreatablemale infertility is inseminated with donor sperm,has been a treatment option for several decades.Donor sperm can also be used in ondition. FurtherThis paper discusses the social context and outlinesthe regulatory framework for gamete/embryo donationand surrogacy in Australia.The Commonwealth of Australia is a country in thesouthern hemisphere with a population of approximately22 million people. Australia comprisessix states: New South Wales (NSW), Queensland(Qld), South Australia (SA), Tasmania (Tas), Victoria(Vic) and Western Australia (WA) and twoTerritories: Australian Capital Territory (ACT) andthe Northern Territory (NT). Legislation relatingto health, including ART, is a matter for individualstates.Australia has a two-tiered health care system. Thenational universal taxpayer-funded Medicare andPharmaceutical Benefit Schemes cover all Australians.Medicare allows access to out-of-hospitalmedical care and treatment in public hospitals atno or limited cost to the individual and the PharmaceuticalBenefit Scheme (PBS) reduces the personalcost of most prescribed medications. In additionto Medicare, private health insurance schemeswhich reduce the financial cost of private hospitalcare can be purchased.ART has a long tradition in Australia where scientistsand clinicians pioneered some of the ARTtechniques in the 1970’s and 1980’s . DI wasThere is no Australia-wide government body orlegislation regulating the provision of ART services.However, all ART clinics are required tocomply with the Code of Practice for ReproductiveTechnology Units developed by the FertilitySociety of Australia’s Reproductive TechnologyAccreditation Committee (RTAC), which performsannual accreditation visits to all ART clinics. The purThe Australian Federal Government, through theNational Health and Medical Research Council(NHMRC), has issued Ethical Guidelines onthe Use of Assisted Reproductive Technology inClinical Practice and Research  and RTACIn addition, four of the six Australian states havelegislation which regulates ART in those states:The Assisted Reproductive Technology Act 2007(NSW) and the associated Assisted ReproductiveTechnology Regulation 2009 in New South WalesThe Assisted Reproductive Treatment Act 1988(SA) in South Australia  The Assisted Reproductive Treatment Act 2008(Vic) and the associated Assisted ReproductiveTreatment Regulations 2009 in Victoria , 17The Human Reproductive Technology Act 1991 (WA) and the associated Human ReproductiveTechnology (Licences and Registers) Regulations1993 in Western Australia .In broad terms, State laws, the NHMRC EthicalGuidelines and the RTAC Code of Practice stipulatethat:Only altruistic gamete and embryo donation ispermissible. Apart from compensation for expensesincurred as a result of donating gametes or embryos,a donor cannot receive any payment or otherinducement.Potential donors and recipients must receiveadequate information about the medical, social,psychological and legal implications of donor proceduresand counselling is mandatory before proceeding.Potential donors must undergo screening for infectiousdiseases.Recipients are entitled to access informationabout the donor’s medical history, physical characteristics,and the number and sex of children bornfrom gametes donated by the same donor.Children born as a result of gamete or embryodonation have the right to access information aboutthe donor, including identifying information.ART clinics are obliged to maintain detailedrecords, including identifying and non-identifyinginformation, of donors, recipients and offspringwhich allow donor-conceived young adults to findout their genetic origins.In some states, ART clinics are obliged to provideinformation about donors, recipients and offspringto a central state register.The donor does not bear legal responsibilities forand is presumed not to be the parent of a child bornas a result of his or her donation.A maximum number of families can be createdwith a donor’s gametes.There is agreement around the world that the social,emotional, medical, legal and ethical complexitiesof donor conception require thorough explorationby those donating and receiving gametesand embryos -23. The The following matters are covered in donor counselling:Circumstances that lead to considering being adonorMedical and practical aspects of the procedurefor the donorPsychological and social aspects of being a donorLegal aspects of being a donor including the possibilitythat a child who is born as a result of thedonation may contact the donor in the futurePossible impact of the donation on the donor’srelationship with his or her intimate partnerPossible impact of the donation on the donor’sown childrenPossible impact of the donation on the donor’srelationship with the recipient if they are knownto each other.Counsellors also gauge prospective donors for theirsuitability to be a donor in terms of their medicaland genetic history, personality characteristics andmotivations for being a donor. People consideringdonating embryos are encouraged to contemplatetheir feelings about donating a potential full geneticsibling to their own child or children.Counselling for recipients aims to ensure that theyconsider the implications of donor conception forthemselves, a future child, their family and socialnetworks, and, if the donor is known to them, theimpact of the donation on their relationship withthe donor.The following matters are covered in recipientcounselling:How a donor was foundThe lack of a genetic tie to one or both parents ofa child born after a donor procedureMedical and practical aspects of the procedurefor the recipientPsychological and social aspects of using a donorto conceiveLegal aspects of using a donor to conceivePossible impact of using a donor to conceive onthe intimate partner relationshipPossible impact of the donation on the recipient’srelationship with the donor if they are known toeach otherThe importance of disclosing the use of a donorto a child born as a result of gamete or embryodonationWhen, how and to whom to disclose the use ofdonor gametes or embryosPossible future interaction between the child andthe donor.In a prospective longitudinal survey of 72 donorsand recipients in Victoria the majority perceivedthe counselling they received before undergoingdonor procedures significantly more helpful thanthey had anticipated it would be. This suggests thatcounselling is beneficial for those contemplating donor procedures and helps potential donors andrecipients become aware of the intricacies of donorconception .In the past, parents were advised not to tell childrenthat they were conceived using donated gametes, 26. HowThe abolition of anonymous donation across Australiahas undoubtedly also contributed to increasedrecognition of the needs and rights of donor-conceivedpeople. Victoria was the first jurisdiction inthe world to implement comprehensive legislationregulating the provision of ART services includingthe establishment of a state donor register , 30,31.To increase the chance of donor-conceived peoplefinding out about the way they were conceived,even if their parents do not disclose this to them,the recently introduced Victorian Assisted ReproductiveTreatment Act 2008  stipulatThe capacity for donor-conceived adults or theirparents to apply for information about their donorvaries from state to state. In Western Australia,amendments to the Human Reproductive TechnologyAct 1991, allow donor-conceived adults toapply for information about their donors if theydonated after December 2004 . In New For states without legislation, guidelines now requireclinics to only use donors who consent tothe release of identifying information to childrenwho are born as a result of their donation. From2006, RTAC guidelines require fertility clinicsacross Australia to only accept donors who areprepared to provide identifying information to anyoffspring who wish to receive information aboutthem on reaching adulthood and to have policiesand procedures in place to support the offspring’sright to know their genetic origins . This ecParents of donor-conceived children and donorconceivedadults who are unable to access informationabout their donor can now use the Internetto search for half-siblings and the donor. Ina recent study, which included participants fromAustralia, 73% of 791 parents of donor-conceivedchildren who were members of the Internet-basedDonor Sibling Registry (DSR) had found siblingsand 18% had found the donor using the DSR website.Australian research shows that although teenagersunderstand that it may be difficult for parentsto ‘disclose such information,’ they believe parentswho have used a donor to conceive should share this information with their children .Though Since 2006, the Victorian Infertility Treatment Authority(now the Victorian Assisted ReproductiveTreatment Authority) has provided advice to parentson ‘how to tell’ children that the family wasformed through the use of a donor. While healthcare professionals such as counsellors in clinicsmay have encouraged openness, parents are oftenanxious about what to say and how their childwill react, and fear that their child may reject themwhen they find out that they are donor-conceived.Practical resources such as children’s books, podcastsof other families who share their experiences,seminars for parents and web-based and written informationassist parents in talking to their childrenand help to increase their confidence. In practice,parents often report that telling their child or youngadult about the way he or she was conceived waseasier than they had anticipated and are relievedonce they have shared the information .Donors may be recruited by the ART clinic and beunknown to the recipient or recruited by the recipientand known and, in some instances, related tothe recipient.Most sperm donors are recruited by ART clinicsthrough advertising campaigns and public awarenessactivities. Some clinics have a sufficientnumber of sperm donors to meet demand. However,the requirement to only use donors who arewilling to provide identifying information to anyoffspring and broadened access to treatment, to includesingle women and same-sex couples in certainstates, has led to a shortage of sperm donorsin some clinics. Occasionally, couples recruit theirown sperm donor, either through social networksor advertising.Occasionally oocyte donors are recruited by ARTclinics and are unknown to the recipient couple.However, due to the shortage of women who areprepared to donate oocytes to someone they do notknow, most recipient couples rely on a friend or arelative to agree to donate oocytes, or attempt torecruit an oocyte donor through advertisements inlocal newspapers or via the Internet.There is a substantial shortage of embryos availablefor donation. At the end of the legal storagetime limit for frozen embryos (five years in moststates), couples are required to decide the fate offrozen embryos that they are not intending to use.Most couples donate these embryos to research orchoose to have them discarded and only 10-15% ofcouples donate their embryos to another infertilecouple , 36. ComEligibility requirements for access to ART servicesvary throughout Australia. In New South Wales,Victoria and Western Australia, access to ARTservices is broad, enabling any woman, regardlessof relationship status or sexual orientation to haveART treatment , 16, 18.ART legislation has been challenged in the courtsin South Australia and Victoria , 40. In People wishing to have ART treatment in SouthAustralia are required to sign a statutory declarationprior to commencing treatment to indicatethat they do not have a criminal history that couldimpact on the health and welfare of a child to beborn. Legislation introduced in Victoria in 2010requires eligible women and their partners (if theyhave one) to undergo criminal record and childprotection order checks prior to treatment and theyare denied access to ART if they have convictionsof violent or sexual offences or have had a childremoved from their custody . VictoriFor many years, surrogacy was legal only in theAustralian Capital Territory and Victoria. In the lastfew years, other states and territories have changedtheir laws and regulations to allow altruistic surrogacyand this is now legal in most parts of Australia.Some states, such as Queensland and Tasmania,still prohibit surrogacy , 43. HowA number of restrictions apply to surrogacy in Australia.For example, surrogates are not able to usetheir own oocytes in a surrogacy arrangement. Instead,the commissioning woman’s oocyte or a donoroocyte may be used. In addition, commercialsurrogacy, where a woman is paid to carry a pregnancy,is banned throughout Australia. However,as in other countries where only non-commercialsurrogacy is legal , reimburMedical expenses associated with the pregnancyand birth that are not recoverable under Medicareor by private health insuranceCost of obtaining legal advice in relation to thesurrogacyCost of counselling associated with the surrogacyTravel costs associated with the pregnancy orbirthLoss of earnings due to leave taken during thepregnancy or around the time of the birthPremiums payable for health, disability or lifeinsurance that would not have been taken out ifthe surrogacy arrangement had not been enteredinto.In Australia, the woman who gives birth to a childis presumed to be the child’s mother and her malepartner, if she has one, is presumed to be the father.Provisions are in place in some Australianstates to transfer parentage to the commissioningcouple. This has been in place in ACT for someyears, has recently been introduced in Victoria andWA, and will be introduced in South Australia bythe end of 2010.In states with surrogacy legislation and under theNHMRC Ethical Guidelines, counselling is mandatoryfor all parties involved in a surrogacy arrangement.In some states, matters to be coveredin counselling are prescribed and may include:Implications of surrogacy for the relationship betweenthe commissioning parent/s, the surrogateand her partner (if she has one) and the donor, ifdonor gametes are usedImplications of the surrogacy on existing childrenof the surrogate or commissioning parent/sPossibility of and attitudes of all parties towardprenatal screening and diagnosis, termination dueto fetal genetic or chromosomal abnormalities orother pregnancy complicationsPossibility of any party deciding not to proceedMatters associated with the health of the fetusduring the pregnancyDispute resolution processesCommissioning parents’ intentions for care ofthe child should one of them diePossible grief reactions of the surrogate and herpartner (if she has one)How to tell the child about the surrogacyAttitudes towards an ongoing relationship between the parties .In all states with surrogacy legislation, participantsare also required to obtain legal advice in relationto the surrogacy arrangement.Eligibility requirements for surrogacy in the stateswhere it can be undertaken vary. State laws and NHMRCEthical Guidelines require that participantsundergo counselling to consider the implicationsof the surrogacy arrangement for all parties and forthe child to be born and that they understand themedical, legal, social and ethical implications ofthe surrogacy arrangement . FurtherIn addition, state legislation may specify a minimumage for surrogates, requirements for the surrogateto have previously carried a pregnancy andgiven birth, and requirements for obtaining legaladvice in relation to the surrogacy arrangementand transfer of parentage to the commissioningparent/s. Both Victoria and Western Australia requirethe surrogate to be over 25 years of age. Insome states, access to surrogacy is restricted to coupleswho are married or in a de facto relationship.In Western Australia, surrogacy is also available tosingle women who cannot conceive or give birthfor medical or genetic reasons  and in VIn Australia, the provision of gamete and embryodonation and surrogacy are regulated in state laws,the Fertility Society of Australia’s ReproductiveTechnology Accreditation Committee’s (RTAC)Code of Practice, and the National Health andMedical Research Council’s (NHMRC) EthicalGuidelines on the Use of Assisted ReproductiveTechnology in Clinical Practice and Research. Theuse of donor gametes and embryos and surrogacyto form a family is socially accepted and legal inmost parts of Australia. The state of Victoria, as thefirst jurisdiction in the world, pioneered ART legislationand paved the way for other states that nowhave laws pertaining to the provision of ART services,including donor procedures and surrogacy.There are some minor differences between thestate laws that govern third-party reproduction.However state laws, the RTAC Code of Practiceand the NHMRC Ethical Guidelines are all built onthe same three main principles:Commercial gamete and embryo donations andsurrogacy arrangements are banned and hencethird-party reproduction can only be undertakenfor altruistic reasons.People considering donor procedures or surrogacyare required to undergo extensive counsellingregarding the medical, social, psychological, ethicaland legal complexities of third-party reproductionbefore proceeding.Donor-conceived children have a paramountright to be informed of their donor origins. Therefore,only donors who are willing to be identifiedwhen a donor-conceived child reaches the age of18 can donate gametes and embryos and parentsare strongly encouraged to disclose to their childrenthat they were donor-conceived. To allowdonors to be traced, ART service providers areobliged to maintain detailed records with identifyingand non-identifying information about donors,recipients and children born as a result of donorprocedures and, in some states, provide this informationto state run donor registers.


Antibodies to glutamic acid decarboxylase (GAD-ab) associate to different neurological syndromes. It is unknown if the diversity in syndrome association represents epitopes in different immunodominant domains or co-existence of antibodies to other proteins of the inhibitory synapsis. We examined the serum and CSF of 106 patients with anti-GAD related syndromes . GAD65-ab titres were quantified by ELISA. Immunoblot was used to determine if the antibody-targeted epitopes of GAD65 and GAD67 were linear. A cell-based assay (CBA) with HEK293 cells expressing the GAD65 N-terminal, central catalytic domain, or C-terminal was used to investigate the immunodominant domains. Antibodies to GAD67, gamma-aminobutyric acid A receptor (GABAaR), glycine receptor (GlyR), GABAaR-associated protein (GABARAP), and gephyrin were determined with CBA. GAD-ab internalization was investigated using cultured rat hippocampal neurons. CSF GAD65-ab titres were higher in patients with cerebellar ataxia and LE compared to those with SPS (p = 0.02). GAD67-ab were identified in 81% of sera and 100% of CSF. GAD65-ab recognized linear epitopes in 98% of the patients and GAD67-ab in 42% (p<0.001). The GAD65 catalytic domain was recognized by 93% of sera, and the three domains by 22% of sera and 74% of CSF (p<0.001). Six patients had GABAaR-ab and another 6 had GlyR-ab without association to distinctive symptoms. None of the patients had gephyrin- or GABARAP-ab. GAD65-ab were not internalized by live neurons. Overall, these findings show that regardless of the neurological syndrome, the CSF immune response against GAD is more widespread than that of the serum and that there is no specific association between clinical phenotype and the presence of antibodies against other proteins of the inhibitory synapsis. High levels of antibodies against glutamic acid decarboxylase (GAD-ab) have been reported in serum of patients with several neurological syndromes, including stiff person syndrome (SPS), cerebellar ataxia, epilepsy, and limbic encephalitis (LE), all of them characterized by neurological dysfunction of the GABAergic system –3. The rThe pathogenic significance of GAD65-ab is controversial. Some studies suggest they play a direct pathogenic role, but several lines of evidence suggest otherwise. First, GAD65-ab-positive neurological syndromes do not respond well to immunotherapy compared to those associated with antibodies against neuronal surface antigens ,8, seconTo address all these questions, we examined serum or CSF of 106 patients with different anti-GAD associated neurological syndromes aiming to determine the repertoires of antibodies against the two GAD isoforms, the main immunodominant regions and linear or conformational structure of the epitopes, the presence of co-existing antibodies to other proteins or receptors of the inhibitory synapses, and whether GAD-ab were internalized by live neurons.Patients were seen by the authors or referring physicians between December 1994 and April 2013. Serum or CSF were examined for autoantibodies in the laboratory of Neuroimmunology at the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clinic, Barcelona, Spain, or in the Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, USA. Inclusion criteria was the presentation of a neurological disorder associated with serum GAD65-ab detected by brain immunohistochemistry (this technique detects GAD-ab with radioimmunoassay (RIA) levels >2000U/mL; patients below these titres have diabetes (T1DM), but almost never neurological symptoms) .Clinical information was obtained directly by the authors or provided by referring physicians, through questionnaires and telephone interviews. SPS included patients with classical forms, defined by stiffness and spasms predominantly involving the proximal aspect of the legs and lumbar muscles, and patients with partial forms, including stiff-limb syndrome or isolated lower or upper extremity stiffness . CerebelAll subjects gave written informed consent for the storage and use of serum and CSF samples for research purposes. Serum and CSF samples used in the study are deposited in the collection of biological samples named "Neuroinmunología" registered in the biobank of Institut d' Investigació Biomèdica August Pi i Sunyer (IDIBAPS), Barcelona, Spain. Animal handling procedures were approved by the Local Ethics Committee (99/1 University of Barcelona) and the Generalitat de Catalunya (1094/99), in accordance with the Directive 86/609/ EU of the European Commission. The study was approved by the Comité Ètic d’Investigació Clínica (CEIC) of Hospital Clinic.GAD-ab immunoreactivity was analyzed  using an avidin-biotin technique on paraformaldehyde-fixed frozen rat cerebellar sections as described previously . To studTo study the presence of intrathecal synthesis of GAD-ab, the GAD-ab titers obtained by immunohistochemistry in paired samples of CSF and serum and the CSF/serum albumin index were used to calculate the index for intrathecal synthesis of GAD-ab as previously reported . Values Levels of GAD65-ab were detected by ELISA  using a commercial kit following manufacturer’s specifications. Since GAD65 titres in neurologic syndromes are high, serums and CSF were titrated to determine the optimal dilution factor. Briefly, ELISA wells were seeded for 1 h with patients’ sera diluted 1:10000 or CSF diluted 1:200, followed by 1h incubation with GAD65 biotinylated protein, and 20 min incubation with streptavidin peroxidase. Colorimetric reaction was developed with 3,3’,5,5’-Tetramethylbenzidine for 20min, stopped with sulfuric acid and read at 450nm in a multiplate reader.Human GAD65 , GAD67 , and GABARAP (Abnova) recombinant proteins were electrophoretically separated and transferred to a polyvinylidene difluoride membrane and strips incubated with the patient's serum (1:1000 dilution), GAD65 , GAD67 , or GABARAP (Abcam) commercial antibodies followed by biotinylated goat antihuman IgG or horse antimouse IgG  and developed with diaminobenzidine tetrahyrochloride . In someTo test the presence of antibodies against different synaptic antigens, HEK293 cells were transfected with plasmids containing human GAD65 or GAD67 , the α1 and β3 subunits (co-transfected 1:1) of the GABAaR, GABARAP, α1 subunit of the GlyR, or gephyrin (co-transfected 1:1 with collybistin) as described ,18–20. ATo validate the CBA for GAD65, 50 sera from patients with different neurological disorders  and 17 from T1DM were used. None of the 50 sera were positive by this CBA and the sensitivity for T1DM depended on the titer: only 10/17 T1DM sera with higher antibody titer (1822–7900 U/ml by RIA) showed reactivity against GAD65. This finding indicates that the CBA for GAD65, like rat immunohistochemistry, is not sensitive enough to detect low levels of GAD65-ab  and that are identified by RIA.We designed plasmids to express the sequence of the N-terminal domain (Nt: aa 1–188), the central region containing the decarboxylase catalytic domain also termed pyridoxal-5’-phosphate (PLP) domain to indicate the cofactor used by GAD (aa 189–464), and the C-terminal domain (Ct: aa 465–585) from GAD65 . Customized sequences were ordered and commercially subcloned with NheI-NotI  into a pCMV6-AC-Myc-tagged plasmid  Resulting plasmids were transfected into HEK293 cells and immunocytochemistry studies were performed as described above. A commercial antibody against the Myc-Tag protein (Myc-Tag (9B11) mouse mAb, Cell Signaling Technology) was used to confirm the correct expression of each construct.Antibodies against neuronal surface antigens were detected by immunohistochemistry of rat brain and immunofluorescence of rat hippocampal and cerebellar neuronal cultures as previously reported ,21,22.To determine if GAD-ab are internalized in vivo, GAD-ab-positive CSF samples diluted 1:25 were incubated with live rat hippocampal neurons for 24h at 37°C. Cells were then washed with fresh neurobasal media and incubated with unlabeled anti-human IgG antibody  at 1:5 dilution for 1h at 37°C . After extensive washing, neurons were fixed for 5min with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated for 1h with Alexa Fluor 488 anti-human IgG. As positive control of the IgG internalization we used a NMDAR-ab-positive CSF. Results were photographed under confocal microscope (Zeiss LSM710) and analyzed with Zen software .2, ANOVA) were violated. As ELISA values did not follow a normal distribution, log-transformation was used to normalize them prior to statistical analysis. Statistical software  was used for the analyses and a p value < 0.05 was considered significant.Non-parametric tests  were used when the application conditions for parametric tests  patients had GAD65-ab and 88% GAD67-ab. No differences were observed in the frequency of GAD67-ab among the cerebellar ataxia, SPS, LE, or epilepsy groups. By contrast all 61 CSF tested (100%) contained both GAD65- and GAD67-ab . There wp = 0.38. The presence of serum GAD67-ab was associated with GAD65-ab titres (7.2 x105 U/ml (3.3–14.0) vs. 2.9 x105 U/ml (1.5–7.8) for patients with and without serum GAD67-ab respectively; p = 0.02; Serum GAD65-ab levels tended to be higher in patients with cerebellar ataxia and LE . This trTo elucidate whether GAD65- and GAD67-ab identify linear epitopes, all 106 serum samples were screened by immunoblot of purified GAD65 and GAD67 recombinant protein. All but two sera (98%) reacted with GAD65 whereas only 39 (42%) of the 93 sera with GAD67-ab assessed by the CBA were positive by immunoblot of GAD67 . No corrp < 0.001) (p = 0.002), whereas epileptic patients showed more reactivity against the Ct domain . We did not find these differences in CSF, since most of the samples recognized all three domains. No other epitope-region differences were seen among clinical phenotypes. The distribution of frequencies of the number of domains recognized by serum GAD65-ab was similar between the group of sera with or without GAD67-ab, indicating that the presence of GAD67-ab was not related to the extent of the reactivity against GAD65.To investigate the antigenic region of GAD65 recognized by GAD65-ab of patients with the different neurological syndromes, we produced constructs of the three different domains of GAD65: Nt, PLP, and Ct. After demonstrating that the subcloning process did not induce a decrease in GAD65 immunoreactivity (data not shown), we then tested the reactivity of all sera and CSF samples on HEK293 cells expressing each of the three constructs. In 46% of patients serum GAD65-ab recognized only one domain, whereas 74% of CSF GAD65-ab recognized all three domains (< 0.001) . The mosAll 106 serum and 61 CSF samples were examined by immunofluorescence with live rat hippocampal neurons , HEK293 cells expressing α1/β3 subunits of GABAaR, α1 subunit of GlyR, gephyrin or GABARAP. We found six patients with GABAaR antibodies  and six with GlyR antibodies . All samples had low titres (<1:200) of the antibodies. No additional antibodies against neuronal surface antigens were detected in neuronal cultures. We did not find any patient with GABARAP or gephyrin-ab. This result was confirmed by immunoblot using GABARAP recombinant proteins that were tested with serum from 32 patients with SPS (not shown).If we postulate that GAD65 antibodies are pathogenic, they should reach the antigen, which is intracellular, and for this the antibodies need to be internalized. For this experiment we first confirmed that the cultured neurons used contained GAD65. Therefore, cultured neurons were first fixed, permeabilized and incubated with a commercial GAD-6 monoclonal antibody along with patients’ CSF. This preliminary study indicated that the neurons contained intracytoplasmic GAD65 and that both, the commercial antibodies and patients’ CSF antibodies were able to react with the antigen when the cell membrane was permeabilized , panel AThe current study provides for first time a comprehensive overview of the humoral immune responses present in serum and CSF of patients with neurological disorders associated with GAD-ab. Our data show several important immunological features; 1) the humoral immune response against GAD is different in CSF and serum. In the CSF the antibodies target more domains of GAD65 and also immunoreact with GAD67, 2) GAD65-ab titres were higher in the CSF of patients with LE or cerebellar ataxia compared to those with SPS, 3) the frequency of additional antibodies against antigens of the GABAergic inhibitory synapses is low across all neurological syndromes, and 4) GAD-ab are not internalized by primary neuronal cultures suggesting that GAD-ab are not directly pathogenic.A novel finding of our study is that the humoral response to GAD was different in CSF and serum. All patients regardless of the associated neurological syndrome had GAD67-ab in the CSF, including 9/61 (15%) patients whose serum was negative for these antibodies. In addition, CSF GAD65-ab usually recognized epitopes present in the three domains examined. These data along with the frequent detection of CSF oligoclonal bands ,25 and sAlthough we did not identify differences in the GAD65 epitope repertoire among the four neurological syndromes examined, CSF levels of GAD65-ab were lower in patients with SPS than in those with cerebellar ataxia or LE which unlike SPS, commonly associate with neuronal loss. In a prAnalysis of GAD65-ab epitopes has been previously done in patients with T1DM and SPS but not in other neurological syndromes. In T1DM, GAD65-ab mostly recognize conformational epitopes in the PLP and Ct domains ,30. InitWe did not find antibodies against gephyrin and GABARAP. Gephyrin is a protein involved in clustering both GlyR and GABAaR  and antiThe recent discovery of antibodies against cell-surface or synaptic receptors in patients with autoimmune encephalitis, along with data supporting their pathogenicity and sometimes coexistence with GAD-ab, led us to investigate whether additional cell-surface antibodies could offer an explanation for the syndrome diversity. In the current study we used the same approach that resulted in the discovery of 9 of 11 cell surface or synaptic receptor autoantigens  and onlyLastly, the pathogenic role of GAD-ab has been subjected to debate . In vitrThe present study emphasizes the importance of studying the CSF of patients with CNS syndromes suspected to be mediated by GAD autoimmunity because the repertoire of antibodies to different immunodominant regions is wider in the CNS than systemically. We did not find a specific immunodominant region or associated antibody that could be used as a biomarker of a particular neurological anti-GAD-associated syndrome. Moreover, GAD-ab are not internalized in live neurons, raising doubts about the direct pathogenicity of the antibodies.


To identify changes in cefoperazone/sulbactam penetration into cerebrospinal fluid (CSF) after craniotomy and to investigate preliminarily whether cefoperazone/sulbactam CSF concentration can reach therapeutic level when administered intravenously after neurosurgical operation.Neurosurgical patients with an indwelling ventricular drainage pipe who received prophylactic cefoperazone/sulbactam for the treatment of intracranial infection were received a cefoperazone/sulbactam 2:1, 3.0-g infusion for 3 hours every 6 hours for 24 h. Venous blood and CSF specimens were collected to determine cefoperazone/sulbactam concentrations.90 of resistant bacteria. If single intravenous administration time was extended to 3 hours, the serum concentrations of drugs were able to meet the PK/PD standard (T> MIC%> 50%) for treating common, highly resistant bacteria.The cefoperazone and sulbactam concentrations in serum were highest at the second hour  and then decreased. The cefoperazone and sulbactam concentrations in CSF were highest at the 4th hour  and then decreased. CSF penetration measured by the ratio of peak concentrations (CSF/serum) was 8.6%±7.2% for cefoperazone and 13.5%±11.9% for sulbactam, CSF penetration measured by the ratio of trough concentrations (CSF/serum) was 13.4%±5.3% for cefoperazone and 106.5%±87.5% for sulbactam. CSF penetration represented by the ratio of area under the curve (AUC) of CSF and serum was 14.5% for cefoperazone and 22.6% for sulbactam. Neurosurgical impairment of the blood–brain barrier may improve the CSF penetration of these drugs, but it is difficult to reach the MICThe CSF penetration of cefoperazone/sulbactam may be enhanced after neurosurgical impairment of the blood–brain barrier. This study is a pilot research of cefoperazone/sulbactam using in neurosurgical individuals, However, it needs to be confirmed by further large-scale studies. Among several major Gram-negative bacteria causing all nosocomial infections, the increased incidences of Acinetobacter spp. and Pseudomonas aeruginosa are significant, and the rate of resistance is high.Acinetobacter baumannii and Pseudomonas aeruginosa, followed by Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae [Acinetobacter baumannii and Pseudomonas aeruginosa. Moreover, sulbactam plays an important role in overcoming an emerging worldwide problem as it is effective against Acinetobacter baumannii [At the same time, an epidemiological survey on hospital-acquired infections in Beijing Tiantan Hospital, Capital Medical University, also showed that, among patients with intracranial infections after craniotomy, the most common pathogenic bacteria are eumoniae . Cefoperaumannii -5. HowevThe study was approved by the Research Ethics Committee in Tiantan Hospital, Capital Medical University . Written informed consent was obtained from all patients or their healthcare surrogates prior to enrollment in the study.Neurosurgical patients (over 18 years old) with an indwelling ventricular drainage pipe after neurological surgery who were treated with cefoperazone/sulbactam for prevention of intracranial infection were eligible. Patients with histories of heart, pulmonary, liver, and renal dysfunctions were excluded.Cefoperazone/sulbactam  in 1.5-g ampoules with cefoperazone 1.0 g/sulbactam 0.5g was given to all included patients. All the patients were given cefoperazone/sulbactam 3.0 g in 50-mL saline by intravenous injection for 3 hours every 6 hours after craniotomy. 1.5mL of venous blood and 1.5 mL of CSF were collected before the start of drug administration and at Hour 1, 2, 3, 4, 6, 12, 15, 16, 18 h after administration. The specimens were centrifuged at a speed of 3500 r/min for 5 min. Then, the supernatant was collected and stored at −70°C for uniform testing.Cefoperazone and sulbactam concentrations were measured with liquid chromatography mass spectrometry (LC-MS/MS).The tools in our study included an Agilent 1200 liquid chromatograph , Chem Station workstations , Thermo LTQ XL mass spectrometer , OHAUS CP214 electronic balance , Mettler Toledo Seven Easy pH meter, and KH-500 ultrasonic cleaner  were used.The cefoperazone sodium reference substance (Pfizer) was dissolved in double-distilled water (1004 mg/L). The sulbactam sodium reference substance was also dissolved in double-distilled water and the concentration was 402 mg/L. The appropriate amounts of stock solution of cefoperazone were taken and diluted with water to 0.1 mg/L, 0.5 mg/L, 1.0 mg/L, 2.5 mg/L, 5.0 mg/L, 10.0 mg/L, and 20.0 mg/L as reference solutions. The appropriate amounts of stock solution of sulbactam were taken and diluted with water to 0.02 mg/L, 0.1 mg/L, 0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L, and 4.0 mg/L as reference solutions. 100 μL of the reference solution was drawn into a 1.5-mL centrifuge tube, and dried in a stream of nitrogen. The 200-μL blank plasma was added into the tube and dubbed in standard drug-containing plasma after swirling and mixing. Subsequently, 100 μL of chloramphenicol solution (50 mg/L) of the internal standard (IS) and 50 μl of HCL (1.0 mol/L) were added and mixed, and then, ethyl acetate was used to extract the drug. After centrifugation (16000 g for 5 min), the supernatant was dried and the residue was dissolved in a 200-μL mobile phase and centrifuged at 8000 g for 5 min. The supernatant was filtered with a 0.2-μm membrane and analyzed by LC-MS/MS. The CSF specimen was handled in the same process. The ratio (Y) of the cefoperazone-sulbactam peak area (AS) and to the IS peak area (AI) was calculated, and the cefoperazone-sulbactam plasma concentration (X) was weighted (1/x2) for regression calculation. The correlation coefficient R of the serum cefoperazone concentration and the serum sulbactam was 0.9958 and 0.9913, respectively. The respective correlation coefficient R of CSF cefoperazone concentration and CSF sulbactam concentration was 0.9921 and 0.9532.The samples were thawed to room temperature and vortex mixed. The supernatant was analyzed by LC-MS/MS, and the peak area was recorded. The peak area of the main component in the sample solution was brought into the working standard curve to calculate cefoperazone/sulbactam content in each sample solution.All the data were analyzed by the Microsoft Office Excel 2007. Mean serum and CSF cefoperazone and sulbactam concentration was calculated at each time point and expressed as means±standard deviation (x±SD). The mean value of CSF penetration of cefoperazone and sulbactam was also expressed as means±standard deviation (x±SD). The drug concentration-time curves were drawn; The area under the curves were further calculated. The relationships of cefoperazone/sulbactam concentrations in CSF and serum at each time point with the MIC50 and MIC90 of corresponding bacteria were examined.From July 2011 to November 2011, 8 cases  were enrolled. Their mean age was 53±15 years (39 to 77 years), and their average weight was 72.5±9.4 kg (60 to 84 kg). Of the 8 included patients, 3 cases underwent meningioma resection, 3 cases of glioma resection, 1 case of hematoma clearance of hypertensive intracerebral hemorrhage, and shunt surgery after carotid artery aneurysm intervention, respectively.Table CSF penetration measured by the ratio of peak concentrations (CSF/serum) was 8.6%±7.2% for cefoperazone and 13.5%±11.9% for sulbactam, CSF penetration measured by the ratio of trough concentrations (CSF/serum) was 13.4%±5.3% for cefoperazone and 106.5%±87.5% for sulbactam. In addition, the CSF penetration can be evaluated by the ratio of the area under the curve (AUC) of CSF and serum, and the ratio was 14.5% for cefoperazone and 22.6% for sulbactam in the group. If the CSF penetration was evaluated by the ratio of average peak concentration , the penetration of cefoperazone and sulbactam was 16.0% and 13.9%, respectively. Table 90 for cefoperazone/sulbactam against Acinetobacter baumannii and Pseudomonas aeruginosa at 64 mg/L as an example (cefoperazone concentration marked), T>MIC% was greater than 50% [50 of 16 mg/L for cefoperazone/sulbactam against Acinetobacter baumannii and Pseudomonas aeruginosa as an example, T>MIC% was about 50% [50 of cefoperazone against Enterobactercloacae at 2 mg/L, Escherichia coli at 1 mg/L, Klebsiella pneumoniaeat 0.5 mg/L as examples, T>MIC% was close to 100% [90 of 16 mg/L for sulbactam against Acinetobacter baumannii as an example, T>MIC% was greater than 50%  of the sulbactam concentrations at different time points over MIC50. A scatter plot of CSF concentration-time for Cefoperazone and Sulbactam were described in Figure Figure  to 100% . With MI9064 mg/L). However, due to the development of drug resistance, the bacteria’s MIC is generally high. Consequently, it is very difficult to achieve this PK/PD standard in the CSF, and a higher dose might be needed to treat intracranial infections. Taking into account the safety of the drug, a daily dose of 4 g daily dose of sulbactam was administered in the study. However, some papers recommended a daily sulbactam dose 6 g for the treatment of hospital-acquired Gram-negative bacterial infections, especially carbapenem-resistant Acinetobacter baumannii [PK/PD studies in recent years have shown that extension of the single-dose infusion time can improve the efficacy of time-dependent antibiotics. The results of the present research consistently suggested that, if cefoperazone/sulbactam single infusion time was extended to 3 hours, the serum drug concentration achieved the PK/PD standard of T>MIC% greater than 50% . Furthermore, extending administration time can improve the PK/PD indices of the two drugs, and further improve clinical efficacy. In addition, destruction of the blood–brain barrier after craniotomy can increase the CSF concentration to a certain extent. However, for intracranial infections caused by highly resistant bacteria, comprehensive treatment may be needed with a higher dosage of antibiotics, otherwise the antibiotics may need to be given through the ventricle and subarachnoid topically.The results of this study suggest that if the single intravenous administration time of cefoperazone/sulbactam is 3 hours, the serum concentrations can meet the PK/PD standard (T>MIC% greater than 50%) for common, highly resistant bacteria such as The CSF penetration of cefoperazone/sulbactam may be enhanced after neurosurgical impairment of the blood–brain barrier. It may be needed enhance doses of sulbactam for reaching the effective concentrations in CSF (e.g. MIC50/MIC90 of Acinetobacter baumannii).This study is a pilot research of cefoperazone/sulbactam using in neurosurgical individuals, However, it needs to be confirmed by further large-scale studies.


Oxygen causes an increase in the longitudinal relaxation rate of tissues through its T1-shortening effect owing to its paramagnetic properties. Due to such effects, MRI has been used to study oxygen-related signal intensity changes in various body parts including cerebrospinal fluid (CSF) space. Oxygen enhancement of CSF has been mainly studied using MRI sequences with relatively longer time resolution such as FLAIR, and T1 value calculation. In this study, fifteen healthy volunteers were scanned using fast advanced spin echo MRI sequence with and without inversion recovery pulse in order to dynamically track oxygen enhancement of CSF. We also focused on the differences of oxygen enhancement at sulcal and ventricular CSF. Our results revealed that CSF signal after administration of oxygen shows rapid signal increase in both sulcal CSF and ventricular CSF on both sequences, with statistically significant predominant increase in sulcal CSF compared with ventricular CSF. CSF is traditionally thought to mainly form from the choroid plexus in the ventricles and is absorbed at the arachnoid villi, however, it is also believed that cerebral arterioles contribute to the production and absorption of CSF, and controversy remains in terms of the precise mechanism. Our results demonstrated rapid oxygen enhancement in sulcal CSF, which may suggest inhaled oxygen may diffuse into sulcal CSF space rapidly probably due to the abundance of pial arterioles on the brain sulci. Oxygen causes an increase in the longitudinal relaxation rate of tissues through its T1-shortening effect owing to its paramagnetic properties, no T2-shortening effect of oxygen is seen CSF acts as a physical cushion for the brain, and plays an important role in its biological waste disposal. This fluid is known to be produced from the choroid plexus in the ventricles, transfers to the cisterns and is eventually absorbed by the arachnoid villi, after exchanging contents with the interstitial fluid of the brain. CSF has been widely visualized using MRI techniques such as MR cisternography OE on MR (OEMR) imaging of CSF has mostly been studied using FLAIR The approval of the ethics committee of Kyoto University  and written informed consent were obtained. Fifteen healthy volunteers  were recruited, and written informed consent was obtained from all volunteers prior to enrolment.IR-FASE: repetition time (TR), 9575 ms; echo time (TE), 48 ms; inversion time (TI), 1915 ms; matrix, 192×192, 1.37×1.37 mm; slice thickness, 7 mm; field of view (FOV), 263×263 mm; flip angle, 90°; bandwidth, 977 Hz/pixel; Number of averaging is two with additional one TR for echo stabilization. FASE: TR, 4500 ms; TE, 48 ms; matrix, 192×192, 1.37×1.37 mm; slice thickness, 7 mm; FOV, 263×263 mm; flip angle, 90°; bandwidth, 977 Hz/pixel; Number of averaging is two with additional one TR for echo stabilization.IR-FASE and FASE images were acquired using a 3-T MRI scanner  using a 13-channel head coil and the following parameters in a single-slice axial acquisition at the level of 15 mm superior to the anterior commissure – posterior commissure line. 2), where subjects breathed normal room air (21% O2) for 5 min; 2) 100% oxygen administration, where subjects breathed 100% O2 at a flow rate of 15 L/min for 5 min; and 3) post-oxygen administration (Post-O2), where subjects breathed normal room air (21% O2) for 8 min. Oxygen was delivered through a non-rebreather mask that was firmly attached to cover the mouth and nose of the subject.Using IR-FASE  and FASE  to track OE of CSF, images were divided into three phases: 1) pre-oxygen administration  of CSF was calculated for each subject from the start of O2 inhalation. Slope of SI (SIslope) was calculated using a differential function for SI curve and maximum SIslope (maxSIslope) of CSF was then calculated for each subject from the start of O2 inhalation. Approximation methods were determined by selecting best R2 value for each approximation: linear curve fitting regression analysis was performed for the Pre-O2 and Post-O2 phases and polynomial curve fitting regression analysis for O2 phase. Mean SI with standard error of CSFs and CSFv signal values in both IR-FASE and FASE independently, and R2 values were calculated for each phase.Normalization was achieved by setting the mean SI of all dynamic images of Pre-Oslope values for each volunteer in CSFs and CSFv between IR-FASE and FASE, with values of p<0.05 considered statistically significant. MedCalc version 12.2.1 software  was used.Paired Student's t-test was conducted to compare maxSI and maxSI2 showed a linear correlation with time . With oxygen administration, signal values of CSF increase in correlation with time, with a better polynomial curve fit for CSFs than for CSFv . Post-O2 signal values decrease with time, showing a linear curve fit  (Pre-Octively) .2 signals showed a linear correlation with time . With oxygen administration, signal values of CSF rose in correlation with time, with a better polynomial curve fit in CSFs than CSFv . Post-O2 signal values decrease with time, showing a linear curve fit  (Pre-Oor CSFv) .2 minus Pre-O2 shows a positive SI difference, while Post-O2 minus O2 shows a negative SI difference in both IR-FASE and FASE images   .slope were significantly higher for IR-FASE than for FASE   .slope than CSFv in both IR-FASE   (CSFs showed significant higher maxSI and maxSIctively)  and FASEctively) .2 administration and maxSI data supported these findings. Rapid OE after O2 administration displayed by SIslope was also demonstrated and represented by maxSIslope. In addition, OE of CSFs was visualized better than OE of CSFv on both IR-FASE and FASE. OE differences between CSFs and CSFv appeared largely compatible with previous studies of FLAIR with 5-min imaging sequence This study demonstrated dynamic tracking OE of CSF on both IR-FASE and FASE, since both methods showed positive signal increases during O2) and CSF oxygen tension, increasing PaO2 levels with 100% O2 administration will lead to increased CSF levels of O2. Oxygen diffuses into the CSF through the blood-CSF barrier, and this exchange occurs more in CSFs than in CSFv, due to the abundance of pial vessels on the surface of the brain compared to intra-ventricular vessels. The larger amount of intra-ventricular CSF might also cause more dilution of oxygen. Another cause might be the leaky areas of the blood-brain barrier near the pituitary gland, which would further facilitate oxygen diffusion into CSFs more than into CSFv. All of these mechanisms might contribute to the difference in OE between CSFs and CSFv CSF is traditionally thought to mainly form from the choroid plexus in the ventricles and is absorbed at the arachnoid villi IR sequences on MRI have been used to study OE of CSF in most previous reports OE-MRI might act as a non-invasive method for delineating pathological lesions occupying CSF spaces Several limitations exist, since these methods require a high degree of compliance from the imaged subject; firm fixation of the head was conducted in this study to reduce minor motions of the head. Second, quantification of OE such as T1 value calculation was not conducted in this study. We focused on higher temporal resolution of OE in this study, but rapid T1 calculation imaging is expected in the near future.In conclusion, rapid oxygen enhancement of CSF can be dynamically tracked with both IR-FASE and FASE, and is observed more in CSFs than in CSFv, probably due to the abundance of pial arterioles on the brain surface compared to the intra-ventricular arterial system.


Real‐time quaking‐induced conversion (RT‐QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt–Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT‐QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160–165 Sc. Once formed, PrPSc can induce PrP to undergo a conformational change and produce more PrPSc in a self‐propagating manner. The PrPSc aggregates, becomes protease resistant, and deposits throughout the brain, leading to spongiform change and neuronal loss.Creutzfeldt–Jakob disease (CJD) belongs to a family of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). TSEs are characterized by the post‐translational conformational change of a normally expressed protein called prion protein (PrP) into a disease‐associated form known as PrPPatients with sporadic CJD (sCJD) present with a rapidly progressing dementia, and death usually occurs within 6 months. Current diagnostic criteria for sCJD rely on clinical features, the results of electroencephalography and magnetic resonance imaging, and the presence of 14‐3‐3 protein in the cerebrospinal fluid (CSF).Sc to convert native PrP into PrPSc in a newly described protein aggregation assay known as real‐time quaking‐induced conversion (RT‐QuIC). This technique uses recombinant PrP (rPrP) as a substrate, which is induced to aggregate by the addition of CSF containing PrPSc. Thioflavin T (ThT) in the reaction binds to the aggregated PrPSc, causing a change in the ThT emission spectrum, enabling the reaction to be monitored in real time.A new approach to the premortem diagnosis of sCJD has been to exploit the ability of small amounts of CSF PrPCSF RT‐QuIC has been shown to be an accurate diagnostic test for sCJD, with a high degree of sensitivity 85–87%) and specificity (99–100%).% and speThis study reports the findings of 2 international ring trials that were undertaken over a 2‐year period. The initial ring trial comprised European participants, whereas the second ring trial was wider and included participants from Australia, Canada, and Japan.CSF samples were provided by the National CJD Research & Surveillance Unit, United Kingdom. These samples are stored at −80 °C, and consent was obtained from the next of kin for their use in research (05/MRE00/67). CSF samples were selected on the basis of having appropriate ethical consent and sufficient volume to ensure that each participant had an adequate volume of CSF for analysis. Each set of CSF samples was sent to each laboratory on dry ice and was analyzed blind to the final diagnosis. The performance of the RT‐QuIC assay was evaluated in an interlaboratory ring trial, where identical CSF samples were analyzed by each of the participating laboratories. Using this approach, it may be possible to identify analytical procedures that affect overall assay performance.A total of 11 laboratories participated in the ring trials: Department of Pathology, University of Melbourne, Melbourne, Australia (Aust); Department of Neurology, Antwerp University, Antwerp, Belgium (Bel); Prion Laboratory Section, Public Health Agency of Canada, Winnipeg, Canada (Can); Laboratory Investigations of Alzheimer's Disease and Prion Diseases, Pitié‐Salpêtrière Hospital Group, Paris, France (Fra); Department of Neurology, University Medical Center and German Center for Neurodegenerative Diseases, Göttingen, Germany (Ger‐Goet); Centre for Neuropathology and Prion Research, Ludwig Maximilian University, Munich, Germany (Ger‐Mun); Department of Neurological Sciences, University of Bologna, Bologna, Italy (It‐Bol); Department of Neurodegenerative Disease, Carlo Besta Neurological Institute, Milan, Italy (It‐Mil); Department of Neurological Sciences, National Institute of Health, Rome, Italy (It‐Rome); Department of Molecular Microbiology and Immunology, Nagasaki University, Nagasaki, Japan (Jpn); and National CJD Research & Surveillance Unit, University of Edinburgh, Edinburgh, UK (UK).Seven laboratories  participated in the first ring trial, and each received 10 CSF samples sent on dry ice. Of these, 1 was from a patient with neuropathologically confirmed sCJD, 4 were from patients with probable sCJD, diagnosed according to the World Health Organization criteria, 2 patients improved and the diagnosis of sCJD was excluded on clinical grounds, 1 patient had a steroid‐responsive encephalopathy, 1 had no neuropathological evidence of CJD at postmortem, and 1 had mixed Alzheimer disease and vascular dementia at postmortem. The age of the patients ranged from 48 to 86 years and included 5 females and 5 males. The disease duration of the patients with sCJD ranged from 2 to 12 months, and the CSF samples were taken between 53% and 94% of the disease duration.Eleven laboratories  participated in the second ring trial, and each received 15 CSF samples sent on dry ice. One laboratory analyzed the CSF samples twice using a BMG LABTECH  Optima and a BMG LABTECH Omega. Of the 15 CSF samples, 5 were from patients with probable sCJD, 3 were from patients with neuropathologically confirmed sCJD, 1 patient improved and the diagnosis of sCJD was excluded on clinical grounds, 1 had seizures, 1 had anti‐immune encephalopathy, 1 had a psychiatric disorder, 1 had Huntington disease, 1 had a non‐CJD dementia, and 1 had normal pressure hydrocephalus. The age of the patients ranged from 55 to 87 years and included 6 females and 9 males. The disease duration of the patients with sCJD ranged from 1 to 26 months and the CSF samples were taken between 57% and 90% of the disease duration.Each laboratory performed the RT‐QuIC analysis using a standard 10mM phosphate buffer pH 7.4), 170mM NaCl  containing 0.1mg/ml rPrP, 10 μM ThT, and 10 µM ethylenediaminetetraacetic acid tetrasodium salt. However, a range of instrumentations, analytical conditions, and types of rPrP were used (Table .4, 170mMThe results of the ring trials are given in Tables None of the laboratories obtained positive RT‐QuIC responses in any of the 5 CSF samples from patients without sCJD. Four of these laboratories used a BMG LABTECH Optima, and the remaining 3 used a BMG LABTECH Omega. However, all the laboratories used the same full‐length hamster rPrP.The second ring trial included a larger number of laboratories with a wider geographical distribution and included a wider range of instrumentation and type of rPrP. Despite this, the results showed complete concordance (see Table In the first ring trial, 6 of 7 laboratories correctly identified all sCJD cases; however, 1 laboratory obtained a negative CSF RT‐QuIC result from an sCJD patient with a disease duration of 12 months. It has been reported that CSF samples from sCJD patients with longer disease durations may have lower seeding efficiencyThe second ring trial took place 18 months later and was expanded to include other European laboratories and participants from Australia, Canada, and Japan. Some of the additional laboratories used alternative types of rPrP and other forms of instrumentation. This enabled a more rigorous assessment of the robustness and transferability of the RT‐QuIC technique.All 11 laboratories accurately identified the 8 CSF samples from sCJD patients, and none detected a positive RT‐QuIC in any of the non‐CJD cases. The agreement between laboratories using different rPrP as a substrate and different forms of instrumentation is encouraging. The accuracy of the results obtained was identical for each of the rPrP substrates used. From this limited number of CSF samples, we have achieved an overall sensitivity of between 85.7% and 100% and a specificity of 100%. This compares well with a previous intralaboratory study, which had fewer participants and reported a sensitivity of 85% and a specificity of 99%.L.I.M. organized both the ring trials, coanalyzed the data, and cowrote the manuscript; A.P., I.P., S.Su., K.G., S.Sh., B.d.V., S.Sa., K.S., K.C., M.C., G.F., and M.E. performed RT‐QuIC analyses; M.S., I.Z., and P.C. developed RT‐QuIC analysis; L.I.M., F.T., R.A., D.K., S.C., S.H., P.P., M.P., and A.G. participated in the design of the study; A.G. coanalyzed the data and cowrote the manuscript.Nothing to report.


Oophaga pumilio) shows strong divergence in aposematic coloration among populations. To investigate whether predation pressure is important for color divergence among populations of O. pumilio we selected four mainland populations and two island populations from Costa Rica and Panama. Spectrometric measurements of body coloration were used to calculate color and brightness contrasts of frogs as an indicator of conspicuousness for the visual systems of several potential predators  and a conspecific observer. Additionally, we conducted experiments using clay model frogs of different coloration to investigate whether the local coloration of frogs is better protected than non-local color morphs, and if predator communities vary among populations. Overall predation risk differed strongly among populations and interestingly was higher on the two island populations. Imprints on clay models indicated that birds are the main predators while attacks of other predators were rare. Furthermore, clay models of local coloration were equally likely to be attacked as those of non-local coloration. Overall conspicuousness (and brightness contrast) of local frogs was positively correlated with attack rates by birds across populations. Together with results from earlier studies we conclude that conspicuousness honestly indicates toxicity to avian predators. The different coloration patterns among populations of strawberry poison frogs in combination with behavior and toxicity might integrate into equally efficient anti-predator strategies depending on local predation and other ecological factors.Natural selection is widely noted to drive divergence of phenotypic traits. Predation pressure can facilitate morphological divergence, for example the evolution of both cryptic and conspicuous coloration in animals. In this context Dendrobatid frogs have been used to study evolutionary forces inducing diversity in protective coloration. The polytypic strawberry poison frog ( A successful predation event consists of two elements: detection of prey and realization of an attack . AposemaPoecilia reticulata, Xiphophorus helleri, Alticus arnoldorum), the evolution of elaborate signals face a trade-off, since they attract both mates and increase the risk of attacks by predators [Parasemia plantaginis, the maintenance of polymorphism is the result of a trade-off between predator selection and mating success: white males had higher mating success while yellow males survived better when confronted with predators [Heliconius butterflies coloration and spotting pattern seem to be optimized for both predator deterrence and mate attraction [Ranitomeya imitator, frogs of different populations use Müllerian mimicry and resemble several other sympatrically occurring toxic frog species in coloration and pattern (e.g. R. ventrimaculata and R. fantastica) [Oophaga granulifera, rather cryptic and rather conspicuously colored populations have been described [Environmental conditions are likely to differ among geographically isolated populations, and in different populations opposing expressions of a signal may be of selective advantage. Interactions of natural selection with sexual selection and stochastic processes might furthermore contribute to divergent evolution of phenotypic traits among different populations of a species. For example, in several fish species  . In the escribed , whose described .Oophaga pumilio), skin coloration and patterning is highly diverse among populations, comprising more than 15 distinct, mostly geographically separated, color morphs [O. pumilio has been proposed to be driven by both sexual and natural selection .O. pumilio has been confirmed by several experiments, predation was suggested to have played a substantial role in this divergence [O. pumilio, the cryptic and aposematic anti-predator strategy was further supplemented by the respective inconspicuous or bold behavior of the frogs [O. pumilio, we expect predation pressures to vary among populations [O. pumilio [Ranitomeya imitator and Dendrobates tinctorius, predation caused by birds was significantly higher on novel aposematic morphs compared to local aposematic morphs, which exerts strong homogenizing selection in favor of local color morphs [O. pumilio [As the aposematic function of conspicuously colored populations of vergence –23. Althvergence , severalvergence . In two he frogs . If natuulations , 26. Sev pumilio , 23, 27  pumilio , 28, 29.r morphs , in partr morphs , and red pumilio , clay mo pumilio . DespiteField work was conducted between December 2008 and June 2011 in six populations of strawberry poison frogs, two populations in Costa Rica and four populations in Panama. To assess the conspicuousness of the frogs on their specific substrate for conspecific and several heterospecific observers we took reflectance measurements. We measured the spectral reflectance of the skin of a total 255 frogs in Sarapiquí , Hitoy Cerere  (both Costa Rica), Río Gloria , Tierra Oscura , Isla Colón  and Isla Solarte  (Panama). Additionally we measured the reflectance of the specific substrate  on which each individual frog was found. Reflectance spectra of the skin and substrate were taken at a distance of 2mm using an Ocean Optics HR2000+ Spectrometer, an Ocean Optics bifurcal optic fiber (R-200-7-UV/VIS) with a fixed outer sleeve to control for the 2mm distance and a deuterium-tungsten lamp (DT-Mini-2-GS). To account for lamp drift we calibrated the measurements with a white standard (WS-1-SS) every other frog. Illumination of the habitat (Irradiance) was measured using an optic fibre (QP400-2-UV-BX) with an Ocean Optics cosine adaptor-head (CC-3UV). Irradiance spectra were taken at the places where we found the frogs and at times when the frogs showed most activity  on two to three different days. The population-specific average irradiance was calculated for each population, including between 192 and 396 irradiance spectra per population.O. pumilio in an earlier study [We calculated average reflectance spectra for dorsal and ventral regions for each frog. For dorsal reflectance spectra four reflectance measurements were averaged . For frogs from the population of Río Gloria and on Isla Colón, where frogs possess a dark spotting pattern on a yellow or green background color on their dorsums, we included two measurements of the dorsal background color and the two head measurements for dorsal average spectra. We neglected the influence of dark spots, because spotting pattern did not affect predation risk on clay models of er study , and birer study –35. For er study , and ourO. pumilio [Sturnus vulgaris, [Uca tangeri, [Masticophis flagellum, [Uca tangeri [O. pumilio include information about the spectral sensitivity of Masticophis flagellum [O. pumilio [Visual models were calculated according to Maan & Cummings and Crothers & Cummings , 37 usin pumilio  were useulgaris, ), the fitangeri, –42) and agellum, , 44). Th tangeri , 42. Thelagellum  and one  pumilio . The avi pumilio ). For thCrothers .Oophaga pumilio is abundant across its range and listed as a species of "least concern" by the IUCN. All sampling and measuring methods are described in detail above. No animals have been sacrificed and all individuals have been released at their capture site after measuring. Approval by an ethics committee was not necessary since all sampling methods and manipulation of the frogs is part of the evaluation of the study by the local authorities (SINAC and ANAM) that authorize the research permits.Our study species is not a species protected by the laws of Panama or Costa Rica. However it is on the CITES Appendix II colourful dendrobatid species are often collected illegally for commercial reasons. Brightness and color contrast (ΔL and ΔS) of the frogs from the study populations were calculated for potential predators and conspecifics. As absolute values of color and brightness contrasts are not comparable among different visual models, i.e. different observers, we normalized the results of each calculated model and calculated means and standard deviation of color and brightness contrasts (ΔS and ΔL) for dorsal and ventral measurements for each population. We applied ANOVAs to test if contrasts differ significantly among populations for different (conspecific or heterospecific) observers. ANOVAs indicated differences in color and brightness contrasts of both body regions among populations for all observers. Therefore, we applied Tukey post-hoc tests to localize the differences among populations. We furthermore visually checked the residuals to justify the use of ANOVAs. Statistical analyses were conducted using STATISTICA 6.0.To evaluate whether frogs of local or nonlocal (i.e. novel) colors are more or less prone to an attack, we conducted predation experiments with clay model frogs. The use of clay models as specimen replicates facilitates assigning damaged models to different predator categories, according to teeth marks or other characteristic imprints preserved in the clay . PreviouThere was no difference in frequency of predation among the consecutive time intervals of the experiment. Hence, for the analyses we summarized the attacks of all intervals. To analyze whether risk of an attack was predicted by the population, the coloration of the clay model frog and/or by its origin , we used Generalized Linear Models (GLM´s), assuming binomial-error distribution and a logit-link-function. Conducting deviance analyses with Chi-square statistics for the GLM´s we tested whether population, coloration and/or origin of the clay model were significant predictors for general risk of attack, and whether predation risk within each population was predicted by the latter two variables. Furthermore, we applied Tukey post-hoc tests to localize between which populations or clay model colors predation pressure differed. All analyses were calculated using R .O. pumilio. We therefore repeated the analyses considering only damages caused undoubtedly by birds (category ‘bird marks’: U- and V- shaped marks) and damages probably caused by birds . The assignment of ‘holes & scratches’ to ‘potential bird marks’ follows Hegna et al. [Considering the assignation of different predation marks to predator categories, our data–as well as previous studies , 22, 23—a et al.  and is sa et al. , who obsWe furthermore tested, if predation pressure is correlated with high levels of conspicuousness  of the local frogs, which would indicate that natural selection contributed to the evolution of aposematic coloration in this species. Correlation analyses were conducted with STATISTICA 6.0.Visual modelling calculations are based on dorsal and ventral reflectance spectra of differently colored frogs. Average reflectance spectra for each studied population are depicted in Dorsal contrasts: Visual modeling revealed that frogs from Isla Solarte had the highest values of dorsal color contrast . These cVentral contrasts: For avian and conspecific vision, the frogs of Isla Solarte showed the highest values for ventral color contrast, which significantly differed from contrasts of frogs of most other populations, followed by contrasts of frogs from Tierra Oscura and Isla Colón. For avian and conspecific vision, frogs from Río Gloria had the lowest contrasts which were significantly different from most other populations. For crab and snake vision frogs from Isla Colón had the highest color contrasts, followed by frogs from Tierra Oscura and Isla Solarte, while frogs from Hitoy Cerere had the lowest contrasts .Dorsal contrasts: Frogs of Sarapiquí displayed the highest dorsal brightness contrasts for all observers, and were significantly brighter than frogs from Hitoy Cerere, Tierra Oscura and Isla Colón, marginally brighter than frogs from Isla Solarte, but not significantly brighter than frogs from Río Gloria .Ventral contrasts: Similar to their dorsal measurements, frogs from Sarapiquí had the highest ventral brightness contrasts for all observers, followed by frogs from Río Gloria. For all observers, brightness contrasts of frogs from Sarapiquí and Río Gloria were significantly higher than contrasts from all other populations. The frogs from Hitoy Cerere, Tierra Oscura and Isla Solarte displayed the lowest ventral brightness contrasts .L and ΔS in a brightness and color contrast space  of the model frogs . OverallO. pumilio  . Total nOur study yielded several interesting results. Strawberry poison frogs from populations differed in their coloration and conspicuousness, i.e. in color and brightness contrast as well as in overall conspicuousness. In predation experiments with clay model frogs, predation risk varied among frog populations and was higher on island than on mainland populations. Birds were the main predators, while attacks of other predators like lizards, snakes, rodents and crabs were rare. Unlike other studies, our results do not provide evidence that the local color morph was better protected than other color morphs. However bird predation was highest on blue frog models . Our finding of higher attacks on blue frog models than on yellow or green models might indicate that frogs of high conspicuousness (yellow) or low conspicuousness (green) were better protected than intermediate color morphs (blue). Moreover, in populations where avian predation on clay frogs was higher, local frogs were dorsally brighter and more conspicuous, which suggests that predation exerted by birds primarily selects for higher conspicuousness in the local prey.O. pumilio visual models for the specific background where each frog was found. For all observers, including the conspecific viewer, frogs from the population of Isla Solarte possess the highest dorsal color contrasts on their substrates from all studied populations but also possess a high brightness contrast and overall conspicuousness. Previous studies revealed that Isla Solarte is one of the most toxic populations of O. pumilio [The results of visual modeling demonstrate that contrasts calculated for each population are very similar among different observers especially between snake and crab and between avian and  pumilio . Being h pumilio , Isla So pumilio , 36. The pumilio , i.e. to pumilio  suggestsAn additional possibility is the involvement of sexual selection. Our data reveal that avian predators and conspecifics, i.e. potential mating partners, perceive frogs as similarly conspicuous when evaluating contrasts against the specific backgrounds and irradiance conditions in which the frogs were captured. While sexual selection often favors high conspicuousness, it entails easy detection by predators. This might imply that sexual selection for brighter colors in strawberry poison frogs evolved in concert with higher toxicity reinforcing aposematism in this species. The interaction between natural and sexual selection for the divergence in warning colors in this and other taxa warrants further study .O. pumilio. Damages caused by crabs or snakes or other unidentified animals were rare. Most predation events on Isla Solarte clearly resulted from birds, but on Isla Colón more attacks were assigned to the category “holes and scratches”. In this regard more research is necessary to confirm the true nature of these predators and to differentiate predator composition among populations. One previous study using clay model frogs to investigate predation pressure in O. pumilio on Isla Colón confirmed that even though overall attack rate was high on the local frogs, unambiguous bird predation was low on this island compared to mainland populations [Our experiments with clay model frogs showed, that predation pressure significantly varies among populations with higher predation rates on islands (Isla Solarte and Isla Colón) compared to mainland populations. These results are contrary to the general assumption of relaxed predation pressure on islands , 48. Forulations . We obtaulations , 31, 32,O. pumilio found that attack rates on red and yellow clay frogs were not affected by the local frog coloration [Dendrobates tinctorius and Ranitomeya imitator) reported that predation rate on local color morphs was lower than on a (conspicuous) novel color morph [R. imitator showed that the initial advantage of the local color morphs disappeared during the experiment and after 72 hours all frog morphs in the experiment were equally attacked. Thus the apparent local color morph advantage is of short duration, which makes a long-term evolutionary effect unlikely.Our data does not provide direct evidence that model frogs of local coloration are better protected than model frogs of non-local coloration. The color which was most attacked in all four mainland populations  was blue, which is a non-local color for all populations except Tierra Oscura. In Tierra Oscura, where local frogs are blue and possess moderate toxicity levels , loloration  or foundloration . On the or morph , 30. An O. pumilio.Overall, our data do not provide evidence for substantial differences of predation pressure on clay model frogs as a function of the local coloration of strawberry poison frogs. Three possible explanations for this result are as follows: First, predation pressure might not be the major force driving divergence in coloration among populations and its contribution to divergence in coloration might be concealed by the impact of other forces . Second, selection for divergence in color patterns cannot be examined properly as predator communities may have changed since color morphs diverged among different populations in the Bocas del Toro archipelago  and elseHigh conspicuousness (e.g. through high brightness contrast) can facilitate detection of prey by predators, but it might also provide protection through predator learning. Interestingly the rate of attacks which were unambiguously caused by birds is highly correlated with brightness contrast and overall conspicuousness of the local frogs for avian eyes. This indicates that bird predation selects for a bright, conspicuous coloration in local prey because the conspicuousness itself facilitates the education of birds for predator avoidance. For instance, unpalatable prey of higher brightness contrast provided greater predator aversion learning in the Chinese mantid than prey of lower brightness contrast . HoweverOophaga granulifera is another dendrobatid frog species which shows color polytypism ranging from green to red populations in natural lowland forests along the pacific coast of Costa Rica and Panama [O. pumilio and exhibits high similarity in its ecology, behavior and morphology. Research on O. granulifera explores the relation between predation pressure, toxicity and conspicuousness to the visual systems of birds with similar methods as in our study [d Panama . This spur study , 14, 58.O. pumilio [O. granulifera, i.e. green frogs were more cryptic but more toxic than red frogs [O. granulifera follows a specific pattern: birds avoided attacking clay models reproducing the local coloration of the frogs (i.e. red in populations of red frogs and green in populations with green frogs), while lizards mostly attacked red clay models which mimic the highly conspicuous but less toxic red frogs.Contrary to  pumilio , toxicited frogs , while ted frogs . WillinkEpipedobates, proposed that there might be a trade-off between conspicuousness and toxicity in some dendrobatid frogs [O. granulifera and different predator taxa might cause opponent selective advantages for phenotypic traits. For O. pumilio there is evidence that conspicuousness is an honest indicator of toxicity [O. granulifera and O. pumilio might furthermore be caused by differences in the ecology of these two species. Oophaga granulifera inhabits more natural, undisturbed habitats [Oophaga pumilio, however, populates primary and secondary habitats, including abandoned agricultural areas (e.g. cocoa plantations).Another study, investigating the link among predator avoidance, visual conspicuousness and toxicity in three species of the genus id frogs . A similtoxicity  for its habitats , where pO. pumilio, further research is necessary. A persisting problem is the lack of knowledge about the actual predators of this species, which is essential for the evaluation of the importance of natural selection for color divergence [Oophaga granulifera and Oophaga pumilio would allow setting the results of predation experiments and visual modeling in relation to toxicity levels. This may allow insights in trade-offs and the interplay between conspicuousness, toxicity and behavioral strategies and how these interactions may have affected color divergence of poison frogs. Finally, similar studies on other aposematic animals would help to achieve a better understanding of the relationship between “honest” or “dishonest signaling” and predator and prey ecology.For a real understanding of the impact natural selection may have on color divergence of vergence . Furthervergence , will heS1 Fig(DOCX)Click here for additional data file.S2 FigMean reflectance curves average spectra of 38 to 52 individuals . Reflectance curves show reflection of the frogs relative to white standard. Reflectances over 100% emerge due to the brilliant surface of the frogs´ skin, while the used white standard has a dull surface. In order to verify accuracy of reflectance curves, all spectra were visually controlled for oversaturation.(DOCX)Click here for additional data file.S3 FigO. pumilio). Circles of different colors represent means of calculated conspicuousness measurements of each clay color in each population for the respective observer.We calculated conspicuousness measurements (color and brightness contrast) of blue, red, green and yellow clay model frogs on all measured substrates of the respective population for four different observers Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Cerebrospinal fluid (CSF) overproduction results from either CSF infection or choroid plexus hypertrophy or tumor, with only a single idiopathic case described so far. We report a unique case of a male infant with Crouzon syndrome who presented with intracranial hypertension, caused by up to 4-fold increase in CSF daily production. Conditions related to CSF overproduction, namely central nervous system infections and choroid plexus hypertrophy or tumor, were ruled out by repeated magnetic resonance imaging and CSF samples. Medical therapy failed to reduce CSF production and the patient underwent several shunting procedures, cranial expansion, and endoscopic coagulation of the choroid plexus. This article thoroughly reviews pertinent literature on CSF production mechanisms and possible therapeutic implications. Hydrocephalus is generally defined as abnormal accumulation of cerebrospinal fluid (CSF) within the ventricles and subarachnoid spaces, and is often associated with dilatation of the ventricular system and increased intracranial pressure (ICP). It can be regarded as a disparity between production and absorption of cerebrospinal fluid (CSF), almost always as a result of CSF flow obstruction either at the ventricular level  or the subarachnoid level . More rarely hydrocephalus is a result of impaired absorption of CSF, due to increased venous pressure in the dural sinuses or at the jugular veins  and evenWe present a unique case of idiopathic CSF overproduction and the specific difficulties arising from its management. This case prompted us to review the literature focusing on the molecular mechanisms of CSF production and the drugs regulating this process.A 6-month-old male infant, born to non-consanguineous parents, presented with macrocrania, irritability, and multiple episodes of vomiting. He also had dysmorphic features, in particular low-set ears, hypertelorism with exorbitism, mandibular prognatism with inverted bite, and broad and varus big toes. The clinical diagnosis of Crouzon syndrome was confirmed by a genetic test, showing FGFR2 gene mutation. Head computed tomography (CT) scan showed a premature fusion of the squamosal and parieto-mastoid suture bilaterally with open sutures at the cranial vault . Brain m and a concomitant “protective” external ventricular drainage (EVD). Transient closure of EVD, in order to challenge the cardio-vascular system to receive such a daily amount of CSF, was associated to an increased blood level of brain natriuretic peptide (BNP). Finally, two CSF shunt devices were concomitantly placed, namely a VPS on the left side and a VAS on the right side, both utilizing a middle pressure Pudenz valve.A few months later, the child suffered again from symptoms of increased intracranial pressure. After surgical revision of both shunting devices, MRI confirmed craniocerebral disproportion with slit ventricles. A biparietal cranial expansion was therefore performed. However, postoperative CSF drainage still ranged between 1400 mL and 1700 mL per day, requiring once again dual shunting. After 5 weeks a new shunt malfunction required exteriorization of both CSF shunts, confirming daily overproduction of CSF (about 1500 mL/d). A pharmacological approach with terlipressin administration was attempted with poor results. Thus, in spite of normal appearance of the choroid plexuses at MRI, endoscopic choroid plexus coagulation was performed bilaterally. Due to small ventricular size, the procedure was performed through bilateral occipital access and using neuronavigation . Since tHydrocephalus resulting from CSF overproduction is a rare disease and is usually caused by pathological conditions affecting the choroid plexus, namely hyperplasia or tumor. In the last decade, endoscopic coagulation of the choroid plexus has become a treatment option for this condition, warranting shunt independency, with lower complication rate compared to surgical plexectomy . In the To our knowledge, a single case of idiopathic CSF overproduction, ie, not related to the conditions described above, has been reported in the English-language literature so far . In thisIn our case, there was no evidence of CSF infection in the patient’s clinical history. The patient with Crouzon syndrome suffered from a communicating hydrocephalus secondary to impressive idiopathic CSF overproduction. In patients with Crouzon syndrome, hydrocephalus is present in up to 40% of cases and is usually related to venous outlet occlusion and small posterior cranial fossa . In the Finally, the endoscopic coagulation of the choroid plexus, though initially contraindicated due to the risk related to the small size of the ventricular cavities, was performed as a last resort option when the other treatment options failed. This procedure resulted in a significant reduction of CSF production, thus confirming its role in complicated cases of hydrocephalus .This unique case of idiopathic CSF overproduction prompted us to review the mechanisms of CSF production, which are still far from being completely understood, in order to identify possible targets of a medical therapy.+, Cl-, HCO3- and H20, while K+ ions are absorbed ((Table 1).CSF production is an active process, which according to the classical theory takes place mainly in the epithelial cells of the choroid plexuses -19. The absorbed ,22  . Among tAlthough the mechanism of ion secretion has been clarified and most of the involved transporters have now been localized to specific membranes ,22, the Distribution of AQP1 at the choroid plexus apical surface suggests its role in CSF production. Studies on AQP1 knockout mice showed a decrease in both CSF production and ICP values compared with wild-type mice, although the latter effect could be related to the decrease in the central venous pressure in AQP1 knockouts due to the effect on the kidneys ,43. A reStudies on AQP4 role in brain edema in knockout mice models showed a protective role of the protein in vasogenic edema development but an active role in cytotoxic edema development ,48. AnimIn conclusion, the traditional explanation that the CSF is produced by the choroid plexuses and reabsorbed by the arachnoid villi has been questioned. A study in children hypothesized the presence of a minor pathway involving the perivascular spaces as a site of CSF reabsorption . The pre(Table 2). One of the corticosteroid action mechanisms for reducing brain edema and ICP is the reduction of CSF production mediated by a decreased activity of Na+-K+ATPase  reduced CSF production, while a chronic intake resulted in ventriculomegaly, due to increased CSF production and potentially associated periventricular leukomalacia . Such anated CBF .3- and H+ from H2O and CO2. Although carbonic anhydrases are not directly involved in ion transport, they are believed to have an important role in CSF secretion on the basis of the effects of their inhibitors  in CSF production in animal models , also known as vasopressin, markedly decreased CSF production in rabbits  and sheeanalysis ,79. On tal edema .The role of endogenous vasopressin in CSF is still debated and is probably related to brain water regulation . It may CSF production is a complex and active process that involves a huge number of transporters, with insufficiently understood regulatory mechanism, which makes the management of idiopathic CSF overproduction a complicated issue. This extremely rare pathological condition could be related to the malfunction of ions or water transporters at ependymal and choroid plexus level or to an imbalance in the CSF production regulatory mechanism. Drug therapy has so far failed to control the CSF production, so that surgical and endoscopic options, in particular choroid plexus coagulation, should be considered. A better understanding of the molecular substrates of the CSF production could lead to the development of targeted drugs that could reduce CSF production and alleviate intracranial hypertension symptoms in hydrocephalic patients and potentially have a role in reducing brain edema related to ischemic and neoplastic lesions.