During endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes.Using a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition.Our results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate. During skeletal development and growth, bone formation occurs either by intramembraneous or endochondral bone formation. In endochondral bone formation, which occurs at the growth plates of long bones, cartilage is formed first, then the chondrocytes undergo a proliferative phase followed by hypertrophy, changes in gene expression, and matrix calcification, after which the cartilage is replaced by bone. Although generally referred to as chondrocyte hypertrophy, cell enlargement is just one manifestation of the more complex process of chondrocyte maturation, which can be considered an end-stage of chondrocyte differentiation. It is important to define the mechanisms that induce chondrocyte maturation, not only to understand bone development, but also to help prevent hypertrophy and ossification during cartilage tissue engineering.Hypertrophic chondrocytes are characterized by their increased levels of alkaline phosphatase (ALP), reduced levels of type II and IX collagens, and the emergence of type X collagen (Col X), which is a specific marker of hypertrophy ,2. AscorIn osteoblastic cells, BMPs and ascorbate have been shown to operate via mechanisms that at least partly involve mitogen activated protein kinases (MAP kinases). For example, ascorbate promotes extracellular matrix production which, in turn, activates the extracellular-signal regulated kinases, (ERK1/2 or p42/ p44) in an osteoblastic cell line . MAP kinIn general, MAP kinase pathways involving ERK1/2 stimulate proliferation, growth and differentiation, whereas those that stimulate p38 kinase lead to differentiation and apoptosis . In earlChick sternal chondrocytes are a popular model for the study of chondrocyte maturation because under normal development chondrocytes from the cephalic portion of the sternum undergo hypertrophy followed by mineralization and bone formation, whereas the caudal portion remains as cartilage ,8. In thStudies with the ERK1/2 inhibitor U0126 indicated that blocking ERK1/2 signaling increased the activity of the type X collagen promoter but had no effect on alkaline phosphatase (ALP) activity in chick cephalic sternal chondrocytes Fig. . In contALP activity was highly variable between cell isolates and is expressed here normalized to BMP-2 treated controls for the purpose of combining experiments, typical ALP values ranged from approximately 0.5–2 nmol/min/μg DNA in controls to between 4 and 12 nmol/min/μg DNA in BMP-2 treated cultures.The effects of altered ERK1/2 signaling on Col X promoter activity in chick sternal chondrocytes was further studied both by transfection with mutant kinases and by treatment with additional kinase inhibitors. Col X promoter activity was increased, both in the presence and absence of BMP-2, when the mitogen-stimulated ERK pathway was suppressed by transfecting chondrocytes with dominant negative ERK-2 Figs. . ConversAs seen with the ERK1/2 inhibitor U0126, treatment with the more specific ERK1/2 inhibitor PD098059 increased b2/640 Col X promoter activity, in the presence of BMP-2 Fig. . Dose reTransfection with dominant negative p38 caused a decrease in Col X promoter activity in BMP-2 treated cephalic chondrocytes, reducing activity to half of that seen in BMP-2 treated controls (p < 0.005) and eliminating the BMP-2 response Figs.  and 2B. Addition of either PI3 kinase inhibitor or PKC inhibitor resulted in similar stimulation of the collagen type X promoter. Calphostin C, a PKC inhibitor, increased activity in BMP-2 treated cells more than 2-fold, an effect similar to that seen with the ERK1/2 inhibitor PD98059 at 10 μM Fig. . SimilarTo assess the possible effects of protein kinase inhibitors on cell proliferation and survival, we measured relative numbers of live cells using a tetrazolium (MTS) assay. The results indicated that all cultures treated with inhibitors, with and without BMP-2 and/or ascorbate, had cell numbers within 10% of untreated controls (data not shown).We examined the effect of 75 μM ascorbate-2-phosphate on the activity the Col X promoter in cultures treated with kinase inhibitors. Col X promoter activity was unaffected by addition of ascorbate, and 4 μM of the ERK1/2 inhibitor U0126 increased promoter activity to comparable levels both with and without ascorbate Fig. .ALP activity in the absence of exogenous BMP was stimulated at least 2-fold in ascorbate-treated cultures without inhibitors, as previously reported, and this stimulation was not significantly affected by addition of either ERK1/2 or p38 inhibitors Fig. . In cultThe present studies demonstrate that ERK1/2 inhibition increases activity of the BMP responsive region of the type X collagen promoter. This indicates that ERK1/2 signaling interferes with the ability of BMP-induced signals to stimulate type X collagen transcription. Interestingly ERK1/2 has also been shown to inhibit type I collagen expression in an osteoblastic cell line  suggestiIn contrast to the stimulatory effects of inhibiting the ERK pathway, p38 inhibition blocked BMP-stimulated Col X promoter activity. Zhen et al.  and BeieWhile the classical pathway for BMP signaling is via activation of R-Smads, there is also evidence for BMP signaling via a TGF-activated kinase (TAK1) leading to p38 signaling -26. HoweInhibiting either protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3 kinase) increased type X collagen promoter activity both in BMP-2 treated cultures and controls. Both PKC and PI3 kinase have been reported to negatively regulate p38  and posiAlthough alkaline phosphatase expression, like type X collagen expression, increases during chondrocyte hypertrophy and is stimulated by BMPs, its regulation clearly differs from Col X in several respects. ERK1/2 inhibition has little effect on the ALP activity induced by BMP-2 or ascorbate acting alone and reduces the ability of BMP-2 to further stimulate ALP activity in ascorbate treated cultures. Inhibiting p38 does not have a clear effect on BMP-stimulated alkaline phosphatase activity in this model although it has been shown to reduce ALP activity in long-term (12–15 day) micromass cultures .Preliminary studies with cyclohexamide indicate that new protein synthesis is required for the up-regulation of alkaline phosphatase mRNA in response to BMP-2. We propose that these differences reflect a direct Smad-mediated effect of BMPs on type X collagen expression and an indirect effect on ALP expression.A mechanism which could account for the observed effects of ERK and p38 signaling on expression of type X collagen and ALP is presented in Fig. Evidence that BMP stimulation of type X collagen requires both activated R-Smads and Runx2 has been previously reported ,7. LittlAs well as its demonstrated role in Col X expression as found here and by ,31, therRetinoic acid ,8, anothElucidating the signaling pathways by which chondrocytes are driven to hypertrophy is necessary in order to better understand skeletal development, cartilage disease and improve the design of tissue engineered cartilage. We showed here that the ERK1/2 pathway inhibits type X collagen production by either directly or indirectly acting at the BMP responsive region of the promoter. p38 kinase signaling stimulates type X collagen transcription at the same promoter region, probably in conjunction with BMP-2 activated Smads. The factor upstream of p38 in this stimulatory pathway is unknown. Alkaline phosphatase activity is likely to be regulated in a different way from type X collagen since MAP kinases do not contribute in the same way to this pathway. Although ascorbate and BMPs both induce hypertrophy in chondrocytes ascorbate does not act at the same region of the Col X promoter as BMPs.The ERK1/2 inhibitor PD98059, which blocks the upstream kinase (MEK1) of ERK1/2, the p38 inhibitor SB203580, and the PKC inhibitor Calphostin C were obtained from Sigma (St. Louis, MO). UO126, also a MEK1 inhibitor, was obtained from Biomol (Plymouth Meeting, PA) and LY294002, a phosphatidylinositol 3- (PI3) kinase inhibitor from Cell Signaling Technology (Beverly, MA). Plasmids were kindly donated as follows: constitutively active MEK1 from Michael Webber (University of Virginia); dominant negative p38  from RogChondrocytes were cultured as previously described . Cephalirenilla luciferase reporter (0.4 μg/well) (Promega) which served as a transfection control [4 precipitation or Fugene transfection reagent at 6 μl/ml (Promega). Since preliminary experiments using green fluorescent protein showed that Fugene was more effective in terms of numbers of cells transfected, this method was used for the majority of the experiments; however, relative outcomes between controls and treated cells were not affected by the transfection method.On day 1 of secondary culture (24 hrs after plating) the cell layer was washed in HBSS and the media changed to DMEM supplemented with 10% FBS in place of NuSerum. Cells were co-transfected with pGL2 plasmid containing the b2/640 type X collagen promoter region attached to a firefly luciferase reporter (0.2–1 μg/well) (Promega, Madison, WI) and pRL null plasmid attached to a  control . When apTransfection proceeded for 5 hrs after which the cell layer was rinsed twice in HBSS and cultured with serum free medium . Some wells were supplemented with 75 μM ascorbate-2-phosphate (Wako, Takara, Japan) or 30 ng/ml human recombinant BMP-2 (Wyeth, Cambridge, MA). Where inhibitors were used they were added at this point and cells incubated for 1 hr before the addition of BMP-2. Cells were cultured for a further 48 hours, then lysed and assayed using a dual luciferase assay kit (Promega).2, 0.1 mM MgCl2 and 1% Triton X-100 for 30 mins at 37°C, followed by overnight storage at 4°C. A sample of the cell lysate was reacted with p-nitrophenyl phosphate substrate in 1.5 M Tris buffer pH 9 with 1 mM ZnCl2 and 1 mM MgCl2. Phosphatase activity was measured specrophotometrically at 410 nm with 1 absorbance unit equivalent to 64 nmol of product. For DNA analysis, cells were trypsinized and a subsample of cell suspension centrifuged, the cell pellet lysed with the CyQUANT lysis buffer and the fluorescent DNA dye added. The resulting solution was transferred to a 96 well plate and DNA assayed fluorometrically. The remaining cells were extracted for the alkaline phosphatase assay as above. Alkaline phosphatase enzyme levels were calculated as nmol p-nitrophenol product per minute normalized to MTS units or μg DNA.For alkaline phosphatase assays, cells were switched to serum free medium on day 1 of secondary culture, inhibitors were added and cells incubated for 1 hr before the addition of ascorbate or BMP-2, as described for the luciferase assay. Cells were cultured for a further 72 hrs and then rinsed twice in HBSS. Cells numbers were assayed either by DNA quantification (CyQUANT cell proliferation assay kit, Molecular Probes, Eugene, OR) or by MTS tetrazolium salt assay of mitochondrial activity (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega). When MTS was used, a 1:10 dilution of MTS was applied in phenol red-free media for 30–60 minutes, 200 μl of media plus MTS was transferred to a 96 well plate and assayed in a 'Multiskan ascent' plate reader (Thermolabsystems, Franklin, MA). The cell layer was then washed twice in HBSS and extracted with 0.15 M Tris, pH 9 with 0.1 mM ZnClStatistics were performed using Minitab™ software. After expressing results as a ratio of experimental/control within each experiment, the data from at least 3 experiments were combined. The combined data was tested for normality using the Anderson-Darling test. Normally distributed or non-parametric data were tested for differences between treatments using two-sample Students t-test or the Mann-Whitney test respectively. Where groups of experiment means were compared a paired t-test was used.ALP – Alkaline PhosphataseBMP – Bone Morphogenetic Proteinbp – base pairsCA – constitutively activecbfa – core binding factor alpha 1Col X – Collagen type XDN – dominant negativeERK – extracellular signal regulated protein kinaseMAP – Mitogen activated proteinMEK – ERK kinaseMTS – 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner saltPI3 – phosphhatidylinositol 3PKC – Protein Kinase CRunx – Runt associated proteinTAK – Transforming growth factor beta activated kinaseThe author(s) declare that they have no competing interests.GCR was involved in experimental design, performed 90% of the experiments, analyzed the data and wrote the first draft of the manuscript. EBG isolated chondrocytes, performed preliminary experiments and contributed to trouble-shooting of the methods. GG-K, performed preliminary experiments and developed methodology. PSL was involved in experimental design and data analysis, wrote portions of and edited the whole manuscript and was the project supervisor.


The exponential development of Patient-Reported Outcomes (PRO) measures in clinical research has led to the creation of the Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID) to facilitate the selection process of PRO measures in clinical research. The project was initiated by Mapi Research Trust in Lyon, France. Initially called QOLID , the project's purpose was to provide all those involved in health care evaluation with a comprehensive and unique source of information on PRO and HRQOL measures available through the Internet.. The first level is free of charge and contains 14 categories of basic useful information on the instruments . The second level provides significantly more information about the instruments. It includes review copies of over 350 original instruments, 120 user manuals and 350 translations. Most are available in PDF format. This level is only accessible to annual subscribers. PROQOLID is updated in close collaboration with the instruments' authors on a regular basis. Fifty or more new instruments are added to the database annually.PROQOLID currently describes more than 470 PRO instruments in a structured format. It is available in two levels, non-subscribers and subscribers, at Today, all of the major pharmaceutical companies, prestigious institutions , dozens of universities, public institutions and researchers subscribe to PROQOLID on a yearly basis. More than 800 users per day routinely visit the database. In clinical research it has become increasingly common to assess the patients' perspective of their symptoms and their impact on their daily life as a tool for determining treatment and a means of evaluating the outcome of the treatment chosen ,2. The aHowever the successful application of PRO studies is dependant on the selection of the appropriate questionnaires for a given application ,8. They The search for the most appropriate instruments is hindered by the substantial increase of PRO questionnaires developed in the past ten years. A recent search on PubMed, matching "quality of life" and "questionnaires" shows a striking growth of 450% between the last two decades Figure .In order to facilitate the selection process the project of a Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID) was initiated by Mapi Research Trust in Lyon, France. Initially called QOLID , the project's purpose was to provide all those involved in health care evaluation with a comprehensive and unique source of information on PRO and Health-Related Quality of Life (HRQOL) measures available through the Internet. In collaboration with Dr. Marcello Tamburini , the developer of the QLMed.org web site, PROQOLID was launched at the beginning of 2002..PROQOLID was created by the systematic collection of over 470 validated HRQOL and PRO instruments and their subsequent ordering into categories (e.g. pathologies, conditions, population). Through the structured presentation of synthesized, reliable and constantly updated data on PRO instruments, the PROQOLID database aims to present an overview of existing PRO instruments including all relevant and updated information on each. By providing this information the PROQOLID database facilitates access to the instruments and their developers and eases the process of selecting the instrument appropriate for a given application. Instruments can be chosen through a powerful interactive search function that will allow for the rapid selection of an appropriate instrument. What follows is an overview of the proper usage of the PROQOLID web site through a series of FAQ's collected directly from users of the web site. The PROQOLID database can be accessed on the Internet at The access to PROQOLID is organized in two levels.• Full name of the instrument and acronym• Author(s)• Objective• Pathology• Disease• Type of instrument: Coping, Disability/physical functioning, Health status, Psychosocial/psychological, Quality of life, Satisfaction, Social functioning, Symptom/functioning, Utility, Work• Population: Adolescent, Adult, All, Caregivers, Female, Geriatrics, Male, Pediatrics, Terminal patients• Mode of administration: Caregiver-administered, Interviewer-administered, Nurse-rated, Physician-rated, Proxy-administered, Self-administered, Telephone-administered• Number of items• Original language• List of existing translations• Existence of a database: Yes / No• Time recallSince 1995, Mapi Research Trust has been a non-profit organization promoting the use and development of Patient Reported Outcomes. In an effort to accomplish this, a significant part of PROQOLID has been made accessible to all users free of charge.In order to further develop and improve the database and provide additional information on the available instruments, Mapi Research Trust requests a financial participation in order to access PROQOLID'S advanced (or members') level. By subscribing to PROQOLID, you are supporting the continuous collection and update of this unique PRO resource. Membership options are available for pharmaceutical or commercial companies, non-profit organizations, universities, individual academic researchers and students. Benefits to members include a greater degree of practical information on the instruments and, when available, includes the review copy of the instrument, its translations and the user manual, most of them in PDF format.• Name of the instrument (full and abbreviated)• Name and contact information of the Authors• Contact person for information on, or permission to use, the instrument in its original language• Copyright information• Detailed conditions of use (e.g. fee, written permission, user agreement etc.)• Review copy of the original instrument (when possible). Depending on the author's wish, original instruments may be used under specific conditions such as an access fee or signed agreement• Bibliographic references of the original instrument• Contact person for information on, or permission to use the translations• Review copy of the available translations (when possible)• Bibliographic references of the available translations (when possible)• Dimensions covered by the instrument• Time for completion• Age range• Scoring: response options, available scores, weighting, score direction and Minimal Important Difference (MID) or Minimal Clinically Important Difference (MCID)• Existence of a user manual and copy of the user manual (when possible)• Link to the PRO database identification form, when available• Methodology of development• Internal consistency reliability• Related websites• Other bibliographic referencesThe web site is available to anyone having an interest in the development, availability and use of Patient-Reported Outcomes (PRO). Through the power of the Internet the PROQOLID project intends to provide this information to the world. Every major pharmaceutical company, non-profit organizations such as the US Food and Drug Administration, the NIH's National Cancer Institute, the Veterans Administration as well as dozens of Universities, researchers and students worldwide subscribe to the advanced level of PROQOLID on a yearly basis. The PROQOLID database is routinely visited by over 800 users per day, thereby educating clinicians, researchers, students, and the world about the availability and proper usage of PRO instruments.The PROQOLID database was created in an effort to provide a means to facilitate the search process for and provide more efficient searches of any given PRO instrument. By organizing instruments in the PROQOLID database by several easy to understand categories, both time and energy are saved by the user. The different categories can be located on the Search page of the web site or by accessing directly from the tool bar at the top of the page. The different categories are as follows:The purpose of the Alphabetical list is to provide an overview of all existing PRO instruments. Over 1000 instruments are listed in alphabetical order according to their abbreviated name (or acronym). Some of the instruments are only listed, and these are displayed in standard font. For the remaining instruments access is available by simply clicking on the green link containing the abbreviated name of the instrument. Instruments can be accessed through an interactive letterbox at the top of the page. For example if the instrument begins with "D", simply click on the "D" at the top of the page and all instruments beginning with that letter will be displayed alphabetically.The generic instruments are listed by alphabetic order on a separate web page.A specific web page is dedicated to each pathology, and the instruments are listed either as generic instruments of the pathology or as disease-specific. The classification is structured based on Medline's Medical Subject Headings (MeSH) to ensure that the concepts are widely accepted. Please note that some diseases may be part of several pathologies. For example the disease "dementia" is part of both the pathologies "Neurology" and "Psychiatry/Psychology".The web page lists the instruments as they apply to specific populations including Adolescent, Adult, All, Caregivers, Female, Geriatrics, Male, Pediatrics, and Terminal patients.Instruments are grouped alphabetically according to the author's name, and as in the alphabetical list a letterbox is provided at the top of the page.You may search for instruments according to 10 criteria, including the name of the instrument, the pathology, the population or the available languages. The various criteria may be crossed referenced using the following Boolean Operators: AND, OR, NOT.The PROQOLID database was developed and is updated in close collaboration with the instruments' developers. It currently describes over 470 PRO instruments according to a structured format. The list is currently growing at the rate of fifty instruments per year. The database also includes review copies of 350 original instruments and 350 translations, most of them in PDF format. In order to determine the available translations simply access the instrument on the web site and all existing translations are conveniently listed. Also available through PROQOLID are over 125 associated User Manuals, and the description of 80 separate PRO databases. A fifth update of the whole database is underway and will include new information for each questionnaire on the reproducibility (or test-retest reliability) and clinical validity. In addition the PROQOLID website contains links to 150 external Internet resources relevant to the field of PRO usage and development. A general question and answer section on PRO is also included as well as on line full text articles on the development, validation, and linguistic adaptation of many of the PRO instruments published in medical journals.To be eligible for inclusion in the database an instrument must be the subject of a publication that describes its development and/or validation. There is no charge to authors who wish to insert their instruments in PROQOLID nor are authors paid for their participation in this program. The Mapi Research Trust in Lyon, France determines ultimate decision for inclusion.Besides the search by categories of instruments listed above , an interactive search engine is included in the PROQOLID web site. All of the instruments contained on the web site can be accessed from this location. Searches can be made by:• Abbreviated Name• Full Name• Author• Pathology• Disease• Type of Instrument• Population• Mode of Administration• Inclusion of a PRO Database• LanguageIn an effort to increase the effectiveness of each search the ability exists to include up to nine (9) sub-parameters per search with the stipulation of "AND" or "OR". For example one could enter Author "=" Anderson J "OR" Author "=" Anderson R "AND" Type of Instrument "=" Quality of Life, "AND" Language "=" French, or any other combination that would suit the users needs. Through this function a user is able to drastically narrow the number of instruments displayed in the results window, thereby saving time and effort. If questions exist on the functioning of the search engine, or any questions about the PROQOLID database a contact page is provided with names and e-mail addresses for Mapi Research Trust and individual site managers in both Europe and North America. Additionally a video including a demo of PROQOLID can be seen on the home page.Mapi Research Trust has maintained the PROQOLID database for three years. The information contained in the website for each instrument is updated at least once a year in collaboration with the instruments' developers and over fifty new instruments are added to the website each year. Mapi Research Trust has listened to the needs of the Pharmaceutical Industry, Industry Regulators, health care professionals, and patients. With the passing of time the organisation has developed into an intricate team of professionals whose single goal is to define and unite the various requirements of each of these groups in order to provide better communication and understanding of each groups needs. PROQOLID achieves to translate these objectives into a concrete application which ultimate goal is the improvement of the patients' quality of life and health outcomes..The Patient-Reported Outcome and Quality of Life Instruments (PROQOLID) database aims to present an overview of existing PRO instruments. PROQOLID currently describes more than 470 PRO instruments in a structured format. It includes review copies of over 350 original instruments, 120 user manuals and 350 translations. Most are available in PDF format. The database is updated in close collaboration with the instruments' authors on a regular basis. Fifty or more new instruments are added annually. By providing this information the PROQOLID database facilitates access to the instruments and their developers and eases the process of selecting the most appropriate instrument for a given application. Instruments can be chosen through a powerful interactive search function. The PROQOLID database can be accessed on the Internet at MPE conceived the database and participated in its implementation and helped to draft the manuscript. CA helped in the conception of the database and drafted the manuscript. LLP has contributed in the design, coordination and follow up of the database. All authors read and approved the final manuscript.


Focal clusters of slow wave activity in the delta frequency range (1–4 Hz), as measured by magnetencephalography (MEG), are usually located in the vicinity of structural damage in the brain. Such oscillations are usually considered pathological and indicative of areas incapable of normal functioning owing to deafferentation from relevant input sources. In the present study we investigated the change in Delta Dipole Density in 28 patients with chronic aphasia (>12 months post onset) following cerebrovascular stroke of the left hemisphere before and after intensive speech and language therapy (3 hours/day over 2 weeks).Neuropsychologically assessed language functions improved significantly after training. Perilesional delta activity decreased after therapy in 16 of the 28 patients, while an increase was evident in 12 patients. The magnitude of change of delta activity in these areas correlated with the amount of change in language functions as measured by standardized language tests.These results emphasize the significance of perilesional areas in the rehabilitation of aphasia even years after the stroke, and might reflect reorganisation of the language network that provides the basis for improved language functions after intensive training. Cerebrovascular stroke is a highly prevalent condition and the major cause of language impairment in adults. Immediately following a stroke about 38% of the affected population experience aphasia . SpontanTo date, the evaluation of impairment and recovery of function, including training-induced improvement in aphasia, has been based mainly on performance in neuropsychological tests. This is now being increasingly complemented by measures of brain function. Different mechanisms and time courses of recovery of language function after brain damage have been discussed. Hemodynamic imaging suggests the involvement of two mechanisms: (1) regression of diaschisis  and (2) functional reorganization of the neuronal networks involved in language processing. Regression of diachisis in perilesional and more distant regions have been shown to contribute to recovery of function particularly in early phases of the recovery process . In contBrain structures in the vicinity of structural lesions produce a larger amount of slow wave activity. This might be due to a loss of afferent input (e.g. from the lesion) or to a primary metabolic change within these perilesional areas .These abnormal slow waves can be detected in the electroencephalogram (EEG) and, due to their focal generators, they can be localized using magnetic source imaging, a magnetencephalogram (MEG) based technique. In Abnormal Slow Wave Activity Mapping (ASWAM) , generatThe mapping of abnormal slow wave activity can be used not only to identify dysfunctional neuronal networks, but also to track changes in the course of recovery or treatment. For instance, de Jongh et al.  reportedThe present study employed ASWAM before and after intensive language training in aphasic patients. If ASWAM qualifies for the evaluation of treatment or training-supported rehabilitation in chronic aphasics, changes in the intensity and distribution of focally generated abnormal slow wave activity should vary with improvement of language function after a specific intervention. Aphasics were recruited from an ongoing project evaluating the effectiveness of an intensive language training program. This program combines the learning principles of shaping and the efficacy of concentrated training ,6 while It was hypothesized that (a) aphasics would display an increased density of slow wave generators in the damaged (left) hemisphere before training, (b) this density would be reduced in the perilesional zone following language training and (c) there would be an improvement of language functions as evaluated by a standardized language test (Aachen Aphasia Test Battery, AAT) .The average test performance of the entire patient group increased after language training, as indicated by the AAT profile (t(27) = 9.85, p < 0.0001, paired t-test, two tailed). Similar improvements were found for the Token Test (t(27) = 6.10, p < 0.0001). The average improvement of the profile score was 2.9 ± 1.3 points and 6.1 ± 5.3 points on the Token Test (T-scores). Twenty-five of the 28 patients improved on at least one subtest (N = 19) or subscale (N = 6) of the AAT.In 26 subjects the maximum activity of delta dipoles was found in the left hemisphere and in the vicinity of the structurally obvious lesion . The coordinates of maximum delta dipole density were exactly the same in eleven patients, while maximum delta activity shifted by one voxel in one of the three cardinal planes in eight patients, and by more than one voxel in nine patients. .Thresholds were significantly higher in the left hemisphere (F(1,54) = 49.03, p < 0.0001). Clusters of voxels with delta activity > 2 SD above the average DDD in a group of 25 healthy controls were found in 26 of the 28 patients in the left hemisphere before training. Such clusters were found in the right hemisphere in only 7 patients. In two patients only, delta activity in the right hemisphere exceeded left hemisphere activity. Average delta activity was significantly more pronounced in the left hemisphere before and after training (for the pre-measurement the main effect HEMISPHERE was F(1,54) = 55.35, p < 0.0001; for the post-measurement, F(1,54) = 46.55, p <0 .0001: Figure Twelve patients showed an increase in left hemisphere delta activity after training, while a decrease occurred in sixteen patients Figure . This di"Magnitude of change" in the left hemisphere was more pronounced in those patients who displayed significant improvement in at least one subtest of the AAT (N = 19) compared to patients with minor improvements  or no improvements  = 4.95, p < 0.05). Magnitude of change of left-hemispheric delta activity varied significantly with improvements in language functions  discloses generators of abnormal slow waves. The mapping of abnormal slow wave activity on to brain structures allows for the identification of areas that are active, but not capable of normal function. This is, to the best of our knowledge, the first report of a re-test after controlled neuropsychological/-linguistic intervention within the same subjects. The comparison between the two measurements indicates a high reliability of peak locations in left hemispheric perilesional areas, even though successful training modified this activity in magnitude and spatial distribution. In almost all patients, the region surrounding the structural lesion continuously and reliably produced abnormal slow waves, whereas only very few patients presented slow wave activity distant from the structurally confirmed lesion. The amount of perilesional slow wave activity was markedly altered in patients who had improved after training, and the magnitude of this change was related to the changes in language functions.Substantial functional improvements were achieved even in chronic aphasic states by shaping procedures, constraint of non-verbal communication and massed practice. This replicates and extends the findings of Pulvermüller et al. . The preor increase of delta activity in perilesional areas. Moreover, the increase of delta activity was related to longer duration of disease. In most of the patients, re-integration of 'spared' brain areas into the language network should be completed in time (in patients exhibiting an increase of slow wave activity, the time-since-lesion averaged 55.4 ± 39.3 months, compared to 35 ± 13.9 months in patients exhibiting a decrease). Therefore, "dysfunctional" delta activity might be related, at least in a subgroup of chronic patients, to functionally more favorable outcomes. The increase of delta activity might be explained by reduced reciprocal exchange of information within functionally intact and permanently impaired network components.In contrast, some patients, though displaying language improvement after training, exhibited an increase of delta activity in the vicinity of the lesion. One explanation for this might be that the functional capabilities of the affected brain area remain disturbed, with no further potential to be restored or re-integrated in the language 'network', and this might inhibit or impair functionally intact regions. Further segregation of these continuously dysfunctional areas from the remaining network might then lead to improved language functions and consequently to increased delta activity. This hypothesis is supported by the correlation between language improvements and either decrease Compared to the more conventional procedures for aphasia treatment in the chronic stage, the present training involved an intense use of language capabilities and a restraining of alternative, non-verbal methods of communication. In our opinion, any training that encourages speech production several hours a day over several days has the potential to be efficacious. Massed practice is likely to produce activity-dependent cortical reorganization, found to result from CI-movement therapy -31. It iTwenty-eight patients suffering from chronic aphasia participated in the training . Training took place 3 hours/day for 10 consecutive days and included (for 18 patients) language exercises with increasing levels of difficulty  or . Language function was evaluated by two sensitive measures of change of aphasia severity: the profile score and the Token Test of the Aachen Aphasia Test . Tests wUsing a 148-channel whole-head neuromagnetometer (MAGNES™ 2500 WH, 4D Neuroimaging, San Diego, USA), MEG-measurements were collected twice: once on the day before training and once on the day after training. MEG was measured in a 5-minute resting period, during which subjects were asked to relax while staying awake, and to not engage in any specific mental activity. MEG recordings were obtained in a supine position. Subjects were asked to fixate a colored mark on the ceiling of the magnetically shielded room throughout the recording in order to avoid eye- and head-movement. A video camera installed inside the magnetically shielded room allowed for a monitoring of the subject's behavior and compliance throughout the experiment. Written informed consent was obtained from subjects prior to each MEG-session and the study was approved by the ethics committee of the University of Konstanz.® Fasttrack prior to each measurement. The subject's head position relative to the pickup coils of the sensor was estimated before and after each measurement. MEG was recorded with a sampling rate of 678.17 Hz, using a 0.1–200 Hz band-pass filter. For artifact control, eye movements (EOG) were recorded from four electrodes attached to the left and right outer canthus and above and below the right eye. The electrocardiogram (ECG) was monitored via electrodes attached to the right collarbone and the lowest left rib using a Synamps amplifier (NEUROSCAN®).The fiducial points, coils, and head shape were digitized with a Polhemus 3SpaceData were reduced by a factor of 16 and digitally filtered for the delta (1.5–4.0 Hz) frequency band using a digital band pass filter (Butterworth filter of the order 6). Artifact-free time segments were determined by visual inspection. Single equivalent current dipoles were fitted for each time point in the selected artifact free segments (distance of time points 24 ms.). Five non-overlapping channel groups over left, right, center, anterior, posterior regions were chosen for dipole modeling. A homogeneous sphere, which gives the best least-squares fit to the digitized patient's headshape below the selected sensors, served as a model for the volume conductor.3, using the AFNI-to3d-software  a dipole moment (q) of 10 nAm < q < 100 nAm; (2) a goodness of fit (GOF) greater than 0.90. These restrictions should ensure that neither artifacts nor small amplitude biological noise would affect the results, and that only dipolar fields that were generated by focal sources were analyzed. Each data-set was divided into 1000 voxels, each of 20 mmroimages ). For earoimages .Whole brain magnetic resonance images (TR = 19, TE = 5,6, Flip angle = 30°, FOV = 256 mm, 1 mm isotropic resolution) were acquired within the 2-week training period across a 256 mm slab from each subject using a Philips Gyroscan 1.5 Tesla scanner . MRIs were aligned to the coordinates of the MEG according to anatomical landmarks, coil positions and head shape information using the AFNI software.A spatial clustering algorithm  was applied to smooth the data. Maximum activity, i.e. the voxel with the highest percentage of delta dipoles, was determined using the AFNI subroutine 3dExtrema. The localization of this maximum was determined on the x- , y- (anterior-posterior) and z- (inferior-superior) plane for the two measurements (pre- & post-training).The localization of areas with high dipole densities (adjacent clusters of voxels) within each hemisphere was analyzed by applying a narrow filter of FWHM 20 mm to the original data. A narrow filter was used to minimize the influence of more distant voxels with low dipole density on the areas of higher density. First, the voxel with maximum activity was determined for each hemisphere in each patient. By setting thresholds according to the following criteria, voxels with high dipole density were extracted and averaged for each hemisphere:a. Only voxels with z-values within one standard deviation below the maximum of each patient were considered for averaging within each hemisphere.b. If the z-value was smaller than 2 standard deviations, voxels were not considered.c. If the peak density was below 2 SD, an iterative process was initiated. Thresholds were lowered until at least one voxel became apparent where the dipole densities were different before and after training.2-T1|) by the mean of both hemispheres before and after training ((T1left+T1right+T2left+T2right)/4). This number was then log transformed  and submitted to statistical analyses. Two patients were excluded from this final analysis .A measure of the "Magnitude of change" was determined to evaluate changes in dipole density relative to changes in language functions pre- and post-treatment. The averaged (absolute) intensity of delta dipoles in voxels above threshold (in each hemisphere and each patient) was scaled by dividing the magnitude of change in each hemisphere (|TChanges in AAT test scores across the two assessments were verified by two-tailed t-tests. Stability of the location of the maximum delta activity was verified by correlation coefficients (Spearman's Rho) for the posterior-anterior, medial-lateral, and inferior-superior axes. Differences in thresholds and average DDD between hemispheres (for both measurements) were verified by means of analysis of variance (ANOVA), as were differences between patients that exhibited an increase or decrease of DDD concerning time-since-onset. Changes in language functions (Token Test, profile score AAT) relative to changes in DDD-magnitude were evaluated by Pearson correlation.MM, TE and BR participated in the design of the study. MM was responsible for conducting the study, performed data analysis and drafted the manuscript. TE and BR participated in the discussion and general conclusions. CW provided knowledge of data analysis and wrote most of the scripts used for data analysis. DD and GB conducted therapy, assisted in collecting the data, and provided experience of therapeutic issues.


Peptides derived from protein tandem mass spectrometry data have been mapped to the human genome sequence forming an expandable resource for the proteomic data. A crucial aim upon the completion of the human genome is the verification and functional annotation of all predicted genes and their protein products. Here we describe the mapping of peptides derived from accurate interpretations of protein tandem mass spectrometry (MS) data to eukaryotic genomes and the generation of an expandable resource for integration of data from many diverse proteomics experiments. Furthermore, we demonstrate that peptide identifications obtained from high-throughput proteomics can be integrated on a large scale with the human genome. This resource could serve as an expandable repository for MS-derived proteome information. The recent definition of the complete nucleotide sequence of the human genome ,2 has moCurrent use of expressed sequence tags (EST) and full coding DNA (cDNA) sequences is extremely helpful in achieving complete genome annotation -9. HowevTo begin annotating the human genome with protein-level information, we have built PeptideAtlas. The generally applicable procedure to annotate eukaryotic genomes with peptide sequences can be applied when datasets are acquired using different experimental protocols. In each case, sample proteins were first proteolytically cleaved into peptides using the enzyme trypsin. The resulting peptide mixture was then subjected to chromatographic separation by strong cation exchange and reverse-phase capillary chromatography. In addition, those experiments using the ICAT (isotope-coded affinity tag) reagent for quantification included an avidin affinity-purification step to select peptides containing biotinylated, stable-isotope-tagged cysteines . The resP ≥ 0.9) of being correct. Each peptide is given a unique and stable identifier with an eight-digit number in the form PAp00000001. We then attempted to map the 26,840 distinct peptides to the human genome sequence using the analysis pipeline shown schematically in Figure Resulting from this, 26,840 distinct peptide sequences were identified from 224,973 spectra with identifications of a high probability  in the atlas is shown in Figure The cumulative number of distinct peptides as a function of the addition of identified spectra  of the 34,091 human Ensembl proteins (version 22.34d.1, 2004-06-02). These proteins represent unique proteins or splice forms from 6,423 genes (27%) of human genes in Ensembl.Some peptides have indistinguishable, perfect protein sequence matches to multiple proteins. These proteins are typically paralogs (protein families), protein isoforms or repeated protein domains in the human genome. We identified 3,718 proteins unambiguously by one or more 'discrete peptides' - peptides that map uniquely to a single protein - in the current build of PeptideAtlas. Those peptides are marked in the genome browser as 'discrete peptide'. 'Degenerate peptides' that map to several protein isoforms are also used to identify proteins. It would thus be more accurate to state that a product of a certain gene, rather than a certain protein, has been identified -22. MoreA significant number of distinct peptides (1,086), assigned by SEQUEST/ProteinProphet from over 5,000 MS/MS spectra, could not be mapped to Ensembl database version 22.34d.1. These peptides were identified by SEQUEST searches against the IPI database  or ABCC The absence of Ensembl matches does raise the question of whether these peptides are false positives or whether real proteins are missing in the Ensembl database. When these peptides were investigated in more detail it was found that nearly 100 were identified 10 or more times in several different experiments, and that many had protein sequence matches for Swiss-Prot entries. They are therefore likely to be true peptide attributions. For example, peptide PAp00000363 (AGKPVICATQMLESMIK) was identified 626 times at different charge states and with different mass modifications in 22 distinct experiments and mapped to KPY1_HUMAN, a pyruvate kinase M1 isozyme. Interestingly, the protein appears to have a likely SNP, which mutates the valine present in the Ensembl genome sequence to the isoleucine observed in PAp00000363.The 9,747 mapped proteins represent 28.6% of the predicted human proteome in Ensembl version 22.34d.1. The distribution of peptide matches to these proteins  are stored, this information could be displayed as well.It is well understood and discussed in the literature  that allInitial datasets of peptide assignments to MS/MS spectra, obtained by searching acquired MS/MS spectra using the database search program SEQUEST, were statistically validated using the computational tool PeptideProphet. For each peptide assignment to an MS/MS spectrum, PeptideProphet computes a probability of its being correct, based on its database search scores, difference between the measured and theoretical peptide mass, the number of termini consistent with the type of enzymatic cleavage used, the number of missed cleavage sites and other factors. Probabilities computed by PeptideProphet have been shown to be accurate in the entire probability range and, therefore, can be used to compute the false-positive identification error rate  resulting from filtering each dataset using any minimum computed peptide probability threshold . The falP ≥ 0.7, 0.9, 0.95 and 0.99. Decreasing the probability threshold increases the number of peptides, both correctly and incorrectly identified, and the corresponding proteins (Table P ≥ 0.99 produced 21,030 peptides with protein sequence matches (4,845 protein identifications), almost 8,400 fewer than the lowest threshold of 0.7 (2,252 fewer protein identifications). The P ≥ 0.9 threshold yielded 25,754 peptides with protein sequence matches at an estimated false-positive rate of less than 7%, and we selected this as an acceptable level for the default PeptideAtlas. The number of false-positive identifications could be reduced by selecting a higher threshold; however, a significant number of correct peptides and proteins would then also be eliminated. The additional peptides resulting from the low-probability threshold were valuable for adding additional peptide evidence in combination with higher-probability peptides corresponding to the same protein . We proDrosophila melanogaster LC-MS/MS experiments. We collected data obtained from cytoplasmic, nuclear and membrane fractions derived from a Drosophila S2 Schneider cell line. The resulting 4,406 different peptides with P > 0.9 were compared to the 18,289 proteins (Ensembl fly database version 18.3a.1, 2003-07-01) using the same pipeline as described for human. From the fly, 3,107 proteins could be validated, representing 1,876 (14%) of the fly's genes. These results show that our method could easily be adapted to other organisms, thus opening up the way for comparative proteome-level evaluations of eukaryotic organisms.To validate our approach for general use in eukaryote genomes, we have extended our methods to peptides obtained from We have annotated the human genome with protein evidence for nearly 10,000 proteins. Although this number only represents a fraction of the genome and still contains some erroneous identifications, it is a first step towards the final goal: to fully annotate eukaryotic genomes via validation of expressed proteins. PeptideAtlas provides a method and a framework to accommodate proteome information generated by high-throughput proteomics technologies and is able to efficiently disseminate experimental data in the public domain. Its significance continues to grow as more data are submitted.Moreover, PeptideAtlas also allows one to address the important question of how big the human proteome is. Due to the technical limitations of current proteomics technologies, it is not possible yet to determine the complete proteome in one experiment. However, if the data from diverse experiments, using different cellular compartments and enrichment methods were combined, the determination of the complete proteome could eventually be achieved. PeptideAtlas offers the framework to answer this question accurately and to determine the size of the complete human proteome using pooled experimental data. Furthermore, PeptideAtlas provides a resource for the development of new avenues of research. The dataset will provide a rich source of data for computational scientists to develop and test new algorithms for proteomic analysis, gene discovery and splice-variant prediction.The methods described here, combined with the ever-increasing power of proteomics and bioinformatics technologies, will facilitate the determination or characterization of protein-coding genes, their features, and their processing and expression in relationship to the sequence of the human genome, thus contributing significantly to our understanding of genome structure.The assembly of experimentally derived distinct peptides is mapped to the human genome in the following way. First, we use BLAST  to matchLC-MS/MS analysis was performed on LCQ, Ion-trap (Thermo Finnigan LCQ) and Q-Tof (Micromass Waters) instruments.To estimate the false-positive error rate on the level of distinct peptide identifications, we first note that there is an almost 10-fold difference between the number of peptide assignments to MS/MS spectra and the number of resulting distinct peptide identifications. This can be explained by the fact that many peptides were sequenced multiple times, with some of the most abundant peptides sequenced more than 1,000 times (for example, peptides PAp00004784, PAp00003568, PAp00026910). While many correct peptide assignments to MS/MS spectra represent the same peptide sequence, the majority of incorrect peptide assignments are expected to be single identifications. As a result, the false-positive error rate on the level of distinct peptides is higher than that on the level of peptide assignments to MS/MS spectra.Second, it should also be taken into account that a considerable fraction of all distinct peptides did not match any Ensembl entry. This is due to the fact that MS/MS spectra were searched against larger databases, such as human IPI, which contained a number of protein sequences not present in Ensembl. The fraction of all distinct peptide identifications that did not map to any Ensembl entry can be estimated using information provided in Table The false-positive error rate among distinct peptides that mapped to Ensembl (peptides with protein sequence match) can then be estimated to be not higher than the maximum possible number of distinct incorrect peptide identifications that have protein sequence matches  divided by the total number of peptides with protein sequence matches. The corresponding estimates are 16%, 6%, 3% and 0.8% in the case of minimum-probability thresholds 0.7, 9, 0.95 and 0.99, respectively (Table The PeptideAtlas pipeline begins with the download of the Ensembl human protein database from . Release


The home is the predominant environment for exposure to many environmental irritants such as air pollutants and allergens. Exposure to common indoor irritants including volatile organic compounds, formaldehyde and nitrogen dioxide, may increase the risk of snoring for children. The aim of this study was to investigate domestic environmental factors associated with snoring in children.A school-based respiratory survey was administered during March and April of 2002. Nine hundred and ninety six children from four primary schools within the Perth metropolitan area were recruited for the study. A sub-group of 88 children aged 4–6 years were further selected from this sample for domestic air pollutant assessment.3), the adjusted ORs of snoring by children with medium (30 – 60 μg/m3) and high exposures (> 60 μg/m3) to NO2 were 2.5 (95% CI: 0.7–8.7) and 4.5 (95% CI: 1.4–14.3) respectively. The corresponding linear dose-response trend was also significant (P = 0.011).The prevalences of infrequent snoring and habitual snoring in primary school children were 24.9% and 15.2% respectively. Passive smoking was found to be a significant risk factor for habitual snoring (odds ratio (OR) = 1.77; 95% confidence interval (CI): 1.20–2.61), while having pets at home appeared to be protective against habitual snoring . Domestic pollutant assessments showed that the prevalence of snoring was significantly associated with exposure to nitrogen dioxide during winter. Relative to the low exposure category (<30 μg/mSnoring is common in primary school children. Domestic environments may play a significant role in the increased prevalence of snoring. Exposure to nitrogen dioxide in domestic environment is associated with snoring in children. Snoring occurs when there is an obstruction to the free flow of air through the airways at the back of the mouth and nose. The prevalence of habitual snoring in children has been reported to vary between 3.2 and 11%. Infrequent snoring is present in 17–27% of all children .Approximately one third of children who snore regularly have obstructive sleep apnea syndrome (OSAS) . A few sNumerous risk factors for snoring and OSAS have been reported including enlarged adenoids and/or tonsils, obesity, allergies or other causes of nasal obstruction, and exposure to environmental tobacco smoke (ETS) -10. HoweNine hundred and ninety-six (996) school children, aged between 4 and 12 years, were recruited from four primary schools within the Perth metropolitan area. Parents/guardians of the children completed a questionnaire related to respiratory health of their children and domestic environments. A sample of 88 children, aged 4–6 years, was then selected randomly to participate in an indoor air quality assessment of their domestic environments. Ethics approval was obtained from the Human Research Ethics Committee of Curtin University of Technology.The survey instrument adopted was taken from a questionnaire on respiratory health and indoor air quality . Some quThe questionnaires were distributed to parents by school teachers and later collected from the classrooms. The survey was conducted between March and April 2002. A consent form was signed by each participating parent or guardian. The response rate was 62.5%.A sample of 88 year one and pre-primary students was randomly selected from participants of the respiratory survey for domestic air pollutant monitoring. Among them, 34 (38.6%) children were snorers . Two home visits were subsequently carried out during the winter of 2002 and summer of 2003 to measure indoor volatile organic compounds (VOCs), formaldehyde and nitrogen dioxide levels.2) were collected by a passive sampling method in both the living room and the child's bedroom for 24 hours. Formaldehyde was analyzed using high-performance liquid chromatography [VOCs were collected in the living room by active sampling using charcoal sorbent tubes. The air-sampling rate was 1 L/min with sampling undertaken for 10 hours during daytime. The analyses were performed using a Perkin Elmer Autosystem XL gas chromatograph equipped with a flame ionization detector. Eleven common compounds were identified and quantified by comparing the retention times: benzene, chlorobenzene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, ethylbenzene, styrene, toluene, m-xylene, o-xylene and p-xylene. Their total amount was expressed as total VOCs (TVOCs). Formaldehyde (HCHO) and nitrogen dioxide  of these variables were calculated after applying a logarithmic transformation. All statistical analyses were performed using the SPSS package Version 10.0.Preliminary data screening and cleaning were conducted prior to statistical analysis. Associations between the prevalence of snoring and environmental and geographic factors and respiratory symptoms were examined using Chi-square tests. Multivariate logistic regression analysis was undertaken to estimate the risk of snoring adjusting for possible confounders. Since the distributions of VOCs, HCHO and NOOf the 996 participants, 985 children (98.9%) had intact records for snoring. There were 248 children  reported infrequent snoring and 151 children  suffered from habitual snoring.Table The prevalences of respiratory symptoms, asthma and other allergic conditions were significantly different among non-snoring, infrequent snoring, and habitual snoring children, with habitual snorers having the highest rates. Results are presented in Table Table To further investigate the impact of passive smoking and pet ownership on snoring, logistic regression analysis was undertaken, controlling for confounders age, gender, asthma and other allergic conditions. The results indicated that passive smoking increased the risk of habitual snoring significantly , while having pets decreased the risk . However, the corresponding effects of passive smoking and pet ownership on infrequent snoring were statistically not significant. The Hosmer-Lemeshow statistic confirmed adequacy of the fitted logistic regression model (P > 0.10).2 concentration in the living rooms of snoring children were higher. In particular, the levels of NO2 in snoring children's bedroom were significantly higher than those in non-snoring children's bedroom during winter.The levels of pollutants exposure were similar between houses of habitual snorers and houses of infrequent snorers. To facilitate analysis, data from the two groups were combined to improve statistical power for comparison with houses of the non-snoring children. Table 2 could be a gas heater and/or gas cooker, we compared NO2 concentration between houses with and without a gas heater in the child's bedroom. The results confirmed that NO2 levels (GM: 49 μg/m3, 95% CI: 37–65 μg/m3) in houses with a gas heater were significantly higher than those (GM: 27 μg/m3, 95% CI: 20–38 μg/m3) recorded in houses without a gas heater.Recognizing that the main source of indoor NO2 during winter and snoring in children.Logistic regression analysis was next conducted to assess the dose-response relationship between bedroom exposure to NO2 distribution, the monitored households were classified as: 'low' exposure (<30 μg/m3), 'medium' exposure (30 – 60 μg/m3) and 'high' exposure (> 60 μg/m3). After adjusting for age, gender, asthma, passive smoking and pet ownership, domestic NO2 exposure level was still positively associated with snoring, the ORs being 2.5 (95% CI: 0.7–8.7) for medium exposure and 4.5 (95% CI: 1.4–14.3) for high exposure. There was also evidence of a linear dose-response relationship (P = 0.011 for trend).Based on the empirical NOSnoring is an important symptom and major risk factor for obstructive sleep apnea . SeveralThe prevalence of snoring among older children was significantly lower than that of younger children. No significant difference in snoring prevalence between boys and girls was observed, which appeared to be consistent with the literature ,18.Strong associations were also found between snoring and respiratory symptoms, asthma and other allergic conditions, as in previous studies ,19,20.In relation to the domestic environment, passive smoking was identified as a major risk factor for habitual snoring and consistent with other studies ,21. An i2 was significantly associated with snoring. It should be remarked that the low exposure threshold was set below the annual value of 40 μg/m3 recommended by WHO [2could increase the risk of snoring by 4.5 times. A previous study reported that children aged 5–12 years had a 20% increased risk of respiratory symptoms and disease for each increase of 28.3 μg/m3 in NO2concentrations (2-week average), when the weekly average concentrations were in the range 15–128 μg/m3 or possibly higher [2 exposure from gas appliances and the prevalence of respiratory symptoms [2 was related to gas heating during winter.Unlike TVOCs and HCHO, it appears that domestic exposure to NOd by WHO , whereasy higher . Anothersymptoms . Our res2 exposure on snoring was significant even after adjustment for asthma, atopy and other confounding factors, caution must be taken when interpreting the NO2 findings and further investigation is required before they can be generalized to the pediatric population at large. A limitation of this cross-sectional study is that only 9% of the study sample was monitored for environmental testing due to budget and other constraints. Nevertheless, this subgroup of children did not differ significantly from the whole sample or other populations of young children in Perth [2 could not be determined because the measurements of exposure and illness were taken at the same time. The assessment of snoring was retrospective in relation to the time of environmental monitoring. Moreover, the significant association between snoring and NO2 exposure in winter may be attributed to NO2 emission from gas heaters in conjunction with low ventilation during the winter season.Although the effect of NOin Perth ,26 with 2 might result in snoring. Although there is evidence to suggest that exposure to NO2 is associated with development of allergic disease [2 exposure and snoring is independent of atopy. Snoring occurs due to upper airway obstruction during sleep. The obstruction commonly occurs at the level of the nasal turbinates as with anterior rhinitis or the nasopharynx due to adenoid hypertrophy. Exposure of airway epithelium in vitro results in the release of inflammatory cytokines and adhesion molecules [2 increases upper airway inflammation, resulting in mucosal oedema and airway obstruction. Alternatively, upregulation of ICAM1 the primary ligand for rhinovirus [2 increases lipid membrane fluidity [2 exposure might produce changes in cell-cell and cell-pathogen interactions that could result in altered upper airway physiology. Given the high prevalence of snoring in our population and the knowledge that snoring is a significant risk factor for obstructive sleep apnea, the mechanisms that might underpin the association between NO2 exposure and snoring require further study.As for potential mechanisms for this association, there is little in the literature that can directly explain how exposure to NO disease , the obsolecules . Therefoinovirus  could influidity  that in In conclusion, the present study shows that snoring is common among primary school children in Perth, and snoring is associated with other respiratory symptoms. Passive smoking increases the risk of snoring in children but pet ownership may decrease the risk. The level of nitrogen dioxide in domestic environment is positively associated with the prevalence of snoring in children.GZ, JS, KR, SS Study design, coordination and managementGZ, JS, KR Field measurement and laboratory workAHL, GZ Data analysis and interpretation of resultsGZ, JS, KR, AHL, SS Preparation and revision of the manuscript


Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming.The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes.This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes. During the last decade, many genomes have been successfully and completely sequenced. Summarized information about the oligonucleotides in genomes provides biologists, who interests in the evolution and growth of genomes, to work in comparative genomics, oligonucleotide probe design, primer design and the analyses of genomic repetitive features.The computation of the occurrences and the frequency of all oligonucleotides in a complete genome is very elaborate and time-consuming, especially when the genome size is very large, such as the human and mouse genomes. A database that summarizes the occurrences and the frequencies of oligonucleotides in complete genomes can facilitate the biological and the statistical analyses of genomes.et al. systematically searched the promoter regions of potentially co-regulated genes for over-represented oligonucleotides which may be transcription factor binding sites [Saccharomyces cerevisiae. Although conceptually simple, the algorithm efficiently extracted the upstream regulatory sequences that had been previously been determined experimentally for most of the yeast regulatory families already analyzed. Other studies [The contents of the database can be used in many biological applications, such as comparative genomics and evolution analyses ,2, the png sites . They pr studies -8,10-12 et al. [Hsieh et al.  investigRepetitive elements, including LINEs, SINEs, LTR and Alu, can be investigated in evolution analysis . It has Another application of the established database is to facilitate the design of primers to amplify specific regions of the genomic sequence. The basic concept is that the sequences of primers from 15 to 40 bps should be unique, unlike the repetitive oligonucleotides in our database. Additionally, our database maintains the repetitive oligonucleotides that facilitate the design of oligonucleotide probes to allow the selection of signature oligonucleotides when identifying different organisms using DNA arrays . The useThe proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The data sources including the complete genomes and the gene annotation information are obtained from GenBank . The repThe occurrences and the frequencies of oligonucleotides from one to 50 base pairs are generated and accumulated from each of the complete genome sequence. Inputting the sequence of the oligonucleotide returns the positions of the oligonucleotides. Additionally, both the occurrences and the frequencies of the repetitive elements such as LINEs, SINEs, Alu and LTR are provided by computationally scanning whole genome sequences. The tandem repeats are computationally detected by the tandem repeat finder . The datVibrio cholerae genome are processed separately to allow the computation of the occurrences of oligonucleotides in each chromosomal sequence. RepeatMasker [A software is implemented to index systematically a complete genome sequence into a suffix-array using a perfect match approach . This inatMasker  and the atMasker , are useatMasker . The TRFThe Tandem Repeat Finder uses seven parameters. These are match score, mismatch score, indel score, probability of match and insertion, minimum score of alignment and the maximum of tandem repeat pattern size. The corresponding values used here are 2, 7, 7, 80, 10, 20 and 500. The values are the default suggestions found in Tandem Repeat Finder documentation.The transposable elements (TEs) are detected by RepeatMasker. TEs in each genome are identified using the complete dataset available from REPbase Updates [Please add the citation]. The sensitivity and the speed of RepeatMasker are set as the default values.Table The web interface enables users to query the occurrences of an oligonucleotide in a genome and the number of occurrences in each chromosome. Figure Figure We have constructed the databases of both the oligonucleotide occurrence locations and their frequencies in the coding and the non-coding regions in complete genomes. The data in flat-file format can be downloaded directly for further analyses in several biological applications. The user may also use the web interface to query and access the database contents. The database also provides a filtering function for retrieving the information about oligonucleotides under search conditions specified by the users. Furthermore, the database provides the occurrences and the frequencies of other repetitive elements, such as LINE, SINE, Alu and tandem repeats in genomes.The database is now available at FML implements the software and refinements the system. HDH conceives of the study and drafted the manuscripts. YCC and JTH participates the design and coordination. All authors read and approved the final manuscripts.Appendix listing the organisms supported in the database.Click here for file


The hardest working molecules in cell biology, proteins abound in a dazzling variety of shapes and sizes to carry out an equally impressive array of tasks. Many proteins function within the cell, while others get shipped out to locations both near and far. Specialized organelles within the cell package the traveling proteins into cargo containers called vesicles. Vesicles move through a highly regulated transportation system until they reach their ultimate destination, either inside or outside the cell, and release their cargo. When vesicles fuse to the plasma membrane and release their cargo outside the cell the process is called exocytosis. Exocytosis allows macromolecules to leave the cell without compromising the structural integrity of its membrane.“Professional” secretory cells specialize in producing copious quantities of their protein product and sending vesicles packed with their customized issue to the plasma membrane. Once there, the vesicles wait for a signal to fuse with the membrane. The signal most often comes in the form of a transient and localized increase in calcium ion levels.Over a decade ago, researchers discovered that calcium-triggered exocytosis also occurs in “nonprofessional” secretory cells. In these cells, the process was thought to be important for healing ruptured plasma membrane, though the identity of the vesicles responsible remained unknown. It has been suggested that these vesicles are lysosomes, enzyme-filled organelles that break down waste and extracellular debris ingested by the cells. Sanford Simon, with his colleagues Jyoti Jaiswal and Norma Andrews, previously confirmed that calcium does specifically trigger exocytosis of lysosomes in the “nonprofessional” secretory cells. Calcium-triggered exocytosis is thought to require the services of a family of proteins called synaptotagmins. But the fifteen members of the synaptotagmin family diverge from this job description in various ways, calling its role into question.PLoS Biology, Jaiswal, Simon, and colleagues investigate the molecular mechanisms underlying calcium-triggered lysosomal exocytosis, focusing on the role of Syt VII.Synaptotagmin VII (Syt VII)—the synaptotagmin member expressed on the lysosomes—is present in most tissues in organisms ranging from worms to humans. This protein functions in processes requiring lysosomal exocytosis and during invasion by trypanosome parasites, such as the one that causes Chagas disease. In this issue of The researchers took cells from two lines of mice: one lacked the functional Syt VII protein and the other produced normal levels of Syt VII. First they labeled the surface and interior cavity of lysosomes in these cells with fluorescent tags and triggered an increase in cells' calcium level; then they watched the behavior of single lysosomes releasing their contents in real time.Most of the lysosomes from normal cells released only a portion of their contents and had very small fusion pores that remained open to the outside of the cell for only a short time. Interestingly, while proteins in secretory vesicle membranes typically diffuse into the plasma membrane during exocytosis, proteins in the lysosomal membrane stayed near the site of fusion.In the cells from Syt VII–deficient mice, Simon and colleagues discovered, to their surprise, that this protein isn't necessary for calcium-triggering of lysosomal exocytosis. Calcium-triggered exocytosis not only occurred in these cells, it happened more rapidly than in normal cells. Plus most ofthese deficient lysosomes fused completely and their membrane proteins fully diffused into the plasma membrane. Simon and colleagues argue that these results show that Syt VII restricts rather than facilitates lysosomal exocytosis. It does so by limiting the formation and size of fusion pores and by preventing lysosomal membrane proteins from integrating into the plasma membrane.


Tumors and viruses have developed many mechanisms to evade the immune system, including down-regulation of target antigens and MHC molecules. These immune escape mechanisms may be able to be circumvented by adoptively transferring T cells engineered to express two different T cell receptors, each specific for a different antigen or MHC restriction molecule.PBMC from the blood of normal healthy donors were stimulated for three days with an antigenic peptide from cytomegalovirus (CMV) pp65. These CMV reactive cultures were transduced with a encoding the TIL 5 T cell receptor (TCR) that mediates recognition of the dominant epitope of the melanoma antigen MART-1. Following selection for transduced cells, the cultures were evaluated for recognition of CMV pp65 and MART-1 expressing targets.+ tumor cells, and maintained their bifunctionality for over 40 days in culture.We were able to rapidly create bifunctional T cells capable of recognizing both CMV pp65 and MART-1 using a combination of HLA-A2 tetramer staining and intracellular staining for interferon-γ. These bifunctional T cells were sensitive to very low levels of antigen, recognize MART-1Bifunctional T cells can be engineered by transducing short term peptide stimulated T cell cultures. These bifunctional T cells may be more effective in treating patients with cancer or chronic virus infections because they would reduce the possibility of disease progression due to antigen and/or MHC loss variants. It has long been established that tumors and viruses have multiple mechanisms for evading the immune system including the inhibition of T cell function through the release of inhibitory cytokines and factors ,2, down-We and others have shown that it is possible to use retroviral vectors encoding TCRs isolated from tumor- or virus-reactive T cell clones to engineer human T cells to recognize any antigen . While eIn the current study, we show that it is possible to rapidly generate T cell populations containing T cells reactive with two defined antigens, CMV pp65 and MART-1. These T cell cultures are highly avid for both antigens and retain their reactivity for at least six weeks. More importantly, this methodology could easily be adapted to closed culture systems making it more attractive for use in clinical trials.+, MART-1+), 624-28 MEL (HLA-A2-, MART-1+), 1300 MEL (HLA-A2+, MART-1+), and SK23 MEL (HLA-A2+, MART-1+) and renal cell carcinoma cell line UOK131 (HLA-A2+, MART-1-) were maintained complete medium (CM) which consisted of RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% heat inactivated fetal bovine serum (Life Technologies), and penicillin (100 U/ml)/streptomycin (100 μg/ml)/L-glutamine (2.92 mg/ml)(Life Technologies) as described [et al [All melanoma and renal cell carcinoma cell lines used in this study were established from surgical specimens obtained from cancer patients undergoing immunotherapy at the Surgery Branch, National Cancer Institute. Melanoma cell lines 624 MEL  and 300 IU/mL recombinant human IL-2 (rhIL-2) (Chiron, Berkeley, CA) in T cell medium (TCM) which consisted of RPMI 1640 supplemented with 10% pooled human AB serum , HEPES (Life Technologies), 2-mercaptoethanol (Life Technologies), penicillin (100 U/ml)/streptomycin (100 μg/ml)/L-glutamine (2.92 mg/ml)(Life Technologies) as described [All peptides used in this study were purchased from Synthetic Biomolecules (San Diego, CA). Peptides used were MART-1:27–35 (AAGIGILTV), Influenza M1:58–66 (GILGFVFTL), CMV pp65:495–503 (NLVPMVATV), or gp100:209–217 (ITDQVPFSV). Each peptide was maintained as a concentrated stock (2–5 mg/ml) in 100% DMSO (Sigma, St. Louis, MO) and diluted in the appropriate medium prior to immediate use.E. coli cells (Life Technologies) as described [Given that it is technically difficult to obtain CMV infected targets for immunologic assays, COS A2 cells were engineered to express a mini-gene encoding the CMV pp65:495–503 peptide epitope (COS A2 CMV). A CMV minigene was constructed using complementary synthetic oligonucleotide primers  that encoded the CMV pp65:495–503 peptide epitope with an ATG translation initiation codon, a Kozak consensus sequence  and Not escribed . Recombiescribed . COS A2 in vitro with 5 μg/ml of CMV pp65:495–503 peptide in TCM containing 300 IU/mL IL-2 for 3 days. T cell cultures were then transduced using a modified Retronectin (TaKaRa, Otsu, Japan) protocol with the A7 retrovirus as follows: 24-well plates were coated with Retronectin then were preloaded with retrovirus according to the manufacturer's instructions. 2.6 × 106 T cells were added to each well in 1.3 ml (2 × 106 cells/ml) of A7 retroviral supernantant supplemented with 300 IU/ml rhIL-2 and the plates were centrifuged for 90 min at 1000 g. The next day the medium was replaced with fresh A7 retroviral supernatant and the centrifugation was repeated. The cells were rested for 24 hours and then transduced cells were selected in 1 mg/ml of G418  for five days. Cultures were assayed for antigen reactivity, cyropreserved, and/or expanded for additional assays.PBMC from healthy donors were stimulated 6 cells/ml in TCM supplemented with 300 IU/ml rhIL-2, and 2 μg/ml anti-CD28 mAb (Becton, Dickenson, and Company, Franklin Lakes, NJ) in 24 well tissue culture plates pre-coated overnight with 10 μg of anti-CD3 mAb (Ortho Biotech, Bridgewater, NJ) for three days prior to transduction. Transduction of R6C12 was carried out as described above for CMV peptide stimulated T cell cultures except 2 μg/ml anti-CD28 mAb was added to the medium and 10 μg anti-CD3 mAb was bound to the culture plates in addition to Retronectin.T cell clone R6C12 was cultured at 2 × 104 T cells were cocultured in a 1:1 ratio overnight in 0.2 ml of CM in duplicate individual wells of a 96-well plate with a panel of stimulators that included T2 cells loaded with 5 μg/mL MART-1:27–35, Influenza M1:58–66, CMV pp65:495–503, or gp100:209–217 peptide and a panel of tumor cells. The amount of interferon-γ released was measured by ELISA as described [The antigen reactivity of each T cell culture (TIL 5 TCR transduced and untransduced) was assayed for MART-1:27–35 and CMV pp65:495–503 or gp100:209–217 reactivity in interferon-γ release assays. 5 × 10+ MART-1+ stimulator cells followed by fluorescence staining with HLA-A2/CMV pp65:495–503 tetramers. 1 × 105 T cells were cocultured in a 1:1 ratio peptide loaded T2 cells or tumor cells for five hours in CM supplemented with 10 μg/ml brefeldin-A. Cells were then collected and stained with PE-conjugated HLA-A2/CMV pp65:495503 tetramers (Beckman Coulter Immunomics, San Diego, CA), fixed in 1% paraformaldehyde (Sigma), permeabilized using 0.5% saponin (Sigma), and then stained with FITC-conjugated anti-interferon-γ . Relative log fluorescence of 104 live cells was measured by flow cytometry using a FACS Scan flow cytometer (BD Biosciences, Mountain View, CA).The existence of CMV pp65:495–503/MART-1:27–35 reactive bifunctional T cells was determined by first staining T cells for intracellular interferon-γ production following coculture with HLA-A2+ MART-1+ tumor cells , 624-28 MEL (HLA-A2- MART-1+ CMV-), or RCC UOK131 (HLA-A2+ MART-1- CMV-) cells. Untransduced CMV peptide stimulated T cells only released interferon-γ when stimulated with CMV pp65:495–503 peptide loaded T2 or COS HLA-A2+ CMV+ cells. These cultures were extremely sensitive to antigen stimulation since significant amounts of interferon-γ were released when stimulated with T2 cells loaded with 5 × 10-4 μg/mL MART-1:27–35 peptide and <5 × 10-7 μg/mL CMV pp65:495–503 peptide .While the antigen reactivity of our T cell cultures was consistent with us having engineered bifunctional T cells, it was necessary to confirm that individual T cells possess the capability to recognize both CMV pp65 and MART-1. To confirm that we had engineered bifunctional cells, each T cell culture was stained with HLA-A2/CMV pp65:495–503 tetramers for anti-CMV reactivity and with intracellular anti-interferon-γ monoclonal antibodies following stimulation with HLA-A2Cells that were double stained with tetramers and for intracellular anti-interferon-γ were considered to be reactive with both antigens and therefore bifunctional. As shown in Figure + MART-1+ tumor cells, 0.65% (1300 MEL) and 0.52% (SK23 MEL) of the T cells were HLA-A2/CMV pp65 tetramer positive and interferon-γ positive indicating that approximately 20% of the peptide reactive T cells were also tumor reactive , compared to 44.6% of our peptide stimulated populations (data not shown). This profound expansion allowed for more efficient transduction, and 2.7% of the resulting culture was measurably bifunctional . This is likely due to the low precursor frequency and/or the state of immunologic tolerance of T cells reactive with antigens such as gp100 or tyrosinase ,22,23. TThe approach for generating bifunctional T cells we describe in this study may be feasible for viral infections and malignancies and may represent a powerful approach for those patients that otherwise would fail immunotherapy due to the accumulation antigen- or MHC-loss variants.PBMC, peripheral blood mononuclear cells; TCR, T cell receptor; TAA, tumor-associated antigen; CMV, cytomegalovirusThe authors declare that they have no competing interests.AL designed the experiments, performed the transductions, carried out the cocultures and flow cytometry and prepared the manuscript. GC engineered the CMV minigene and the CMV-expressing COS cells, and edited the manuscript. MN conceived of the study, oversaw design and execution of the experiments, and finalized the manuscript.


Lung cancer causes approximately one million deaths each year worldwide and protein p53 has been shown to be involved in the intricate processes regulating response to radiation and/or chemotherapeutic treatment. Consequently, since antibodies against p53 (anti-p53 antibodies) are associated with mutations within the p53 gene it seems likely that these antibodies could, hypothetically, be correlated with prognosis.Serum samples from patients with non-small cell lung cancer (NSCLC) admitted to the Department of Oncology, University Hospital, Uppsala, Sweden, during 1983–1996 were studied. Anti-p53 abs were measured using a sandwich ELISA (Dianova, Hamburg, Germany).The present study included 84 patients with stage IIIA-IV (advanced NSCLC). At least three serum samples from each patient were collected and altogether 529 serum samples were analysed for the presence of anti-p53 antibodies. The median value of anti-p53 antibodies was 0.06 (range 0 – 139.8). Seventeen percent of investigated NSCLC first serum samples (n = 84) expressed elevated levels of anti-p53 antibodies. Anti-p53 antibodies were not correlated to tumour volume or platelets.Survival analysis showed that anti-p53 antibodies were not associated with survival as revealed by univariate analysis (p = 0.29). However, patients with adenocarcinoma had a significantly poorer survival if they expressed anti-p53 antibodies (p = 0.01), whereas this was not found for patients with squamous cell carcinoma (p = 0.13). In patients where the blood samples were collected during radiation therapy, a statistically significant correlation towards poorer survival was found (p = 0.05) when elevated anti-p53 antibodies levels were present. No correlations to survival were found for serum samples collected prior to radiation therapy, during chemotherapy, or during follow-up. When anti-p53 antibodies were measured continuously, no increase in median anti-p53 values was observed the closer the individual patient come to death.The result of the present retrospective study indicates that anti-p53 antibodies are not suitable for predictions concerning selection of patients with a more favourable outcome. Further prospective studies are, though, needed to fully elucidate this issue. Lung cancer causes approximately one million deaths each year worldwide . TreatmeThe continuous progress in the field of lung cancer biology has resulted in gradually increased insights into the intricate processes resulting in development and progression of malignancy. One of the first proteins whose occurrence was thought to affect prognosis was p53. This protein was identified during the late 1970s . EndogenThe question if mutations within the p53 gene are correlated to radiosensitivity is not defined. In an in vitro study from our group, we found that p53 mutations located in exon 7 were associated with significantly higher radiosensitivity than in those cell lines expressing p53 mutations in other exons . In a clAntibodies against p53 can be detected in sera from patients with cancer and a correlation exists between mutations in the p53 gene and antibodies against p53 in sera . In a stIn the present study, we included 84 patients with stage III-IV, donating 529 serum samples with the intention to investigate if predictions concerning outcome can be made and to investigate whether or not increased amounts of anti-p53 antibodies develop during disease progression, a question that, according to our knowledge, has not been explored previously.Serum samples from patients with NSCLC admitted to the Department of Oncology, University Hospital, Uppsala, Sweden, during 1983–1996 were studied. All patients gave informed consent prior to the collection of blood samples and the samples were stored at -70°C until analyzed. The study was reviewed and approved by the research ethics committee, Uppsala University, Uppsala, Sweden. The inclusion criteria were: a verified histology of NSCLC, advanced NSCLC defined as stage IIIA-IV according to TNM and a minimum of three serum samples donated from each patient during progression of the disease. The patients included were all followed from admittance until death.The first serum samples from 67 patients included in this study had previously been investigated for the presence of antibodies against p53 . In the The clinical parameters investigated were: age, gender, histology, performance status, weight reduction, smoking, tumour volume (according to RECIST criteria), objective response, subjective response  and the presence of anti-p53 antibodies. Complete information concerning clinical parameters could not be obtained due to inadequate information in the clinical charts.Blood was collected in 7 ml serum tubes without additive (367609, Becton Dickinson, Rutherford, NJ). Anti-p53 abs were measured using a sandwich ELISA (Dianova, Hamburg, Germany). Human recombinant p53 was bound to microtiter plates. Standards and samples were pipetted into the wells. After incubation and washing, a horseradish peroxidase conjugated polyclonal goat anti-human IgG was added. After renewed incubation and washing, a chromogenic substrate was added and the colour intensity was measured at 450 nm in a Titertek Multiskan. A relative index for patient sera was calculated as follows: E450 (sample) - E450 (low control)/E450 (high control) - E450 (low control). The ELISA assays were performed without knowledge of clinical data. According to the manufacturer's instructions, serum samples with an anti-p53 antibody index >1.1 were considered positive, whereas a serum sample with an index <0.9 was considered negative. Serum samples with an index between 0.9–1.1 were considered intermediately positive.Survival was estimated using the Kaplan-Meier product limit method, where univariate analysis was performed using a log-rank test. Cox regression analysis was performed to investigate if certain continuous factors had a significant effect on survival or to perform multivariate survival analyses. Spearman's rank order correlation was utilised for tests of associations between factors. The survival analysis together with the descriptive statistics is based on the first serum sample collected for each patient, whereas the correlation analyses were performed using all sera samples.In order to investigate if the levels of anti-p53 antibodies increased during the progression of the disease a statistical model was designed. In the model, time zero was set to be the date of pathological diagnosis and time one was set to time of death, i.e. the time to death was standardized for all patients. Using a fixed effect leased square estimator to allow for individually different starting values of anti-p53 antibodies, the effect on anti-p53 antibodies from diagnosis to death was calculated.In the descriptive statistics, range was defined as the minimum and maximum. Throughout the paper a 5% significant level was used.The median value of anti-p53 antibodies was 0.06 (range 0 – 139.8). Seventeen percent of the investigated NSCLC first serum samples expressed elevated levels of anti-p53 antibodies according to the manufacturer's instructions. From the time of pathological diagnosis until the time of death, no statistically significant effect on levels of anti-p53 antibodies was found (p = 0.8).Anti-p53 antibodies were not correlated to tumour volume (p = 0.19) or platelets (p = 0.27). A numerically higher median value of anti-p53 antibodies was found for adenocarcinoma patients (index level: 0.12) than for squamous cell carcinoma patients (index level: 0.08).The median anti-p53 antibody level, prior to therapy, was statistically significantly elevated in comparison with serum samples were collected during follow-up p < 0.001) .Descriptive data including survival, median and range for anti-p53 antibodies, as well as univariate analysis (based on the first serum sample for each patient), are shown in Table Analysis concerning histology and the presence of anti-p53 antibodies showed that patients with adenocarcinoma had a significantly poorer survival if they expressed anti-p53 antibodies (n = 23, p = 0.01). This was not found for patients with squamous cell carcinoma (n = 59, p = 0.13).Survival analysis based on when the first serum sample was collected in relation to therapy revealed that anti-p53 antibodies collected prior to radiation therapy (continuous variable) were not associated with survival (n = 53, p = 0.14). In patients from whom blood samples were collected during radiation therapy, a statistically significant correlation towards poorer survival was found (n = 13, p = 0.05). However, no correlations to survival were found when the first serum sample was taken during chemotherapy (5 patients), or during follow-up (n = 12, p = 0.68).Survival analysis showed that increased amounts of anti-p53 antibodies were not associated with survival according to univariate analysis p = 0.29) In the present study, anti-p53 antibodies have been investigated and correlated to various clinical parameters with the intention of studying if predictions concerning a more favourable outcome can be made for NSCLC patients, based on the presence or absence of these antibodies.Correlation analysis showed that anti-p53 antibodies were not correlated with tumour volume (measured according to RECIST criteria). These results support results of a previous study from our group, in which we neither did find a correlation between anti-p53 antibodies and tumour volume in patients with NSCLC prior to thoracic surgery .The clinical utility of anti-p53 antibodies in NSCLC patients with advanced disease has not yet been defined. In a previous study by our group, the presence of anti-p53 antibodies did not correlate to survival but the presence of anti-p53 antibodies prior to radiation therapy resulted in increased survival for anti-p53 positive patients . In the In the present study, the presence of anti-p53 antibodies was not correlated to survival when analyzed prior to radiation therapy, possibly because only patients with stages IIIA-IV were included, whereas our previous study also included patients with stages I-II. In patients where the first serum sample was collected during radiation therapy, a correlation to poorer survival was found (p = 0.05). Naturally, since the number of investigated patients was small, the obtained results should be interpreted with caution.The issue of using anti-p53 antibodies in monitoring NSCLC patients has not yet been determined. Zalcman et al. used anti-p53 antibodies in NSCLC patients during follow-up in order to detect relapse , and conThe result of the present retrospective study indicates that anti-p53 antibodies are not suitable for predictions concerning selection of patients with a more favourable outcome. Further prospective studies are, though, needed to fully elucidate this issue.The authors have fulfilled the criteria for authorship according to rules stated and used in medical journals.None declared.The pre-publication history for this paper can be accessed here: