Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements.The induction of metalloprotease encoded by V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 μg/ml mucus (NSSM), 3M , or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer.Two strains of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm.The data demonstrate that protein extracts of  Vibrio anguillarum is the causative agent of vibriosis, one of the major bacterial diseases affecting fish, bivalves, and crustaceans [staceans -3. Vibristaceans . Vibriosstaceans ,4,5.Vibrio cholerae [Vibrio vulnificus [V. anguillarum strains NB10 and M93Sm have been cloned and sequenced by Milton et al [V. anguillarum shares significant sequence homology with other known proteases. Although the role of the EmpA metalloprotease is not completely understood, we showed previously that V. anguillarum empA mutants are either avirulent or attenuated for virulence in Atlantic salmon depending on the route of inoculation [Extracellular metalloproteases are important virulence factors for many pathogenic bacteria including cholerae ,7 and Vilnificus -10. The on et al  and Denkon et al , respectculation . It can Vibrio harveyi luxR (LuxRVh) homologue in V. anguillarum, vanT, was cloned, sequenced, and shown to be required for protease activity [Vh dependent regulation of luminescence in V. harveyi occurs at the promoter of the lux operon [Vh in V. harveyi is not similar to the LuxRVf protein in V. fischeri which also regulates luminescence [Vh binds to the lux box and activates the expression of the luciferase genes. Since LuxRVh functions as a transcriptional activator in V. harveyi and VanT is required for protease expression in V. anguillarum, we wanted to determine whether any proteins bind to the regulatory regions (e.g. the lux box-like element and promoter) immediately upstream of empA.Recently, the activity . LuxRVh x operon ,15. Whilnescence , it is tV. anguillarum cells grow rapidly in Atlantic salmon intestinal mucus [empA is strongly induced when cells are incubated at high density in mucus [empA transcription is RpoS-dependent [empA in V. anguillarum [lux box-like element about 70 bp upstream of the translational start of empA, which we subsequently showed to span the transcriptional start site [empA in two strains of V. anguillarum (M93Sm and NB10). Differences in empA expression were investigated with regards to various growth conditions, the presence or absence of DNA-binding proteins, and putative regulatory proteins.We have previously shown that al mucus ,18 and tin mucus . Furtherependent . Our finuillarum . Additiouillarum  previousart site . In thisempA, were examined using a digoxigenin (DIG) labeled oligomer containing the lux box-like element and empA promoter region. The location of double stranded oligomer relative to the promoter and coding sequences is shown in Fig. V. anguillarum M93Sm and NB10 cells incubated in LB20 and NSSM were mixed with the DIG-labeled oligomer  to protein extracts did not affect the gel mobility shift of the DIG-labeled oligomer caused by protein extracts from NB10 cells incubated either in LB20 or NSSM. In contrast to protein extracts from NB10 cells grown in LB20 or NSSM causing a gel shift, protein extracts from M93Sm cells incubated in either condition did not show any binding to the DIG-labeled oligomer  were incubated in NSSM for 3 h, a small amount of protease activity was detected (5 and 13% of the wild-type levels, respectively)  and their parental wild-types (M93Sm and NB10, respectively) were also examined for vanT transcription by RT-PCR  were examined for a empA promoter region binding protein. In all conditions, incubation with NB10 protein extracts resulted in a gel mobility shift to the DIG-labeled oligomer. In contrast, M93Sm extracts did not show any protein binding to the labeled DNA in any conditions tested. This was interesting because NB10 protease production occurs in all conditions, while M93Sm protease activity is detected only when the cells are incubated in mucus. Recently, VanT, the LuxRVh homolog in V. anguillarum, was identified by Milton et al [vanT mutants in both M93Sm and NB10 and tested them for vanT expression, protease activity, and DNA binding. These two mutants, M02 and NB02, did not produce detectable vanT mRNA. However, both exhibited low levels of protease activity in mucus - about 5% and 13% of wild type activity in mucus, respectively. For NB02, no protease activity in LB20 was observed. These results demonstrate that vanT is required for protease activity in a non-mucus containing medium (V. anguillarum NB10 in LB20), confirming the result reported by Milton et al [vanT mutants still exhibit some protease activity, but VanT is required for full protease activity in both M02 and NB02 cells.In this study, two strains of on et al  and showvanT mutant of V. anguillarum for the possibility of VanT binding to and activation of empA expression. Since the V. anguillarum NB10 strain expresses empA during stationary phase in all conditions tested and protein extracts from these conditions show DNA binding to the empA promoter region, it was hypothesized that VanT binds to this region. The vanT mutant, NB02, was analyzed for gel shift using protein extracts from cells incubated in LB20 and NSSM. Protein extracts from NB02 cells incubated in each condition still caused a band shift. This result suggests that VanT, the LuxRVh homolog in V. anguillarum, either does not bind to the empA promoter region containing the lux box-like element or perhaps VanT binds to a second protein that binds to this DNA sequence. The former possibility is more likely since the gel shift observed in NB02 is identical to that observed in NB10 extracts. Further, since the M93Sm vanT mutant, M02 still exhibits about 13% of the wild type protease activity in NSSM and does not exhibit protein binding in the gel shift analysis, it is probable that VanT does not bind to the empA promoter-lux box region. However, Croxatto et al. [V. anguillarum NB10 promoter regions for empA, vanOU, and vanT. Examination of their data shows that VanT binds more strongly to the promoters for vanOU and vanT (binding observed with 0.3 μg purified VanT per 2 ng DIG-labeled DNA) than to the empA promoter (binding observed with 2.5 μg VanT per 2 ng DIG-labeled DNA). It should be noted that in our study only 5 μg of total protein extract was used in the gel shift analysis. That binding to the empA promoter is still observed in vanT mutants strongly suggests that an unidentified protein binds to the promoter-lux box region.We tested a o et al.  demonstrempA in V. anguillarum M93Sm is positively regulated by at least two components of salmon gastrointestinal mucus [empA in both M93Sm and NB10 [rpoS mutants of M93Sm (M03) and NB10 (NB03) exhibit no protease activity when cells are incubated in either LB20 or mucus during stationary phase. Extracellular factors present in conditioned media showed both positive and negative effects on empA expression [luxS mutant, M01added to wild-type cells results in a more rapid induction of protease activity, which suggests that AI-2 may act as a negative regulator of empA.Previously, we have shown that al mucus . In thisand NB10 . This wapression . LB20 coempA expression in NB10 when incubated in LB20; however, when NB10 or M93Sm cells are incubated in NSSM, a small percentage of protease activity remains in the vanT mutants. Our data support the observation by Croxatto et al [empA expression. Further, the role of VanT as a transcriptional activator of empA via the lux box-like element has not been established. Our data show that VanT does not bind to the empA promoter lux box-like region, but is required for maximum protease activity. In addition, we show that in NB10 an unknown protein binds to the empA promoter-lux box region that may increase protease activity in all conditions. In contrast, M93Sm protein binding to this region is not observed and protease activity is detected only when cells are incubated in mucus. In conclusion, the expression of the empA virulence gene in V. anguillarum is regulated transcriptionally by the alternative σ-factor RpoS (σS) and the transcriptional activator VanT (a LuxRVh homologue). Additionally, an unknown DNA binding protein binds specifically to the empA promoter lux box-like region in NB10 cells, but not in M93Sm cells. We hypothesize that this protein permits empA expression in the absence of the inducing factors in fish gastrointestinal mucus. A better understanding of how and why these two wild-type strains of V. anguillarum exhibit different levels of empA expression will further reveal how virulence genes are regulated in this fish pathogen.In this study we confirmed that VanT is required for to et al  that VanempA expression between the two strains of V. anguillarum, M93Sm and NB10, correspond with the production of an unknown regulatory protein that binds to a 50 bp oligomer identical to the promoter-lux box region of empA. For NB10, protein binding to the oligomer correlated with the expression of empA in all incubation conditions. However, M93Sm, which expresses empA only in mucus, did not exhibit protein binding in this condition. Additionally, a null mutation in vanT (luxR homologue) results in severely decreased or no empA expression, but does not abolish protein binding (in NB10) to the oligomer. Our data suggest that while empA expression in both V. anguillarum M93Sm and NB10 is dependent upon RpoS and VanT, the two strains differ in production of an unknown protein that binds to the promoter-lux box-like region of empA. We also hypothesize that the presence of this DNA binding protein permits NB10 cells to express empA in the absence of fish gastrointestinal mucus.Differences in V. anguillarum strains were routinely grown in Luria-Bertani broth + 2% NaCl (LB20) [Escherichia coli SM10 was grown in LB10 (LB + 1% NaCl) on a rotary shaker at 37°C. Experimental media included: LB20, 3M  [V. anguillarum were grown in LB20, centrifuged (9,000 × g, 10 min), and pelleted cells washed twice with NSS [600). Antibiotics were used at the following concentrations: streptomycin (Sm), 200 μg/ml and chloramphenicol (Cm), 5 μg/ml.All bacterial strains and plasmids used in this report are listed in Table l (LB20) ,27 suppll media) ,28, ninel media) . Gastroil media) . Overnigwith NSS . Washed V. anguillarum M93Sm and NB10 from E. coli SM10 by conjugation using the procedure described by Milton et al. [V. anguillarum M93Sm or NB10 and E. coli SM10 containing the pNQ705-1 vanT clone, pNQVanT, were prepared and mixed using ratios of 1:1 or 3:1 (recipient: donor) in NSS plus 10 mM MgSO4. The cell suspension was vacuum filtered onto a 0.22 μm nylon membrane, which was placed on an LB15 (LB + 1.5% NaCl) agar plate and allowed to incubate overnight at 27°C. Following incubation, the cells were removed from the filter by vigorous mixing in NSS plus 10 mM MgSO4. The cell suspension (100 μl) was plated on LB20 Sm200 Cm5 (for M93Sm mutants) or on TCBS Cm5 (for NB10 mutants) and allowed to incubate at 27°C until V. anguillarum colonies were observed .Plasmids were introduced into n et al. ,29,30. BvanT. Primers (Table vanT sequence for V. anguillarum NB10 (accession # AF457643). A 320 bp region from vanT was PCR amplified using Qiagen Taq DNA polymerase and cloned into the suicide vector, pNQ705, using SacI and XbaI restriction endonucleases to yield pNQVanT. The presence of the 320 bp empA-derived insert was confirmed by both PCR amplification and restriction analysis using SacI and XbaI. The mobilizable suicide vector, pNQVanT, was transferred into V. anguillarum by conjugation with E. coli SM10. E. coli SM10 contains the λ pir protein that is required for replication of pNQ705. Chloramphenicol resistant colonies were selected and screened for insertion within vanT. PCR and Southern blot analysis were used to confirm the incorporation of pNQVanT. For PCR analysis, a primer described previously by Milton et al. [vanT-Forward (Table vanT rendering the mutants resistant to chloramphenicol at 5 μg/ml. The resulting V. anguillarum vanT mutants were designated M02 (derived from M93Sm) and NB02 (derived from NB10) . Briefly, the cells were pelleted from the experimental conditions by centrifugation at 9,000 × g for 10 min, 4°C. In addition, cells were lysed by multiple freeze-thaw cycles in crushed dry ice or in liquid nitrogen and then heated at 37°C. Insoluble cell debris was centrifuged at 12,000 × g for 10 min, 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad).empA promoter region and putative lux box. These oligomers were synthesized and purified by HPLC . The oligomers were annealed, labeled, and used in the gel shift reactions according to the manufacturer's instructions (Roche). An 8% native polyacrylamide gel in 0.25 × TBE (Tris-borate-EDTA buffer) was prepared and used for electrophoresis (in 0.5 × TBE) of the gel shift reactions. Blotting was performed using a Biorad electro-blotting system (model Trans blot) according to the manufacturer's instructions. Chemiluminescence detection of DIG-labeled DNA-protein complexes on the nylon membranes was detected using Hyperfilm ECL (Amersham Pharmacia). Gel mobility shift fluorograms were examined quantitatively using a densitometer . All gel mobility shift experiments were repeated three times and the data presented are representative examples of each experiment.The digoxigenin (DIG) gel shift kit for 3'-end labeling of oligonucleotides (Roche Applied Science, Indianapolis, IN) was used for protein-DNA binding assays. The 50 bp oligomers Table  used con3. Culture supernatant was prepared by centrifuging 1 ml of cells at 12,000 × g (10 min, 20°C). Supernatant was removed and filtered through a 0.22 μm pore size cellulose-acetate filter. Filtered supernatant (100 μl) was incubated at 30°C with 100 μl of azocasein solution. Incubation time of 30 min was sufficient for assays of supernatants from cell suspensions of ≥ 5 × 108 cells/ml. Reactions were terminated by addition of trichloroacetic acid (TCA) (10% wt/vol) to a final concentration of 6.7% (wt/vol). The mixture was allowed to stand for 1 to 2 min and centrifuged (12,000 × g, 4 min) to remove unreacted azocasein, and supernatant containing azopeptides was suspended in 700 μl of 525 mM NaOH [V. anguillarum M93Sm supernatant (100°C, 10 min). TCA  was added to the blank control supernatant immediately after the addition of azocasein. The mucus used was also boiled (10 min) to destroy any inherent protease activity. Protease activity units were calculated using the following equation: 1 protease activity unit = [1000 (OD442)/CFU] × (1 × 109)] [Culture supernatants were assayed for proteolytic activity using our previously described modification of the method by Windle and Kelleher ,32. Brie × 109)] .V. anguillarum cells as previously described [vanT-F2 and vanT-R2 . Sequencing was performed on a Beckman-Coulter CEQ 8000. The Dye Terminator Cycle Sequencing (DTCS) quick start kit was used for the sequence reactions that were prepared according to the manufacturer's specifications and run in a thermal cycling program. DNA samples were mixed with the appropriate primer Table  and thenS.M.D. carried out the experimental part of the study and drafted the manuscript. D.R.N. conceived of the study, participated in its design and coordination, and edited the manuscript. P.S. participated in the experiment design and assisted with gel shift analysis. All authors have read and approved the final manuscript.


The correct final concentrations for this buffer are as follows:After the publication of this work  it was bHEPES 50 mMNaCl 100 mMNaF 10 mMEDTA 5 mMNa3VO4 0.5 mMNEM 2 mMTriton 0.1%Complete protease inhibitors (Roche).We regret any inconvenience that this inaccuracy may have caused, and thank Dr. Bräuer for bringing it to our attention.


Women report fear of pain in childbirth and often lack complete information on analgesic options prior to labour. Preferences for pain relief should be discussed before labour begins. A woman's antepartum decision to use pain relief is likely influenced by her cultural background, friends, family, the media, literature and her antenatal caregivers. Pregnant women report that information about analgesia was most commonly derived from hearsay and least commonly from health professionals. Decision aids are emerging as a promising tool to assist practitioners and their patients in evidence-based decision making.Decision aids are designed to assist patients and their doctors in making informed decisions using information that is unbiased and based on high quality research evidence. Decision aids are non-directive in the sense that they do not aim to steer the user towards any one option, but rather to support decision making which is informed and consistent with personal values.We aim to evaluate the effectiveness of a Pain Relief for Labour decision aid, with and without an audio-component, compared to a pamphlet in a three-arm randomised controlled trial. Approximately 600 women expecting their first baby and planning a vaginal birth will be recruited for the trial.The primary outcomes of the study are decisional conflict (uncertainty about a course of action), knowledge, anxiety and satisfaction with decision-making and will be assessed using self-administered questionnaires. The decision aid is not intended to influence the type of analgesia used during labour, however we will monitor health service utilisation rates and maternal and perinatal outcomes. This study is funded by a competitive peer-reviewed grant from the Australian National Health and Medical Research Council (No. 253635).The Pain Relief for Labour decision aid was developed using the Ottawa Decision Support Framework and systematic reviews of the evidence about the benefits and risks of the non-pharmacological and pharmacological methods of pain relief for labour. It comprises a workbook and worksheet and has been developed in two forms – with and without an audio-component (compact disc). The format allows women to take the decision aid home and discuss it with their partner. Making evidence-based decisions in clinical practice is not always straightforward: patients and their healthcare providers may need to weigh up the evidence between several comparable options, the evidence for some treatments may be inconclusive, and the information needs to be tailored to each patient's clinical context and personal preferences ,2. Good To assist patients and their doctors in making informed decisions, information must be unbiased and based on current, high quality, quantitative research evidence. However, patient information materials are often outdated, inaccurate, omit relevant data, fail to give a balanced view and ignore uncertainties and scientific controversies ,5. It isDecision aids are "interventions designed to help people make specific and deliberative choices among options by providing (at minimum) information on the options and outcomes relevant to the person's health status" . AdditioInternationally decision aids have been evaluated in a variety of health and clinical settings. Although their use in pregnancy and birth has only just begun to be explored, this is an area in which consumers are known to want to participate actively in decision making . A surveThe pain of labour is a central part of women's experience of childbirth and is a constant feature of antenatal discussion groups . Most woHowever satisfaction with childbirth is not necessarily contingent upon the absence of pain . Many wo• Continuous caregiver support for a single individual should be available to women in labour• Midwives must involve women in decisions about analgesia and recognise the value of promoting personal control• Maternity services should ensure access to written and verbal information on pain relief and should support women in their choices for pain relief• Maternity services should respect women's wishes to have some control over their pain relief• Improved public information and data on pain and analgesiaIn Australia over 250,000 women give birth annually and the increasing use of epidural analgesia means some 75,000 women have an epidural in labour each year . Among pRandomised controlled trials have shown epidural analgesia provides the most efficacious pain relief for labour, but the adverse consequences include prolonged labour, restricted mobility, use of oxytocin augmentation and an increased incidence of instrumental delivery ,18. ConsAlthough not as effective as epidural, randomised trials show inhalational analgesia (e.g. 50% nitrous oxide in oxygen) and systemic opioid analgesics (e.g. pethidine) can provide modest benefit to some patients during labour or supplement an unsatisfactory epidural . Both thA number of women prefer to avoid pharmacological analgesia if possible . The wisWomen report fear of pain in childbirth and often lack complete information on analgesic options prior to labour . For exaDickerson stresses the importance of discussing preferences for pain relief before labour begins . A womanThe management of pain in labour is a clinical decision that fulfils Eddy's criteria for a decision in which patients' values and preferences should be included . The outDuring 2003 and 2004, we developed an evidence-based decision aid about the management of pain in labour for women having their first baby. This followed a needs assessment that collected data on the attitudes, preferences and knowledge of nulliparous women who were making plans about pain relief for labour and childbirth. The needs assessment found that women's knowledge of pain relief options was limited and these women would benefit from a decision aid for labour analgesia.In developing the decision aid we utilised the NHMRC guideline "How to prepare and present information for consumers of health services"  and the The decision aid was designed for women to use at home or in the clinical setting, and takes about 30 minutes to complete. After working through the decision aid, women should take the completed worksheet to their next antenatal appointment to discuss their preferences with their health care provider. The worksheet is also useful for the practitioner, who can see rapidly from it what evidence the patient has considered, what her values and preferences are and which way she is leaning in her preferences for analgesia during labour.The decision aid was developed, pilot tested and revised with extensive consumer involvement, as outlined in the NHMRC guideline on preparing information for consumers . The conA number of draft decision aids (including workbook, audio transcript, and worksheet), were developed and each subjected to pilot testing and revision as we obtained feedback. The process of testing and revising started with the study project group. The next phase included a review by a group of national and international content experts, including decision aid experts, obstetricians, midwives, perinatal epidemiologists, parent educators and psychologists. Once we were convinced that the content was accurate the decision aid was pilot-tested amongst consumers. There were several rounds of consumer review and refinement.Initially we aimed to compare the Decision Aid (workbook and audio-component) with usual care and counselling however preliminary work led us to alter our original study design. We could find no studies that compared Decision Aids with and without an audio-component. As the audio-component adds considerable complexity to the development and cost of the Decision Aid we decided to have two intervention arms: a Decision Aid with an audio-component and a Decision Aid without an audio-component. Further in pilot testing we found that women in the usual care arm were disappointed to not receive any information. Thus, to minimise refusals and losses to follow-up we decided to issue the women in the control group with a pamphlet called "Pain relief during childbirth – A guide for women" This pamphlet is published by the Royal Australian and New Zealand College of Obstetricians and Gynaecologists, is publicly available and includes information about methods of pain relief during labour . These cTo compare the relative effectiveness of the Pain Relief for Labour Decision Aid with a pamphlet on women's decisional conflict, knowledge, expectations, satisfaction with decision making and anxiety, and examine its impact on service utilisation and perinatal outcomes (as secondary outcomes).The primary study hypotheses are:Use of the Pain Relief for Labour Decision Aid by women expecting their first baby:1. Reduces decisional conflict (uncertainty about the course of action)2. Increases knowledge of labour analgesia3. Increases satisfaction with their decision making4. Reduces anxiety.The secondary hypotheses of the study are:Use of the Pain Relief for Labour Decision Aid by women expecting their first baby will not influence:1. The type of analgesia women use for labour2. Maternal and infant outcomes.We will conduct a randomised trial with the following study groups to assess the impact of the decision aid:Group 1: The pamphlet, "Pain relief during childbirth – A guide for women" Group 2: Decision aid with an audio-componentGroup 3: Decision aid without an audio-componentAn Australian tertiary obstetric hospital with a full range of non-drug and anaesthetic options for pain relief in labour. Epidurals are available 24 hours a day from anaesthetic staff designated to labour ward. All forms of antenatal care (clinic, birth centre, private, shared care with a family physician) will be included in the study.Primiparous women in late pregnancy (≥36 weeks gestation) who are expecting to have a vaginal birth of a single infant will be eligible for the study. Primiparous women were selected because previous pregnancies have a strong impact on decision making and analgesia use in labour ,16. ExclThe study procedure draws on the usual schedule of weekly antenatal visits in late pregnancy Figure . We planBrief baseline data will be collected to assess comparability of the study groups. The baseline assessment will include age, brief socio-demographic data, highest level of education achieved, anxiety as assessed by the state component of the short Spielberger anxiety scale , and infThe aim of the decision aid is to assist preference elicitation, and not to influence the direction of the decision taken. Women in each study group will be given the opportunity to review the intervention they are allocated (decision aid or pamphlet) while in the antenatal clinic and/or to take home, which ever is most convenient. Many women will also want to discuss their preferences with their partner. At the next antenatal visit, women will be contacted by the research nurse to discuss the information materials and any questions they may have had.All participants will be given a follow-up questionnaire prior to their next antenatal consultation. (See Outcome Measure details below).After a study participant delivers, the midwife who provided the labour care will complete a brief questionnaire to assess the impact of the decision aid on the management of labour analgesia. Information will also be collected on caregiver support in labour, birthplace (delivery suite or birth centre), use of non-drug analgesic options and stage of labour at admission.At 12–16 weeks postpartum all participants will be mailed a second follow-up questionnaire. This will assess women's satisfaction with the decisions made and the decision-making processes. (See Outcome Measures below). Questionnaires will be mailed with reply paid envelopes, with up to two reminder telephone prompts to non-responders.We will conduct in-depth interviews to explore the impact of the decision aid on women's experiences in labour and childbirth. A sub-sample of 30 women will be purposively selected, to reflect heterogeneity of experience of labour. The interviews will provide an understanding of the complexities of analgesic preferences, management, expectations, satisfaction, and psychological health following delivery. This data will enable examination of unpredicted and subtle effects of the decision aid on psychosocial outcomes that may not be captured using quantitative methods. Interviews will be face-to-face and conducted in women's homes or at a clinic, according to participants' preferences. Interviews will be recorded and transcribed. Data will be analysed using thematic analysis.As with many obstetric interventions blinding is virtually impossible. The main outcomes of this study are self-reported and the women are clearly not blinded to their treatment allocation. However, we will institute a number of measures aimed at keeping antenatal staff blind to the treatment allocation and preventing contamination of the control group:• Women will review the decision aid with the research nurse and complete the first questionnaire (primary outcome measures) prior to their next antenatal consultation• Usual antenatal care providers will be blinded to the exact content and format of the decision aid• Regular in-service  for the antenatal care providers to explain the trial protocol and to make clear the potential effect of unmasking or contamination.• Monitoring decision aid distribution and keeping them locked up and only accessible by the research nurse• Asking participants not to reveal their treatment allocation, or share their decision aid material with antenatal staff or other women. If participants do not want to keep their decision aid they will be asked to return it.The primary outcomes of this study will be:Decisional conflict (uncertainty about which preference to choose) will be assessed by the Decisional Conflict Scale which has established reliability, good psychometric properties and is short (16 items) [6 items) . It has is short 6 items [Measures of knowledge and realistic expectations about labour analgesia options and the benefits and risks of these options will be specific to this project. Thus we will need to develop, and test these measures as part of the project.Anxiety will be measured by the state component of the short Spielberger anxiety scale which has been extensively used and validated [alidated ,32. We dSatisfaction with analgesia decisions will be assessed using the Satisfaction with Decision Scale – a very brief six item scale with high reliability was developed specifically to assess satisfaction with health care decisions [ecisions .Satisfaction with the decision and anxiety will be measured again at 12–16 weeks postpartum. This interval was chosen to avoid the potential bias arising from questioning women still in the hospital who may feel a disloyalty to their caregivers by a critical appraisal and whose opinions have been shown to be more positive and short-lived than those obtained further out from the birth itself . At thatThe aim of the decision aid is to assist preference elicitation, and not to influence the direction of the decisions taken. Nevertheless, it is important to collect service utilisation and pregnancy outcome data so we will record and compare the pain relief methods used by women in all arms of the study, as well as recording and comparing rates of pregnancy complications and perinatal outcomes. The latter will be obtained (with informed consent) from the existing computerised obstetric database and include: medical or obstetric complications, induction or augmentation of labour, mode of delivery , enrolment to delivery interval, gestational age, birthweight, Apgar scores, perinatal deaths, Neonatal Intensive Care Unit admission and length of stay.The planned sample size is 600 women, with approximately 200 women to be recruited to each arm of the trial. Based on data for 2001 from the tertiary obstetric hospital where the study will be conducted, about 1500 primiparous women give birth to singleton infants after 36 weeks gestation and 92% use some form of analgesia. We anticipate that at least 50% of women will be both eligible and willing to participate.The sample size calculations for the trial (significance 0.05, power 0.8) are based on the mean difference in the decisional conflict scale between any two arms of the trial. The effect of decision aids on this scale is documented and effect size data are available . Meta-anApproximately 20% of primiparous women have a caesarean section (6% before labour and 14% after labour has commenced) . Some ofIf there are no significant differences in outcome for the two decision aid groups (with or without the audio-component), the decision aid groups will be pooled giving two women with the intervention for each woman in the pamphlet group thereby increasing the power to detect differences between the decision aid and the pamphlet.Analyses will be by intention to treat, including withdrawals and losses to follow-up firstly of all women randomised and then excluding women who lose their options for analgesia. Study groups will be compared in terms of baseline characteristics. As this is a randomised trial, we would anticipate minimal differences in baseline characteristics. If however, important differences are found, these potential confounders will be adjusted for in the analysis of outcomes. For the primary outcomes, the mean score for each measure for each group will be compared using t-tests. If adjustment for confounders is needed a multiple linear regression model will be used. The secondary outcomes will be compared using chi-squared tests of significance for categorical data and t-tests for continuous data. If adjustment for confounding is necessary logistic regression and multiple linear regression will be used respectively.This work involves the development of a decision aid for the management of pain in labour and childbirth. Women must decide between a range of non-pharmacological and pharmacologic methods of pain relief. However this decision must be made in the context of the likely analgesic effects of each option, the risk of complications and adverse obstetric effects, and maternal preference for relief of pain. There are currently no evidenced based materials available. We therefore expect this project to be beneficial for participating women. A systematic review of decision aids found they improved knowledge without increasing anxiety. Nevertheless we will measure anxiety levels at baseline and follow-up to document any adverse effects. A trained research nurse will interview all women and obtain written informed consent. Women will be encouraged to discuss any concerns/anxiety with the research nurse and/or with their usual antenatal care provider. Women will be reassured that they are able to withdraw from the study at any time with no adverse effects on their pregnancy management. Participation will require women to complete self-report questionnaires during and after pregnancy. Working through the decision aid will take approximately 30 minutes and review of their preferences or outstanding questions will be at a routine antenatal visit. Therefore we do not consider this to be an excessive burden on their time.The study has been approved by the Central Sydney Area Health Service Ethics Review Committee (Protocol no. X02-0247) and the University of Sydney Human Ethics Committee (Ref No. 3419). This project is funded by a nationally competitive peer-reviewed grant from the Australian National Health and Medical Research Council (No. 253635).Participants in the trial will be identified by a study number only, with a master code sheet linking names with numbers being held securely and separately from the study data. To ensure that all information is secure, data records will be kept in a secure location at the University of Sydney and accessible only to research staff. As soon as all follow-up is completed the data records will be de-identified. De-identified data will be used for the statistical analysis and all publications will include only aggregated data. The electronic version of the data will be maintained on a computer protected by password. All hard copy patient identifiable data and electronic backup files will be kept in locked cabinets, which are held in a locked room accessed only by security code and limited staff. Data files will be stored for seven years after completion of the project as recommended by the NHMRC. Disposal of identifiable information will be done through the use of designated bags and/or a shredding machine.The author(s) declare that they have no competing interests.CR, CRG, LT and KM were involved in the conception and design of the study. CR, NN and CRG were responsible for the drafting of the protocol. All authors have read and given final approval of the final manuscript.The pre-publication history for this paper can be accessed here:


Chaperones (CH) play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression.Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH.A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS.The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity. Drosophila busckii. Since then, efforts from a large number of investigators have shown that the heat shock response is ubiquitous and highly conserved. It is observed in all organisms from bacteria to plants and animals. CH form an essential defense mechanism for protection of cells from a variety of harmful conditions, including temperature elevation or heat shock, decrease in pH, hypersalinity, alcohols, heavy metals, oxidative stress, inhibitors of energy metabolism, fever or inflammation [Chaperones (CH) and heat shock proteins (HSPs) play important roles in tumor biology and still are holding centre stage. The heat shock response was discovered in 1962 by Ritossa [ammation . This brammation .Genetic studies showed that most HSPs are essential to life. They are believed to play an indispensable role in the conformational maturation of a nascent polypeptide chain in prokaryotic and eukaryotic cells. Traditionally, HSPs are grouped into five major families according to molecular weights. They were designated HSP90 (heat shock protein of apparent molecular weight 90 kDa), HSP70 (70-kDa HSPs), HSP60 (60-kDa HSPs), HSP40 or DnaJ (40-kDa HSPs), and the small heat shock proteins (sHSPs) -7.The connection of HSPs with tumor immunity was discovered in the 1980s . It was The contribution of HSPs to tumorigenesis may be attributed to their pleiotropic activities as molecular chaperones that provide the cancer cell with an opportunity to alter protein activities, in particular components of the cell cycle machinery, kinases and other proteins implicated in tumor progression. HSP70 chaperone activity may also influence tumorigenesis by regulating the activity of proteins that are involved in the cell cycle machinery .Clinically, in a number of cancers such as leukaemia, breast cancer and endometrial cancer, an increased level of HSP27, relative to its level in non-transformed cells has been detected . In addiMany studies have focused on the critical role of chaperones in protein folding, their relevance in protein conformational diseases and tumorigenesis. There is, however, no systematic information available on their expressional pattern in individual tumor cell lines in a wide range of tumors.This study addresses the question of differential chaperone expression studied in ten different tumor and three normal cell lines using 2-DE and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF/TOF) allowing concomitant determination of many CH at the protein chemical level rather than by immunochemical methods, independent of antibody availability and specificity.Chaperone proteins were taken from the list of all identified proteins from ten tumor and three normal cell lines using a predetermined list of expected CH based upon our own experiments, databases and literature. Identified CH proteins made up approx. 12% of all identified proteins in all cell lines studied. All major housekeeping proteins (cytoskeleton and metabolic) expected to occur in any cell lines were present in all cell lineages studied (data not shown).I values and molecular weights with theoretical value. The observed pI was represented in Table 2 , heat shock protein-beta (HSP90β) and endoplasmin (GRP94) . The twoAll cell lines except adenocarcinoma cells (CaOv-3) expressed endoplasmin (GRP94) protein that functions in the endoplasmic reticulum  and the Heat shock protein 75 kDa is named tumor necrosis factor type 1 receptor associated protein or TRAP1. The 2.4-kilobase TRAP-1 mRNA was variably expressed in skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta. TRAP-1 mRNA was also detected in each of eight different transformed cell lines . At the The HSP70 family constitutes the most conserved and best studied class of HSPs. Human cells contain several HSP70 family members including stress inducible HSP70, constitutively expressed HSC70, mitochondrial HSP75, and GRP78 localised in the endoplasmic reticulum . HSP70 hThe 105-kDa protein shows high similarity with HSP90 on peptide mapping with trypsin digestion. Except for the molecular mass, the physicochemical properties of the 105-kDa protein are similar to those of HSP90, although it has a HSP70 domain. This is detected in the brain, but not in the liver, lung, spleen, kidney, ovary, or uterus, in contrast to the wide distribution of HSP90 . In caseHSP70 kDa protein 1(HSP70-1) stabilises preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. HSP70-1 in mitochondria and endoplasmic reticulum plays an additional role by providing a driving force for protein translocation. They are involved in signal transduction pathways in cooperation with HSP90 and participate in all these processes through their ability to recognise non-native conformations of other proteins. Eight cell lines except SK-N-SH and CaOv-3 cells expressed HSP70-1.Heat shock 70 kDa protein 4 was observed in SK-N-SH, HL-60 and A-673 cells and is represented in Figure Stress-70 protein (GRP75) was proposed to be implicated in the control of cell proliferation and cellular aging  and was Heat shock-related 70 kDa protein 2 and 150 kDa oxygen-regulated protein were detected in SK-N-SH and MCF-7 cells.The best characterised GRP is a 78 kDa protein known as GRP78, which is identical to BiP, the immunoglobulin heavy chain binding protein . Since GProteins with a Cpn60_TCP1 domain are involved in chaperonins, which belong to the 55–64 kDa family of HSP or stress proteins . MammaliThe chaperonin-containing T-complex polypeptide has many subunits. Among these, alpha, beta, gamma, epsilon and zeta units as well as the isoform of epsilon subunit were shown in 2-DE gels of tumor cell lines. T-complex protein 1, zeta subunit was detected in all ten tumor cell lines.Among numerous co-chaperones for HSC70 , the DnaProtein disulfide isomerase, protein disulfide A3 and A6 containing 2 thioredoxin domains are members of the protein disulfide isomerase family and rearrange both intra-chain and inter-chain disulfide bonds in proteins to form the native structures. Protein disulfide isomerase was expressed in CaOv-3, A549 and HL-60 cells. Protein disulfide isomerase A3 was detected in nine cells except Hela cell and protein disulfide isomerase A6 was observed in HL-60, A-375, A-673 and Hela cell lines.Stress-induced-phosphoprotein 1 showing 9 TPR domains was expressed in all cell lines except Saos-2 cell. Hsc70-interacting protein with 3 TPR domains was detected in HL60 and A-375 cell. FK506-binding protein 4 has 3 TPR and 2FKBP_C domains. This protein was observed in SK-N-SH, A549, HL-60, A-375 and MCF-7 cells.Heat shock 27 kDa protein (HSP27) is involved in the stabilisation of cytoskeletal proteins and in the protective mechanisms against oxidative stress by abolishing the burst of intracellular reactive oxygen species (ROS) . HSP27 iPeptidyl-prolyl-cis-trans isomerase A contains a Pro_isomerase domain and accelerates folding of proteins. It was observed in SK-N-SH, A549, HL-60, A-375 and A-673 cell lines.ERP29_C has been recently characterised as a novel 29 kDa endoplasmic reticulum protein that is widely expressed in rat tissues. It plays an important role in the processing of secretory proteins within the ER . SK-N-SHPrefoldin subunit 2 has a KE2 domain but profoldin subunit 3 has a prefoldin domain. Two of them were expressed in Hela cells and profoldin subunit 2 was also detected in SK-H-SH cell. The protein DUF704 is an activator of 90 kDa heat shock protein ATPase homolog 1. This protein was observed in bone marrow neuroblastoma (SK-N-SH) and Hela cells.BAG-1 binds the ATPase domains of HSP70 and Hsc70, modulating their chaperone activity and functioning as a competitive antagonist of the co-chaperone Hip. The human BAG-1, BAG-2, and BAG-3 proteins bind with high affinity to the ATPase domain of Hsc70 and inhibit its chaperone activity. All these proteins contain a conserved 45-amino acid region near their C termini (the BAG domain) that binds Hsc70/HSP70, but they differ widely in their N-terminus . This prGrpE protein homolog1 with GrpE domain and HSPC015 with DcpS domain were observed in A-673 and HL-60 cell exclusively.Major differences between the ten tumor and the three normal cell lines were observed: 21 CH were observed in tumor cell lines only and vice versa, two CH were expressed in three normal cell lines exclusively see  and 2.The main outcome of the study is the generation of tumor cell specific patterns of chaperone and heat shock protein expression. The results form the basis for designing chaperone protein expression studies needed to evaluate the role of these structures in tumor biology by providing an analytical tool for the concomitant determination of CH, unambiguously identifying CH by a protein chemical rather than an immunochemical technique, independent of antibody availability and specificity.It must be mentioned that only high abundance CH have been detected by this method and that the generated individual maps show relative abundance proteins only. A series of relatively abundant CH were presented by more than one spot indicating the presence of splicing variants or posttranslational modifications including glycosylation, phosphorylation, methylation, oxidation, truncation, to name a few and the molecular diversity is currently subject of detailed studies using advanced proteomic tools as MS-MS sequencing and Q-TOF instrumentation. Carbamylation during sample preparation may have been contributing to electrophoretic shifts as wellMethodologically, identification of proteins by MS-MS is a sound approach and the major (inherent) problem with the interpretation may be at the cell culture level: the standard protocols for cultivating the individual cell lines according to the supplier were followed, but the conditions for the individual cell cultures vary.Different antibiotics or fungizides used may well lead to differences in CH expression and indeed, the use of geldanamycin has been already reported to induce heat shock expression in brain tissue . Other tBasically, differences between individual CH expression patterns may be due to different functional roles in individual cells and the presence of specific proteins in the individual cell lineages: there are CH to specifically protect individual proteins as e.g. specific chaperoning of tubulin beta 1 by the TCP complex  and of pThe main focuses in CH protein research maybe now to investigate detection of more CHs, probably by prefractionation into different compartments , to charTen different tumor cell lines were purchased from American Type Culture Collection (ATCC). The cell lines and their ATCC numbers are given in Table 3 see .SK-N-SH (bone marrow neuroblastoma) and Hela (cervix carcinoma) cell lines were grown in Minimum Essential Medium (Eagle) with 2 mM L-glutamine and Earle's Basic Salt Solution (BSS) adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate, with 10% fetal bovine serum (FBS). The same conditions were used to culture the MCF-7 cell line except for supplementing 10% FBS with 0.01 mg/ml bovine insulin. HCT 116  and Saos-2 (osteosarcoma) cell lines were cultured in McCoy's 5a medium with 90% 1.5 mM L-glutamine and 10% FBS. Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 10% FBS were used for the A549 (lung carcinoma) cell culture. HL-60 (acute promyelocytic leukaemia) cells were cultured with Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 80% 1.5 g/L sodium bicarbonate and 20% FBS. A-673 (rhabdomyosarcoma), Caov-3  and A-375  cell lines were cultured in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose with 10% FBS.Three normal cell lines, fibroblasts, lymphocytes and kidney cells were used:6 cells per ml in 96 well plates.Fibroblasts, Hs 545 SK ATCC CRL-7318 (Manassas, VA), were obtained from human skin and grown in monolayers in Dulbecco's modified Eagle's medium (DMEM) containing 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, with 10% fetal bovine serum (FBS), Penicillin and Streptomycin (GIBCO BRL) according to standard techniques Lymphocyte cell line 3610 is a spontaneously EBV transformed cell line from a patient with osteosarcoma and was obtained from the St. Anna Kinderspital-Forschungsinstitut (Vienna, Austria). The cell line was established from peripheral heparinized blood by a density gradient centrifugation using Ficoll-Paque  and grown in RPMI 1640 with 10% FBS, 70 μM gentamicin sulfate and 2 mM glutamine at a density of 2 × 10HK-2 cells (Human Kidney 2) are proximal tubular epithelial cells derived from normal kidney and were purchased from ATCC CRL-2190. Cells were cultured in Keratinocyte-Serum Free Medium (GIBCO-BRL 17005–042) with 5 ng/ml recombinant epidermal growth factor  and 0.05 mg/ml bovine pituitary extract.2 in air at 37°C and logarithmically growing cells were harvested by trypsinisation.All cell cultures were maintained in a humified atmosphere of 5% (v/v) COHarvested cells were washed three times with 10 mL PBS  (Gibco BRL, Gaithersburg, MD, USA) and centrifuged for 10 min at 800 g at room temperature. The supernatant was discarded and the pellet was suspended in 1 ml of sample buffer consisting of 40 mM Tris, 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO, USA), 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma, St. Louis, MO, USA), 65 mM 1,4-dithioerythritol (Merck, Germany), 1 mM EDTA (ethylenediaminetetraacetic acid) (Merck, Germany), protease inhibitors complete (Roche, Basel, Switzerland) and 1 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was sonicated for approximately 30 sec in an ice bath. After homogenisation samples were left at room temperature for 1 h and centrifuged at 14,000 rpm for 1 h. The supernatant was transferred into Ultrafree-4 centrifugal filter units (Millipore, Bedford, MA), for desalting and concentrating proteins. Protein content of the supernatant was quantified by the Bradford protein assay system . The staI values were used as given by the supplier of the immobilised pH gradient strips . Excess of dye was washed out from the gels with distilled water and gels were scanned with an ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop and Microsoft Power Point softwares.Samples prepared from each cell line were subjected to 2-DE as described elsewhere . 1 mg pr+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1–17 and clip 18–39). Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analysed by PMF from MALDI-TOF were additionally analysed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analysed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd, London, UK). Database searches, through Mascot, using combined PMF and MS/MS datasets were performed via BioTools 2.2 software (Bruker). A mass tolerance of 100 ppm and 1 missing cleavage site for PMF and MS/MS tolerance of 0.5 Da and 1 missing cleavage site for MS/MS search were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification.Spots visualised by Colloidal Coomassie Blue staining were excised with a spot picker , placed into 96-well microtiter plates and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure  ,47. BrieThe algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere .CH chaperoneHSP heat shock protein2-DE two dimensional gel electrophoresisMALDI-MS matrix-assisted laser desorption ionisation mass spectrometryOGP octyl-β-D-glucopyranosideCHCA α-cyano-4-hydroxy-cinnamic acidPMF peptide mass fingerprintGRP glucose regulated proteinTCP t-complex proteinThe author(s) declare that they have no competing interests.JKM did data mining and contributed to the preparation of the manuscript. LAS performed protein extraction, two-dimensional electrophoresis and data handling. MFC carried out MALDI-TOF-TOF analyses. IS developed methodology for studying proteins from cell lines. GL initiated and planned the study developing the concept, supervised 2-DE, mass spectrometry, creating data and the manuscript. All authors have read and approved the final manuscript.Table 1. Identified proteins in different human tumor cell lines: Saos-2, SK-N-SH, HCT 116, CaOv-3, A549, HL-60, A-375, A-673, MCF-7 and Hela.Click here for fileTable 1-1. Identified proteins in different normal cell lines: Kidney, Lymphocyte, FibroblastClick here for fileI, observed pI, total score and peptide matched of molecular chaperones in tumor cell linesTable 2. Theoretical molecular weight, theoretical pClick here for fileTable 3. The list of tumor cell linesClick here for file


Ischemia within the optic nerve head (ONH) may contribute to retinal ganglion cell (RGC) loss in primary open angle glaucoma (POAG). Ischemia has been reported to increase neurotrophin and high affinity Trk receptor expression by CNS neurons and glial cells. We have previously demonstrated neurotrophin and Trk expression within the lamina cribrosa (LC) region of the ONH. To determine if ischemia alters neurotrophin and Trk protein expression in cells from the human LC, cultured LC cells and ONH astrocytes were exposed to 48 hours of oxygen-glucose deprivation (OGD). Also cells were exposed to 48 hours of OGD followed by 24 hours of recovery in normal growth conditions. Cell number, neurotrophin and Trk receptor protein expression, neurotrophin secretion, and Trk receptor activation were examined.Cell number was estimated using an assay for cell metabolism following 24, 48 and 72 hours of OGD. A statistically significant decrease in LC and ONH astrocyte cell number did not occur until 72 hours of OGD, therefore cellular protein and conditioned media were collected at 48 hours OGD. Protein expression of NGF, BDNF and NT-3 by LC cells and ONH astrocytes increased following OGD, as did NGF secretion. Recovery from OGD increased BDNF protein expression in LC cells. In ONH astrocytes, recovery from OGD increased NGF protein expression, and decreased BDNF secretion. Trk A expression and activation in LC cells was increased following OGD while expression and activation of all other Trk receptors was decreased. A similar increase in Trk A expression and activation was observed in ONH astrocytes following recovery from OGD.In vitro conditions that mimic ischemia increase the expression and secretion of neurotrophins by cells from the ONH. Increased Trk A expression and activation in LC cells following OGD and in ONH astrocytes following recovery from OGD suggest autocrine/paracrine neurotrophin signaling could be a response to ONH ischemia in POAG. Also, the increase in NGF, BDNF and NT-3 protein expression and NGF secretion following OGD also suggest LC cells and ONH astrocytes may be a paracrine source of neurotrophins for RGCs. Glaucoma is an optic neuropathy defined by characteristic optic nerve head and associated visual field changes. Nearly 67 million people worldwide are believed to have glaucoma, including an estimated 2.2 million in the USA ,2. PrimaNeurotrophins are polypeptide growth factors involved in the development and maintenance of neurons, as well as non-neuronal cells. Included in this family of trophic factors are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4 (NT-4). Neurotrophin signaling occurs via two types of receptors including (a) tyrosine kinase high affinity Trk receptors and (b) the low affinity p75 receptor . The TrkIn addition to their localization at axon terminals, Trk receptors have been localized at neuronal cell bodies, at dendritic projections, and along axons -27. DiscThe administration of exogenous NTs has been shown to protect neurons from ischemic damage ,39 suggeIn POAG, the laminar plates of the LC are compressed and bow backward from the sclera producing an excavated and exaggerated optic cup . BecauseGiven that ischemia can be a component of ocular hypertension, we used oxygen-glucose deprivation (OGD) as an acute model of in vitro ischemia and examined the expression of NTs and their receptors by cultured LC cells and ONH astrocytes following OGD. We are aware that the model used (oxygen and glucose deprivation) is not a perfect model for what occurs in the glaucomatous optic nerve head. However, this acute model was an attempt to mimic the end results of a chronic condition, and as a "first step" we felt this model was adequate for examining the response to injury in these cell types.The LC and ONH astrocyte cell lines used in this study have been previously characterized and described . PrelimiTo determine an exposure of OGD that would result in cellular changes while allowing a majority of cells to remain viable, we examined cell number at various time points of OGD using an assay that estimates cell number based on cell metabolism. The oxygen level within the anoxic incubator was determined to be below detectable levels as measured using an Oxygen Test Kit (Bacharach Inc., Pittsburgh, PA). Lamina cribrosa and ONH astrocyte cell metabolism/cell number following OGD is shown in Figure The expression of NT protein in LC cells and ONH astrocytes following OGD is shown in Figures As seen in Figure Following 48 hours of OGD, ONH astrocytes Figure  demonstrFigure The expression of Trk receptor protein by ONH astrocytes following OGD and recovery from OGD is shown in Figure The expression of phosphorylated Trk receptor protein following OGD and recovery from OGD is shown in Figure Overall, exposure to OGD resulted in decreased phospho-Trk protein expression by LC cells and ONH astrocytes. Expression of the 148 kDa phospho-trk isoform was decreased to a statistically significant level in both LC cells and ONH astrocytes, as was the 120 kDa and 80 kDa isoforms in ONH astrocytes. The only increase in phospho-Trk receptor protein expression following OGD was observed in LC cells. Protein expression of the 120 kDa phospho-Trk receptor isoform was increased 60% over the controls in LC cells, which was statistically significant.Recovery from OGD resulted in increased protein expression of phospho-Trk receptor isoforms in LC cells and ONH astrocytes when compared to OGD alone, with the exception of the 120 kDa isoform in LC cells, which decreased toward control levels. Although its expression increased compared to OGD alone, expression of the 148 kDa isoform was still decreased to a significant level. Interestingly, the 120 kDa phospho-trk isoform was again the only isoform whose expression was increased above control levels. This 100% increase was observed in ONH astrocytes following recovery from OGD and was statistically significant.The secretion of NGF and BDNF following OGD and recovery from OGD is shown in Figure In this study we examined the protein expression of NTs and Trk receptors by LC cells and ONH astrocytes following OGD, an in vitro model of ischemia. Lamina cribrosa cell and ONH astrocyte responses to OGD and recovery from OGD are summarized in Tables Ischemia due to elevated IOP during POAG may cause changes within the ONH and contribute to RGC loss -11. As aNeurons, including RGCs, express Trk receptors not only at the axon terminal and cell body, but also along their axons -27 suggeThe expression of Trk receptors (both full length and truncated) by LC cells, ONH astrocytes or other cells within the LC could limit NT availability to RGCs -24. TherThere is evidence that reperfusion following ischemia is actually more detrimental to cells than ischemia itself ,46. To dIn conclusion, we have demonstrated that LC cells and ONH astrocytes increase the expression of NGF, BDNF and NT-3 protein following OGD, which may be neuroprotective for RGCs. Neurotrophins expressed by LC cells and ONH astrocytes following OGD or recovery from OGD bind and activate Trk receptors expressed by these cells. Increased NT signaling within LC cells and ONH astrocytes could increase cell survival following ischemic injury, but may compromise RGC survival during reperfusion. Further studies examining the expression of NTs and Trk receptors following hypoxia or transient ischemic insults would provide a better model for ischemic injury in POAG. Using this model, RGCs could be co-cultured with LC cells or ONH astrocytes to determine the neuroprotective effects of NTs during ischemic injury. By better understanding NT signaling within the LC under normal and injurious conditions, new strategies involving these factors could be developed to better treat patients with POAG.Lamina cribrosa cells and ONH astrocytes respond to conditions that mimic ONH ischemia by increasing NGF, BDNF and NT-3 protein expression and NGF secretion.Increased protein expression of Trk receptors and phosphorylated Trk receptors by LC cells and ONH astrocytes following OGD and recovery from OGD respectively suggest paracrine and/or autocrine NT signaling occurs within the ONH following ischemic injury.Lamina cribrosa cells and ONH astrocytes may be a paracrine source of NGF, BDNF and/or NT-3 for RGCs, especially during ischemic injury within the ONH throughout POAG progression.® Aqueous Non-Radioactive Cell Proliferation Assays and Emax™ ImmunoAssay Systems specific for NGF, BDNF, NT-3 and NT-4 were purchased from Promega Corporation, Madison, WI.DMEM and fetal bovine serum (FBS) were purchased from HyClone Labs, Logan, UT. The following materials were purchased from Gibco BRL Life Technologies, Grand Island, NY; glucose free DMEM, L-glutamine, penicillin/streptomycin and fungizone (amphotericin B). Costar 96-well plates and Nunc ELISA/EIA 96 well Maxisorp plates were purchased from Fisher Scientific, Pittsburgh, PA. Polyclonal antibodies to Trk A, Trk B, Trk C, truncated Trk B (Trk B.T) and phosphorylated Trk were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, CA. CellTiter 962/95% air at 37°C and media was changed every 2 to 3 days. Characterization of these cells was performed as described previously [Lamina cribrosa and ONH astrocyte cell lines were obtained from human LC explants from separate donors as described previously . Cells weviously . Cells eeviously ,33,36. Ceviously -36. Botheviously ,33,36. A2/5% CO2) for 48 hours. The oxygen level within the anoxic incubator was measured using a Oxygen Test Kit (Bacharach Inc., Pittsburgh, PA) and was determined to be below detectable levels. Recovery following OGD was achieved by placing cells in OGD conditions for 48 hours and then allowing them to recover in growth media (Ham's F-10 Media or DMEM plus 10% FBS) and an aerobic environment (95% air/5% CO2) for 24 hours. Cells cultured in growth media and an aerobic environment for served as controls.Preliminary studies examining cell survival following anoxia, hypoxia, hypoglycemia or serum withdrawl demonstrated LC cells and ONH astrocytes were resistant to hypoxia and serum withdrawl alone (data not shown). To approach what is occurring in vivo, we used the more acute oxygen-glucose deprivation (OGD) model to examine NT and trk expression and NT signaling in cells from the ONH. Preconfluent, age-matched adult LC cells and ONH astrocytes were treated with serum free media for 24 hours. Oxygen-glucose deprivation (OGD) was achieved by culturing LC cells and ONH astrocytes in glucose free serum free DMEM in an anoxic incubator (95% N2) for 24 hours. Cells cultured in growth media and an aerobic environment for served as controls.Adult LC cells and ONH astrocytes were trypsinized, counted using a hemacytometer and plated into Costar 96-well plates at a density of 1,000 cells/well. Cells were allowed to attach overnight and were then placed in serum free media for 24 hours. Lamina cribrosa cells and ONH astrocyte were exposed to OGD as described above for 24, 48 or 72 hours. Recovery following OGD was achieved by placing cells in OGD conditions for 24 or 48 hours and then allowing them to recover in growth media and an aerobic environment . Metabolically active cells convert MTS into formazan, which is soluble in aqueous solutions. The quantity of the formazan product measured by the amount of absorbance at 490 nm is therefore directly proportional to the number of living cells. Cell metabolism/cell number per well was calculated from a standard curve generated using known amounts of cells per well. A standard curve was generated for each cell line assayed. Three LC cell lines and three ONH astrocyte cell lines were assayed. The entire experiment, including standard curves, was repeated twice. Changes in cell metabolism/cell number following OGD and recovery from OGD were reported as a percent of the control ± SEM.Cell number was estimated using the CellTiter 96c Protein Assay System (Bio-Rad Laboratories, Richmond, CA). Cellular lysate (50 μg) was separated on denaturing polyacrylamide gels and then transferred by electrophoresis to nitrocellulose membranes. Blots were processed using primary antibodies and the Western Breeze Chemiluminescent Immunodetection System (Invitrogen, Carlsbad, CA). Blots were then exposed to Hyperfilm-ECL (Amersham, Arlington Heights, IL) for various times depending on the amount of target protein present. The density (O.D. × mm2) of unsaturated bands was measured using the Discovery Series scanner and the Diversity One program from pdi (Huntington, NY) and a digital Venturis FP466 computer (Compaq, Houston, TX). Western blots for NTs and trks were stripped and re-probed for β-actin to ensure equal loading.Cellular protein was collected in lysis buffer modified from Watson et al.  [20 mM TConditioned media was collected and concentrated using Millipore Centriplus YM-3 Centrifugal Filter Devices (Millipore Corporation, Bedford, MA). Emax™ ImmunoAssay Systems specific for NGF, BDNF, NT-3 and NT-4 (Promega) were performed according to manufacturer's instructions. Conditioned media was added to Nunc ELISA/EIA 96 well Maxisorp plates coated with anti-NT polyclonal antibodies. Secreted NT was detected by treating the plates with the respective NT monoclonal antibody followed by a horseradish peroxidase conjugated secondary antibody. Enzyme substrate was added to generate a color product whose absorbance was read at 450 nm. A NT standard included in each assay was used to generate a standard curve that was used to calculate the amount of secreted NT per well. The amount of secreted NT per sample was normalized to total protein per sample. Samples were assayed in triplicate. Conditioned media from three LC cell lines and three ONH astrocyte cell lines were assayed. Each immunoassay was repeated twice. Changes in NT secretion following OGD and recovery from OGD were reported as a percent of the control ± SEM.® statistical package, version 7.4.41 [Cell metabolism/cell number, western blot densitometry and immunoassay data were analyzed using one way analysis of variance (ANOVA) followed by validation using Student-Newman-Keuls tests. The MedCalcn 7.4.41  was usedn 7.4.41 .WL carried out tissue culture, cell proliferation assays, Western blotting, and immunoassays. WL, AC, and RW participated in design of the study, interpretation of the results, and in the writing and revision of the manuscript. All authors read and approved the final manuscript. This study is taken in part from a dissertation submitted to the UNT Health Science Center in partial fulfillment of the requirements for the degree Doctor of Philosophy for WL.


In particular, we compared the migration activity of mature Dendritic Cells (mDC) with that of immature Dendritic Cells (iDC) and also assessed intradermal versus subcutaneous administration.Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissues, where they have the task of priming naïve T-cells. In the present study 99mTc-HMPAO or 111In-Oxine, and the presence of labelled DC in regional lymph nodes was evaluated at pre-set times up to a maximum of 72 h after inoculation. Determinations were carried out in 8 patients .DC were labelled with It was verified that intradermal administration resulted in about a threefold higher migration to lymph nodes than subcutaneous administration, while mDC showed, on average, a six-to eightfold higher migration than iDC. The first DC were detected in lymph nodes 20–60 min after inoculation and the maximum concentration was reached after 48–72 h.in vivo provide preliminary basic information on DC with respect to their antitumor immunization activity. Further research is needed to optimize the therapeutic potential of vaccination with DC.These data obtained  These iDC possess functional characteristics typical of this maturation status, such as phagocytosis, macropinocytosis, receptor-mediated endocytosis and antigen processing [2 may considerably increase migration, inducing CCR7 expression on the surface of DC. Penetration may be limited to the peripheral zones of lymphoid tissue when the DC are still immature, or may reach the deeper T-cell zones, where a greater number of naïve T-cells are present, when DC are mature and activated.Dendritic Cell (DC) vaccination is one of the most promising tools of immunological therapy for cancer. Administration of DC, generated and loaded with tumor antigens olerance ,2. A larocessing ,4. Afterocessing . Followiocessing ,7. The mSurface antigen CCR7, present on the cell membrane of DC, strongly influences migratory capacity through its interaction with transporter molecules, TREM-2, LTC4, LTD4, etc. -10. The in vitro-generated vaccines have shown that mature, but not immature DC, induce an effective antitumor response [Recent studies on cancer patients evaluating the efficacy of response -18. Animresponse . Similarresponse -23.in vivo migration ability of DC by labelling them with 99mTc-HMPAO or 111In-Oxine. In particular, migratory activity was assessed in iDC and mDC in terms of time required for migration to lymph nodes, duration of activity, and number of cells that migrated. Migratory capacity was further evaluated by comparing subcutaneous and intradermal administration.In the course of a vaccination trial using DC pulsed with autologous tumor lysate (ATL) in cancer patients, we evaluated the In vivo migration was assessed using a part of the DC obtained for one of the therapy cycles. Three of the 8 patients were evaluated twice.The case series consisted of a subset of the 19 patients enrolled onto a phase I/II vaccination trial for advanced melanoma and renal cell carcinoma in which the first 9 patients were treated with iDC and the remaining 10 received mDC, both pulsed with autologous tumor lysate and keyhole limpet hemocyanin (Biosyn, Fellbach, Germany). In the present study 8 patients were analyzed  for a total of 11 treatments. Two melanoma patients were treated with iDC (one of whom twice), while 4 other patients with melanoma and 1 with renal cell carcinoma (treated twice) received mDC. The remaining melanoma patient was treated with iDC and subsequently with mDC.The clinical trial was approved by the Italian Ministry of Health and by the Ethical Committee of Forlì Health and Social Services . All patients gave written informed consent.g) and the supernatant was passed through a 0.2-μm filter. Protein contents were determined and aliquots were stored at -80°C until use.Tumor samples surgically removed from the patients were immediately placed in PBS. Adjacent non malignant tissue was removed by scalpel and tumor cells were dispersed to create a single-cell suspension. Cells were lysed by incubation in sterile distilled water. Lysis was monitored by light microscope. Larger particles were removed by centrifugation . From days 2–6, IL-2  was administered subcutaneously at a dose of 3 million IU/die. This procedure was repeated after two weeks and once a month until progression occurred.DC were prepared from peripheral blood monocytes (PBMC) obtained by leukapheresis without previous mobilization. 5–9 liters of blood were processed in each collection. PBMC were purified on Ficoll-Paque. An aliquot of PBMC was utilized immediately for DC generation and the rest was frozen in bags for use at a later date (4–5 bags/1 collection).6 cells/ml for 2 h. The non-adherent cells were discarded and the adherent cells were incubated in CellGro DC Medium containing 1000 IU/ml rhIL-4 (Cell Genix) and 1000 IU/ml rhGM-CSF  for 7 days to generate a DC-enriched cell population. On day 6 DC were pulsed with autologous tumor lysate (100 mg/ml) and with KLH (50 mg/ml) and incubated overnight. On day 7, they were defined as iDC. After eliminating the previous culture medium, pulsed iDC were cultured for a further 2 days with a cocktail of cytokines . On day 9 they were defined as mDC. iDC or mDc were removed, washed and suspended in sterile saline for therapeutic infusion into the patient.PBMC were incubated in tissue culture flasks with CellGro DC Medium (Cell Genix, Freiburg, Germany) at 10 × 106) were resuspended in platelet-poor autologous plasma (CFP1) and incubated for 15 min at room temperature with 99mTc-HMPAO (20 mCi)  111In-Oxine (1 mCi) . After two washes to eliminate the unbound isotope, the cells were resuspended in a total volume of 1.5 ml of CFP1. Radiolabelling of the DC and of the culture supernatant was evaluated with a gamma counter, after which DC were inoculated intradermally into the patient near healthy lymph nodes and in the contralateral zone not used for therapeutic vaccination . The patient then underwent serial acquisitions with gamma-camera positioned at the site of inoculation, with a field of view that included all the lymphatic regions of interest. The first acquisition was performed with a dynamic study of 20 min, followed by 10-min static acquisitions carried out every 30 min for the first 4–6 h and from 18 to 28 h. Other static determinations were made at 36, 48 and 72 h. The maximum duration of observation of DC migratory activity, which depended on the half-life of the radioisotope used, was 72 h for 111In-Oxine and 36 h for 99mTc-HMPAO.Labelling of DC was performed according to the methods described for leucocyte radiolabelling -26. A paThe identification of lymph node stations involved in the migratory activity was initially visual, after which we carried out a semiquantitative evaluation of the percentage of DC that migrated to lymph nodes from the inoculation site and an assessment of the speed of DC migration, expressed by activity/time curves obtained through the compartmental mathematical model.99mTc-HMPAO and the other was labelled with 111In-Oxine. The labelled cells were then suspended in CellGro DC Medium, divided into 4–5 culture flasks for each labelling molecule and incubated for 0 h, 4 h, 21 h, 24 h (99mTc-HMPAO) and 0 h, 4 h, 21 h, 24 h, 48 h (111In-Oxine). The DC from one flask were removed and centrifuged. The supernatant containing the free molecule, and the pellet containing the labelled cells, were then measured with a gamma counter.DC obtained from the culture of frozen PBMC were divided into two parts: one was labelled with 5 cells were suspended in 100 μl of buffer (PBS, 2% FCS, 1% sodium azide) and incubated for 30 min at 4°C with 10 μl of appropriate fluorescein isothiocyanate or phycoerythrin-labelled monoclonal antibodies (mAbs). The cells were then washed twice and resuspended in 500 μl of assay buffer. The fluorescence was analyzed by a FACS Vantage flow cytometer . mAbs specific for human CD1a, CD14, CD80, CD86, (Becton Dickinson) CD83 (Immunotech, Marseille, France) and CCR7  were used.iDC and mDC phenotypes were determined by single or two-color fluorescence analysis. 3–5·10At each pre-set time the supernatant was collected and stored at -80°C until analysis was carried out using commercially available ELISA Kit (Biosource, Nivelles, Belgium) to measure the production of IL-12 + p40 (bioactive heterodimer of IL-12) and IL-10 by DC.Single cell-based measurement of endocytosis was carried out as described 27). Dendritic cells were incubated for 30 min at 37°C with 0.5 mg/ml FITC-Dextran . DX-FITC (average MW 42,000) was centrifuged before use to remove aggregates. As negative control, cells were incubated with DX-FITC at 4°C. The cells were washed with cold PBS containing 2% FCS and 2 nM sodium azide to exclude dead cells and were then analyzed on a FACS Vantage flow cytometer (Becton Dickinson) [. DendritAll the patients  had advanced disease and all but one had undergone previous treatment. Median age was 49 years (range 46–52 years). Three patients were HLA-A1, 3 were HLA-A3, 1 was HLA-A2 and 1 was HLA-A11  are reported in Table in vitro stability of DC labelled with 99mTc-HMPAO and 111In-Oxine was evaluated using DC cultured from frozen PBMC. 99mTc-HMPAO-labelled DC showed a 75% loss of activity 4–24 h after the beginning of in vitro culture. 111In-Oxine-labelled DC showed a higher labelling stability (50%) that lasted for up to 24 h  was evaluated by comparing radioactive uptake in axillary lymph nodes. The intradermal route showed a threefold higher migration than that observed for the subcutaneous route Table . Evaluat111In-Oxine or 99mTc-HMPAO. A simple numerical analysis shows that the maximum uptake ratio between 99mTc-HMPAO-labelled mDC and iDC varies from 2 to 35, with an average of 8.4. The same ratio for 111In-Oxine-labelled cells varies from 4 to 7, with an average of 6. 99mTc-HMPAO labelling is influenced by the very low iDC uptake due to its greater binding instability and to the short half-life of the radioisotope, which does not permit the acquisition of reliable counts beyond 24–36 h.iDC and mDC migration was evaluated in all 8 patients (4 iDC and 7 mDC treatments) (Table 99mTc-HMPAO and 48–60 h with 111In-Oxine). A curve fitting analysis also seemed to indicate a progressive increase in uptake after the first 60 h, but the number of patients evaluated is too low for any definitive conclusions to be drawn.A lymph node uptake can be observed in all patients within the first two hours of inoculation, reaching a maximum uptake after 12 h in 7 patients Fig. ,5. In th99mTc-HMPAO alone in the same inoculation sites used for the study, no labelled hot spots were observed. This would seem to suggest that pure tracers move through lymphatic vessels without accumulating inside lymph nodes.After injection of DC-based immunotherapy has undergone a remarkable transformation in its development from basic research to clinical application ,28. Howe2 is essential for activating DC chemotaxis through the expression of CCR7 on the cell surface [2 may therefore prove to be important for increasing migration activity and DC efficacy. However, it has also been observed that PGE2 inhibits IL-12 production, resulting in a weaker in vivo activation of T-cells [in vivo experimentation.Recent published data have shown that the choice of maturation stimulus may be crucial for therapeutic success. In particular, it has been seen that PGE surface ,32. This T-cells ,34. Thes99mTc-HMPAO and 111In-Oxine) iDC and mDC [In the present study we aimed to clarify some issues concerning DC migration activity in a clinical vaccination trial utilizing radiolabelled ; it was observed that intradermal administration had a threefold higher migration to lymph nodes than the subcutaneous route. Although it remains to clarify the extent to which this migratory capacity is active or passive, it is clear that DC must be administered intradermally to obtain a higher migration.A crucial phase of the study was the comparison between the migratory activity of iDC  and that of mDC . The result was once again unequivocal, showing a greater progressive concentration of mDC that was, on average, six-eightfold higher than that of iDC, in accordance with data reported by other authors ,23 and i111In-Oxine-and 99mTc-HMPAO-labelled DC in succession and in the same site, to follow their migratory course.Notwithstanding the results obtained from the present study, many issues remain to be clarified. It has yet to be determined whether the increased activity detected in lymph nodes corresponds to an effectively greater migratory capacity or whether it is the result of a more effective adhesion capacity between surface molecules. Both hypotheses could even prove to be correct. We also do not know how long DC remain in lymph nodes. The increase in activity in lymph nodes is high in the first few determinations but tends to diminish or stabilize after around 36 h. It remains to be seen whether this presumed stabilization is the result of a sort of saturation or whether it can be attributed to the attainment of a dynamic equilibrium. The former hypothesis would indicate the need for an optimization of the number of DC to administer, as only a limited number would be functionally active. The latter would highlight the importance of the timing of administration and perhaps also the degree of DC maturation. To further investigate this, we plan to administer in vitro transiently stimulated DC (semimature DC). The therapeutic use of this type of DC, which have already begun the process of maturation and may be capable of reaching lymph nodes before their functional exhaustion, could increase the duration of their activation and stimulation. If these semimature DC prove to be equipped with a good migratory capacity, further improvement in the therapeutic use of DC may be possible.Finally, we aim to assess the migratory capacity of The migration activity of DC to regional lymph nodes is one of the many critical factors that influence the therapeutic result of antitumor vaccination. In the present study we used radioisotope-labelled DC and demonstrated that a better migration activity is obtained using intradermal than subcutaneous administration and that mDC show, on average, a six-to eightfold higher migration than iDC. Numerous other issues on DC functionality have yet to be clarified before antitumor therapeutic efficacy can be improved. The next important step will be to closely monitor the quantity and quality of responses observed in T-cells, and it is hoped that a consensus will be reached on standardized criteria for the definition and validation of clinical results obtained.DC, dendritic cell; iDC, immature dendritic cell; mDC, mature dendritic cell; ATL, autologous tumor lysate; PBMC, peripheral blood monocytes.RR and LR participated in the design of the study and were responsible for the clinical side of the study. AR, MP, LF and MS participated in the design of the study and were responsible for the biological part of the study. GM performed the apheresis collections. RG, AM and GF carried out DC labelling and migration evaluation. GG performed the mathematical and statistical analysis. All authors read and approved the final manuscript.None declared.