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 . 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 • 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  .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  = 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  = 55.35, p < 0.0001; for the post-measurement, F = 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 . 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 , 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  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  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 . 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 , 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 , almost 8,400 fewer than the lowest threshold of 0.7 . 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  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 . 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  in houses with a gas heater were significantly higher than those  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 . 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 , 1300 MEL , and SK23 MEL  and renal cell carcinoma cell line UOK131  were maintained complete medium (CM) which consisted of RPMI 1640 medium  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)  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 . 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  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  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  in 24 well tissue culture plates pre-coated overnight with 10 μg of anti-CD3 mAb  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 , 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 .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 .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 . Anti-p53 abs were measured using a sandwich ELISA . 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 . This was not found for patients with squamous cell carcinoma .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 . In patients from whom blood samples were collected during radiation therapy, a statistically significant correlation towards poorer survival was found . However, no correlations to survival were found when the first serum sample was taken during chemotherapy (5 patients), or during follow-up .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:


An increasing body of evidence implicates both brain inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). The relevance of their interaction in vivo, however, is unknown. Previously, we have shown that separate pharmacological targeting of these two components results in amelioration of the amyloidogenic phenotype of a transgenic mouse model of AD-like brain amyloidosis (Tg2576).In the present study, we investigated the therapeutic effects of a combination of an anti-inflammatory agent, indomethacin, and a natural anti-oxidant, vitamin E, in the Tg2576 mice. For this reason, animals were treated continuously from 8 (prior to Aβ deposition) through 15 (when Aβ deposits are abundant) months of age.At the end of the study, these therapeutic interventions suppressed brain inflammatory and oxidative stress responses in the mice. This effect was accompanied by significant reductions of soluble and insoluble Aβ1-40 and Aβ1-42 in neocortex and hippocampus, wherein the burden of Aβ deposits also was significantly decreased.The results of the present study support the concept that brain oxidative stress and inflammation coexist in this animal model of AD-like brain amyloidosis, but they represent two distinct therapeutic targets in the disease pathogenesis. We propose that a combination of anti-inflammatory and anti-oxidant drugs may be a useful strategy for treating AD. Alzheimer's disease (AD) is the most common, complex and challenging form of neurodegenerative disease associated with dementia in the elderly. Neuropathological examination of the AD brain shows extensive neuronal loss, accumulation of fibrillar proteins as extra-cellular amyloid β (Aβ) plaques, and as neurofibrillary tangles (NFTs) inside neurons . HoweverIn the present study, we examined whether the simultaneous administration of an anti-oxidant, vitamin E, with an anti-inflammatory drug, indomethacin, would exert an additive anti-amyloidogenic effect in the Tg2576 mouse model of AD-like Aβ brain amyloidosis, one of the most extensively studied mouse models of AD . Signifiin vivo and significantly reduced brain inflammation [The genotype and phenotype features of the heterozygote Tg2576 mice that we studied here have been described in earlier reports on these mice from our group . Mice weammation . The higammation . During Animals were anesthetized and euthanized following procedures recommended by the Panel on Euthanasia of the American Veterinary Medical Association. They were always perfused intra-cardially for 30 min with ice-cold 0.9% phosphate buffer saline (PBS), containing EDTA (2 mM/L) and BHT (2 mM/L), pH7.4. Brains were removed and one hemisphere was fixed by immersion in 4% paraformaldehyde in 0.1 M PBS (pH7.4) at 4°C overnight, blocked in the coronal plane, and embedded in paraffin as previously described for immunohistochemistry ,17, The 2α-VI by ion chemical ionization gas chromatography/mass spectrometry assay, as previously described [2 and TxB2 by a standardized ELISA kit following the manufacturer's instructions (Cayman Chem. Com.). Briefly, extracts were diluted with acetate buffer and purified through an affinity column. The purified samples were evaporated, re-dissolved in the assay buffer and applied to 96-well plates pre-coated with goat anti-serum IgG and incubated with PGE2 or TxB2 monoclonal antibodies. The plates were rinsed with washing buffer and developed using Ellman's reagent for 60–90 min at room temperature with gentle shaking. Specific concentrations were determined spectrophotometrically and expressed as pg/mg tissue.Tissue samples were minced and homogenized, and total lipid extracted with ice-cold Folch solution . Lipids were subjected to base hydrolysis by adding aqueous 15% KOH and then incubated at 45°C for 1 hr for measurement of total iPFescribed ,16,17. IIL-1β levels were measured by a standardized sandwich ELISA kit following the manufacturer's instructions (Endogen Pierce). Briefly, equal amounts of sample were loaded onto 96-well plate pre-coated with monoclonal antibody against mouse IL-1β overnight at 4°C. The plates were rinsed three times with washing buffer and developed with streptavidin-horseradish peroxidase (HRP) . SpecifiTotal protein carbonyls in tissue were determined by using the Zenith PC test kit according to the manufacturer's instructions (Zenith Tech.) . BrieflyAn aliquot of brain homogenates was electrophoresed on a 10% acrylamide gel under reducing conditions. Protein were transferred to a polyvinylidene membrane before blocking in 10% nonfat dry milk for 2 hr. Blots were incubated with monoclonal antiboby against glial fibrillary acidic protein (GFAP) (2.2B10) , or an anti-beta actin  antibody overnight at 4°C. After three rinses, blots were incubated with HRP-conjugated goat anti-mouse for 45 min before development with chemiluminescent detection system using ECL (Amersham). Bands were quantified using densitometric software . The anti-GFAP is a monoclonal antibody, and its characterization has previously been published  . Subsequently, sections were incubated with a biotinylated antibody against Aβ (4G8) , at 4°C overnight [Serial 6-μm-thick paraffin sections were cut throughout each brain, and mounted on APES-coated slides. Sections were deparaffmized, hydrated, rinsed with PBS and pre-treated with formic acid (88%) for 10 min to antigen retrieval, and with 3% Hvernight ,17. Sectvernight ,16,17. At test corrected for multiple comparisons. Significance was set at p < 0.05.Data are expressed as mean ± standard error of mean (S.E.M.), analyzed by analysis of variance (ANOVA), and subsequently by student unpaired 2-tailed 2 and suppression of TxB2 levels in tissue homogenates from total cortex and hippocampus (Table 2α-VI (a reliable biomarker of lipid peroxidation), as well as protein carbonyls (known biomarkers of protein oxidation) were both significantly decreased  added to their diet, plus indomethacin (l0 mg/liter) in their water, and they were treated until they were 15 months old. Notably, at 8 months of age, the Tg2576 mice show elevated brain levels of soluble and insoluble Aβ as well as isoprostanes, relative to their non-transgenic littermates, but they show no evidence of any brain Aβ deposits, while following the initial onset of mature plaque-like brain deposits at about 11–12 months of age, the Tg2576 mice show abundant Aβ deposits and higher levels of isoprostanes in neocortex and hippocampus a 15 months of age ,15,20. AWestern blot analysis was used to determine the effect of the drug treatment on GFAP levels, a marker of astrocytosis . These lNext, we assessed the effect of indomethacin and vitamin E on brain levels of soluble and insoluble Aβ1-40 and Aβ1-42. As expected for their age, Tg2576 mice on placebo showed elevated levels of both forms of these peptides in their cortex and hippocampus Figure , whereas2-isoprostanes (lipid peroxidation); protein carbonyls, nitrotyrosine (protein oxdidation); 8-hydroxy-2'-deoxyguanosine (DNA oxidation) [Caernorhabditis elegans model, which over-expresses Aβ1-42 [There is substantial evidence implicating both oxidative stress and inflammatory mechanisms in AD pathogenesis. Evidence for oxidative stress derives from both human (post-mortem and living patients) studies, and transgenic mouse models of the disease. There is a long list of surrogate markers of reactive oxygen species-mediated injury that have been found increased in the brain and cerebrospinal fluid of AD patients. It includes, just to mention a few, malondialdehyde, 4-hydroxynonenal, Fidation) -5. Transidation) ,21. Oxids Aβ1-42 ,22,23. Fs Aβ1-42 ,25. TakeInflammatory mechanisms are also operative in the AD brain and significantly contribute to the pathophysiology of the disease. Although classical defined inflammation, including such features as edema and neutrophil invasion, is not seen in the AD brain, hallmark of innate immune response are constant elements of the neuropathology associated with brain degeneration in AD . FurtherLim et al. first reported that chronic administration of the NSAID ibuprofen to Tg2576 reduces total Aβ levels, amyloid burden and brain inflammation . More reIn the present study, we extended these previous observations by examining whether administration of indomethacin in combination with vitamin E would result in a better anti-amyloidotic effect. Our findings show that soluble Aβ1-40 and Aβ1-42 levels were reduced by ~65%, while the insoluble fractions were decreased by ~55%. Consistently, we observed a better and more diffuse effect also on the amount of amyloid deposited in the brain at the end of the study. Finally, the two drugs together produced an additive affect on brain inflammation and oxidative stress ,17.Our results confirm previous observation where low-dose curcumin, a drug with reported both anti-oxidant and anti-inflammatory activities, reduced total Aβ and plaque burden . HoweverIn our study, we used two different drugs with a more restricted therapeutic target to provide further evidence that both oxidative stress and inflammation are indeed functionally relevant in the development of the phenotype of these animals. However, we also provide new information on the critical issue of the in vivo relationship between these two events. Thus, our results suggest that brain inflammation and oxidative stress are two separate events, which work in concert to modulate the development of this AD-like brain Aβ amyloidosis model. Previously, we have shown that a full dose of indomethacin alone despite a significant reduction in brain inflammation had only a marginal effect on brain oxidative stress in the Tg2576 mice . This fiIn summary, our findings support the hypothesis that oxidative stress and inflammation represent important but distinct therapeutic targets in AD-like amyloidosis. We conclude that a combination of therapeutic agents targeting these different disease-modulating mechanisms might be rationally evaluated in the prevention or therapy of AD in humans.AD: Alzheimer's diseaseAβ: Amyloid β peptideTg: Transgenic mouse modelNSAIDs: Non-steroidal anti-inflammatory drugs2: Prostaglandin E2PGE2: Thromboxane A2TxBGFAP: Glial fibrillary acidic proteinIL-1β: Interleukin 1-β2α-VI: Isoprostane F2α-VIIPFThe authors declare that they have no competing interests.Yuemang Yao and Cinzia Chinnici have made substantial contribution to the acquisition of data and biochemical analyses. Hanguan Tang contributed to the immunohistochemical analyses. John Q. Trojanowski and Virginia M-Y Lee have been involved in the interpretation of data, and the critical revision of the manuscript for intellectual content. Domenico Praticò has been involved in the conception and design of the studies, interpretation of data, drafting and critical revision of the manuscript.


In this study, we derive an expected value for f, starting from a nearly neutral model of weak selection, and then assess the biological interpretation of this factor with evolutionary simulations.The f = 0.5  is derived for sequences that are evolving under the nearly neutral model of this study. However, this expectation is sensitive to violations of its underlying assumptions as illustrated with the evolutionary simulations.An expected value of f. However, as f is affected by a number of factors that limit its biological interpretation, this factor should normally be estimated as a free parameter rather than fixed a priori in a +gwF analysis.This study illustrates how selection, drift, and mutation at the population level can be linked to the rate matrices of models for sequence evolution to derive an expected value of  Violations of the weak selection model were incorporated in the simulations by: (1) heterogeneous sequences with sites drawn from different equilibrium base frequencies; (2) populations in disequilibrium due to changing Ne; and (3) an accelerated C to T substitution rate. Estimates of f for the simulated sequences were made with the K2P+gwF model. Forty simulations were run for each test condition, with the results for the f estimates summarized as their means and twice their standard errors. In the first set of simulations, six categories of sites with different equilibrium distributions were considered . In contrast, for the simulated heterogeneous sequences , their values of f varied significantly in either direction from 0.5. Analogous results were obtained for the simulations of homogeneous and heterogeneous sequences under the HKY model (with f = 0.0 instead of 0.5). Thus, the value of f can vary considerably when heterogeneous sequences are analyzed with a +gwF model. Here, such deviations are a consequence of using a single rate matrix to analyze sequences that were derived from two or three different ones.Evolutionary simulations were conducted to examine the effects of violating certain assumptions in the above model of weak selection. Unless otherwise noted, these simulations were based on the K2P+gwF model with Ne was kept constant until the time of the most recent common ancestor for the four simulated sequences. Then, Ne was either left unchanged or was suddenly changed by a certain factor. The latter was done by replacing the rate matrix derived from equation (4), resulting in new equilibrium frequencies of the nucleotides. When Newas kept constant, the selective pressures and drift were left unchanged, thereby maintaining the same starting equilibrium frequencies throughout the phylogeny. Thus, the corresponding f estimates did not significantly differ from 0.5 (Fig. Ne lowered the value of f as the efficiency of selection was increased relative to drift [In the second set of simulations, 0.5 Fig. . In contequation , resultif in either direction from 0.5, even though their sequences were simulated in equilibrium (Fig. f can vary considerably when the rates for reciprocal mutations are unequal.In the third set of simulations, an acceleration in the C to T substitution rate was incorporated, thereby modeling an increase in their mutation rate due to the deamination of methylated C's in CpG pairs . The intium Fig. . Thus, tf parameter and rate matrices of the +gwF variants for sequence evolution. Our weak selection model relies on the fixation probabilities of mutant alleles with additive genie selection and equal mutation rates for reciprocal substitutions. What is now needed are additional studies that link other population genetics models to the +gwF variants [f parameter and +gwF rate matrices.This study illustrates how selection, drift, and mutation within a population can be linked to the variants . For exavariants , which ff parameter is complex and can be influenced by a number of different factors [f ill need to be estimated as a free parameter rather than fixed beforehand.Collectively, the three sets of simulations highlight that the  factors . This coGoldman and Whelan  focused Both authors contributed to the conception and design of this study and to the writing, reviewing, and final approval of this article. B.K. performed the simulations and parameter estimations.


The complement system forms the central core of innate immunity but also mediates a variety of inflammatory responses. Anaphylatoxin C3a, which is generated as a byproduct of complement activation, has long been known to activate mast cells, basophils and eosinophils and to cause smooth muscle contraction. However, the role of C3a in the pathogenesis of allergic asthma remains unclear. In this review, we examine the role of C3a in promoting asthma. Following allergen challenge, C3a is generated in the lung of subjects with asthma but not healthy subjects. Furthermore, deficiency in C3a generation or in G protein coupled receptor for C3a abrogates allergen-induced  responses in murine models of pulmonary inflammation and airway hyperresponsiveness. In addition, inhibition of complement activation or administration of small molecule inhibitors of C3a receptor after sensitization but before allergen challenge inhibits airway responses. At a cellular level, C3a stimulates robust mast cell degranulation that is greatly enhanced following cell-cell contact with airway smooth muscle (ASM) cells. Therefore, C3a likely plays an important role in asthma primarily by regulating mast cell-ASM cell interaction. H2 cytokine producing T cells, mast cells and ASM cells play central roles in the pathogenesis of asthma [Asthma, a complex airway inflammatory disease, is characterized by bronchoconstriction, airway hyperresponsiveness (AHR) and remodeling. Current consensus suggests that Tf asthma -7. The cf asthma ,9. A rolf asthma .C3a levels are elevated in bronchoalveolar lavage (BAL) fluid after segmental allergen challenge in asthmatic but not healthy subjects -14. FurtH2 cytokine production and eosinophil recruitment in BAL when compared to wild-type mice. Additionally, guinea pigs with a natural defect in C3aR expression were also protected from AHR in response ovalbumin to challenge with no effect on airway inflammation [Studies with animal models provided compelling evidence for C3aR activation in the development of AHR and inflammation. Humbles et al., , showed ammation . These iammation ,17,18.Aspergillus fumigatous and challenged intranasally with allergen, these mice experienced substantial decreases in both AHR and airway eosinophilia relative to wild-type mice. Furthermore, BAL levels of TH2 cytokines , IgE titres and mucous production were all significantly reduced in C3aR (-/-) mice. Allergen-challenged C3 (-/-) mice also display diminished AHR, lung eosinophilia and TH2 cytokine production when compared to wild-type mice [Recent studies using C3aR (-/-) mice provided new insights on the role of C3a on both AHR and airway inflammation . When seype mice . These fAspergillus fumigatous and mast cell tryptase also activate the complement pathway directly [Increased level of C3a in BAL of subjects with asthma implies a potential role for this apaphylatoxin in promoting airway inflammation. However, the cells responsible for C3a generation and the airway effector cells stimulated by C3a remain unknown. Plausibly, antibody generated during antigen sensitization may interact with allergen to activate the classical complement pathway. Additionally, airway epithelial cells and pulmonary macrophages secrete both C3 and several components of the alternate pathway of complement  -23. Thusdirectly ,24-26. TH2 cytokine response to intraperitoneal ovalbumin sensitization. Furthermore, C3a had no effect on TH2 cytokine production in response to T cell receptor ligation. Further, Taube et al., [H2 cytokine production and lung inflammation. Additionally, a small molecule antagonist of C3a receptor, when administered after sensitization but before challenge also caused significant inhibition of airway inflammation [Both antigen-presenting cells (APCs) and activated T cells express C3aR -30, rais et al.,  showed tammation . These f+-derived primary mast cell cultures [4 generation . These findings suggest that allergen induces mast cell degranulation by at least two mechanisms: cross-linking of FcεRI and via C3a generation following complement activation by allergen protease , which induce mast cell chemotaxis, survival and differentiation ,48. Inteytokines -52. ThusC3a has long been recognized as an agent that evokes force generation in smooth muscle. In guinea pigs, C3a-induced contraction of lung parenchyma may involve indirect effects of histamine and arachidonic acid metabolites . In miceRecently, investigations showed that human mast cells but not human or murine ASM express C3aR . InteresH2 lymphocyte and their cytokines IL-4, IL-5 and IL-13. These cytokines play key roles in the recruitment and activation of eosinophil, mucous production and IgE synthesis. Allergen challenge of sensitized C3 (-/-) and C3aR (-/-) mice decreased production of TH2 cytokines in BAL and substantially reduced recruitment of T cells, eosinophils and neutrophils in lung tissue [H2 effector function in murine model of allergen-induced inflammation. Accordingly, in human mast cells, C3a stimulates the production of MCP-1, RANTES [H2 cytokine and chemokine production in asthma  declare that they have no competing interests.